Behavioral Phenotyping of Rats Selectively Bred for Differential Levels of 50 Khz Ultrasonic Vocalizations

BEHAVIORAL PHENOTYPING OF RATS SELECTIVELY BRED FOR DIFFERENTIAL LEVELS OF 50 KHZ ULTRASONIC VOCALIZATIONS

Kelley M. Harmon

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF ARTS

December 2006

Committee:

H. Casey Cromwell, Ph.D., Advisor

Jaak Panksepp, Ph.D.

Dara Musher-Eizenman, Ph.D.

Kelley M. Harmon

ABSTRACT

H. Casey Cromwell, Advisor

In rats, the rates of 20 kHz and 50 kHz ultrasonic vocalizations (USVs) can be used as a selective breeding phenotype and variations in this phenotype can be an indicator of positive affective states and developmental differences. The 50 kHz USV is elicited by positive or rewarding stimuli (e.g., food, sex, drugs of abuse) and therefore can be an indictor of a positive affective state. Conversely, the 20 kHz USV is elicited by aversive stimuli (e.g., foot shock, presence of a predator, social defeat) indicating a negative affective state. In this study, we tested the effect of selectively breeding for 50 kHz USVs on a variety of social / emotional behaviors across the animal’s lifespan. These social / emotional measures consisted of observations of pup retrieval latency and maternal care behaviors, measurement of isolation distress calls and conditioned odor preference as pups (age 1-12 days), play behavior, and social investigation as juveniles (age 21-32 days) into adulthood (90+ days of age). Cross fostering was utilized to determine if the differences in behaviors were primarily a result of maternal care or genetic expression. Results indicate that animals selected for low levels of 50 kHz USVs show the greatest alterations in social behaviors compared to the random “control” line animals. The low line animals showed a slight decrease in maternal/pup bonding, increased isolation distress calls, and failed to show a preference for a maternally associated odor, a marginal decrease in dorsal contacts during rough-and-tumble play behavior, and significantly more investigation in the social investigation paradigm after isolate housing. The social behaviors of the high line animals did not consistently vary from the random line animals with the exception of more investigation iv

during social port testing. Based on cross fostering results, maternal care does not appear to explain the differences observed in these selectively bred lines. These results provide

implications for the study of genetics underlying emotional states, as well as contribute to the

research underlying the emotional changes in developmental disorders such as autistic spectrum

disorder.

“Laughter seems primarily to be the expression of mere joy or happiness.”

-Charles Darwin vi

This manuscript is dedicated to my family, both immediate and extended, who have provided a

great deal of support and encouragement during this project. vii

ACKNOWLEDGMENTS

First and foremost, I would like to acknowledge my advisor, H. Casey Cromwell for all of the planning and preparation that went into organizing this project, as well as the countless hours of reading and revising needed to prepare this manuscript. A sincere thank you for all of your efforts!

In addition, members, Jaak Panksepp and Dara Musher-Eizenman, who served on this committee, contributed invaluable insight and expertise to this project. Their efforts are greatly appreciated.

Jeff Burgdorf, one of the pioneers of this project, spent countless hours teaching and training me to make sure I was equipped with the knowledge and skills needed for this work.

Not only was he a mentor for this project and an invaluable resource, but a good friend who I look forward to working with in the future. Thank you, Jeff, for your patience and advice both

professionally and personally.

Acknowledgements must be given to Nic Baldwin and Kazusa Ako for all of their

assistance in data analysis. This work was financially supported by a J.P. Scott Center for

Neuroscience Fellowship to K.H. and by Research Incentive Grants from the Sponsored

Programs and Research Office at Bowling Green State University to H.C.C. I, as well as

everyone who worked on this project, would also like to thank the Department of Psychology at

Bowling Green State University and the individuals in the animal facilities for their support of

this project.

viii

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

General Introduction to Vocal Signals...... 1

Vocalizations in Rats as Signals ...... 1

Using Selective Breeding as a Tool...... 4

Selective Breeding According to Vocal Output...... 5

Behavioral Tests to Access Socialness of Selectively Bred Lines ...... 6

Pup Retrieval...... 7

Maternal Care Observations ...... 7

Isolation Distress Vocalizations...... 9

Conditioned Odor Preference ...... 10

Play Behavior...... 11

Social Investigation...... 13

CHAPTER I. SPECIFIC AIMS...... 15

CHAPTER II. METHODS ...... 17

Subjects ...... 17

Cross Fostering Technique ...... 18

Pup Retrieval ...... 18

Maternal Care Behaviors ...... 19

Isolation Distress Calls ...... 20

Conditioned Odor Preferences (COP) ...... 20

Play Behavior ...... 22 ix

Social Investigation...... 23

CHAPTER III. ANALYSIS...... 25

Pup Retrieval ...... 25

Maternal Care Behaviors ...... 25

Isolation Calls ...... 25

Conditioned Odor Preference ...... 26

Play Behavior ...... 26

Social Investigation...... 27

Correlation Analysis...... 28

CHAPTER IV. RESULTS...... 29

Pup Retrieval ...... 29

Maternal Care Analysis...... 29

Isolation Distress Vocalizations...... 31

Conditioned Odor Preferences...... 35

Play Behavior ...... 45

Social Investigation...... 61

Correlation Results...... 66

CHAPTER V. DISCUSSION...... 73

Social Development and Paradigm Comparison ...... 73

Major Findings for Selective Breeding...... 77

Influence of Maternal Care: Cross Fostering Technique...... 78

Neurobiology of Social Behavior ...... 79

Bipolarity of Positive and Negative Affect...... 83 x

Clinical Implications...... 85

Conclusions and Future Studies...... 87

REFERENCES ...... 89

APPENDIX A. INSTRUMENT ...... 104

APPENDIX B...... 105

APPENDIX C...... 106

Table C1 ...... 106

Table C2 ...... 107

APPENDIX D...... 108

Table D1 ...... 108

Table D2 ...... 109

Table D3 ...... 110

Table D4 ...... 111

Table D5 ...... 112

Table D6 ...... 113

Table D7 ...... 114

Table D8 ...... 115

Table D9 ...... 116

Table D10 ...... 117

Table D11 ...... 118

Table D12 ...... 119

Table D13 ...... 120

Table D14 ...... 121 xi

Table D15 ...... 122

Table D16 ...... 123

Table D17 ...... 124

Table D18 ...... 125 xii

LIST OF FIGURES

Figure Page

1 Pup Retrieval...... 30

2 Total Occurrences of Nest Building during the Light and Dark Cycles on

Postnatal Days Four and Eight...... 32

3 Total Occurrences of Nursing Behaviors during the Light and Dark Cycles

on Postnatal Days Four and Eight...... 33

4 Mean Number of USVs for Each Genetic Line During the Isolation Distress Test.. 34

5 Mean Number of USVs for Each Genetic Line After the High

and Low Lines Were Cross Fostered, During the Isolation Distress Test...... 36

6 Mean Number of Total Line Crosses During COP...... 38

7 Mean Number of Total Line Crosses During COP with

the High and Low Line Animals Having Been Cross Fostered...... 39

8 Mean Amount of Time Spent on Each Side of the COP Chamber...... 40

9 Mean Amount of Time Spent on Each Side of the COP

Chamber for High and Low Genetic Lines Consisting of

Cross Fostered Animals and the Random Line...... 41

10 Mean Amount of Time in Seconds Each Genetic Line,

Divided into Conditioning Group, Spent on Each Side of the COP Chamber ...... 43

11 Mean Amount of Time in Seconds High and Low Line

Cross Fostered Animals, Divided into Conditioning Group,

Spent on Each Side of the COP Chamber...... 44

12 Random Line Data for Each of the Play Sessions Separated by Gender...... 46 xiii

13 Play Behavior for each Genetic Line...... 47

14 High Line Animal Data for Each of the Play Sessions Separated by Gender ...... 51

15 Low Line Animal Data for Each of the Play Sessions Separated by Gender...... 52

16 Play Data for Cross Fostered High Line Animals, Cross Fostered Low

Line Animals, and for the Random Line Animals...... 53

17 Cross Fostered High Line Animal Data for Each of the Play

Sessions Separated by Gender ...... 55

18 Cross Fostered Low Line Animal Data for Each of the Play

Sessions Separated by Gender ...... 56

19 Mean Percentage of Dorsal Contacts and Pins During Play

Activity in the Random Line Animals (N=54) ...... 58

20 Mean Percentage of Dorsal Contacts and Pins During Play

Activity in the High Line Animals (N=28)...... 59

21 Mean Percentage of Dorsal Contacts and Pins During Play

Activity in the Low Line Animals (N=36) ...... 60

22 Baseline Investigatory Behavior in Social Port Apparatus...... 62

23 Mean Number of Nose Pokes in the Social and Nonsocial Ports for Each Day...... 65

24 Mean Number of Nose Pokes in the Social and Nonsocial

Ports for Each Day for Cross Fostered Animals...... 67 xiv

LIST OF TABLES

Table Page

1 Pearson Correlation Matrix for the Random Line Animals

for the COP Paradigm...... 48

2 Pearson Correlation Matrix for the Random Line Play

Behavior Data across All Six Testing Sessions ...... 63

3 Pearson Two-Tailed Correlation Matrix for the Random

Line from the Social Port Investigation Paradigm...... 68

4 Pearson Two-Tailed Correlations between Testing

Paradigms for the Random Line Animals...... 69

5 Pearson Two-Tailed Correlations between Testing

Paradigms for the High Line Animals ...... 71

6 Pearson Two-Tailed Correlations between Testing

Paradigms for the Low Line Animals...... 72

Genetics and Social Reward 1

INTRODUCTION

General Introduction to Vocal Signals

Emotions can be indicated by vocal and behavioral signals. Laughing and crying are two

prominent vocal signals in humans that provide an indication of emotional states (Provine,

2001). Historically, many researchers and philosophers like Aristotle, Darwin, Freud, and Kant speculated about the meaning and significance of laughter. Laughter indicates a positive emotional state and is correlated with social behaviors (Pearce, 2004). Crying or sobbing is an indicator of a negative affective state (Miceli and Castelfranchi, 2003). Although the exact role of vocalizations is unknown, vocalizations are known to reduce stress and anxiety. For example, vocalizations may function as an anxiolytic, perhaps by reducing elevated levels of stress hormones like serum cortisol and catecholamines, while also enhancing immune system functioning (Fry, 1992).

Many mammals have vocal signals that relate to emotion, and significant advances in emotional research have been made by using these vocalizations as a quantitative measure of emotion. Some examples of the vocal affective responses include laughter in humans, monkey vocalizations (Zoloth and Green, 1979), and rat vocalizations that are very high in frequency

(ultrasonic). It is possible that the evolutionary mechanisms involved in the production of human laughter also underlie the production of these nonhuman emotional response vocalizations

(Panksepp and Burgdorf, 2003), but this hypothesis needs to be validated through psychobiological human research and comparative approaches.

Vocalizations in Rats as Signals

Ultrasonic vocalizations (USVs) in rats are chirp-like ultrasonic vocalizations and are elicited under various contexts that involve both negative and positive emotional states (Sales Genetics and Social Reward 2

and Pye, 1974). Three different USVs are clearly separated in sonograms based on sound

frequency and duration (Brudzynski, Bihara, Ociepa, and Fu, 1993; Miczek, Tornatsky, and

Vivian, 1991) and each USV seems to have a specific context dependency. The first is a 40 kHz

USV that occurs during infancy in response to isolation from the dam and littermates (Miczek et

al., 1991; Sales and Pye, 1974), commonly referred to as an isolation distress call. The second

type of USV is a 20 kHz USV emitted by juvenile and adult rats in response to exposure to

predators (Blanchard, Blanchard, Agullana, and Weiss, 1991), exposure to pain (Tonoue, Ashida,

Makino, and Hata, 1986), during intermale fighting (Thomas, Takahashi, and Barfield, 1983),

and during the refractory period after copulation (Barfield and Geyer, 1975a). The third type of

USV is a 50 kHz USV that is emitted in juvenile and adult rats in response to solicitation of play

(Knutson, Burgdorf, and Panksepp, 1998), male approach and ejaculation during copulation

(McIntosh and Barfield, 1980), male and female social exploration (Blanchard, Yudko,

Blanchard, and Taukulis, 1993; Brudzynski and Pniak, 2002), male agonistic behaviors during

fighting (Sales and Pye, 1974), and during tickle-induced reward (Panksepp and Burgdorf,

2000).

Since each type of USV is elicited under a different and specific environmental condition,

it has been proposed that each type of USV is indicative of a different affective state. For

example, the 20 kHz USV is elicited under threatening situations; it is a potential indicator of

negative affect akin to anxiety evoked by a real or experimental aversive situation (Brudzynski,

2001; Miczek et al., 1991; van der Poel and Miczek, 1991). The infantile distress vocalization is

also elicited under aversive or threatening conditions such as maternal isolation and is thought to

be an indicator of a negative affective state. It has been posited that 40 kHz distress calls seen in

young rat pups are the precursor call to the adult 20 kHz call and that there is a linear function of Genetics and Social Reward 3

call frequency with larynx size (Knutson, Burgdorf, and Panksepp, 2002). However, this

relationship has yet to be shown empirically. The 50 kHz USV is emitted during situations of

potential reward, and researchers postulate that it is an indicator of positive emotional states

similar to excitement or eagerness (Knutson, Burgdorf, and Panksepp, 1999).

There are three current hypotheses for the function of the various USV types in rats. The

first suggests the USV emission is a mechanical by-product of thermoregulation or locomotor

activity (Blumberg and Sokoloff, 2001). This hypothesis purposes that vocalizations are a result

of a physiological process the pups undergo in an attempt to maintain warmth. The second hypothesis states USVs act as affective expressions (Knutson et al., 2002). The third hypothesis,

which is not mutually exclusive from the second hypothesis, posits that USVs enable or inhibit

specific types of social interactions (Brudzynski and Chiu, 1995; Geyer, McIntosh, and Barfield,

1978). Previous research has tested the efficiency of each hypothesis in its ability to explain the

function of USVs. Experiments concurrently measuring 50 kHz USVs and locomotor activity

have concluded that changes in locomotor activity cannot account for 50 kHz USV changes

(Burgdorf, Knutson, and Panksepp, 2000). Researchers have also investigated the role of USVs

in social signaling and have determined that social stimuli do not need to be present in order to

elicit USVs. Nonsocial rewards and punishments can elicit USVs, and therefore social

interaction is not necessary for production. However, current research supports the notion that

USVs are indicative of affective states that can affect social settings (see Knutson et al., 2002 for

review). Our general goal is to examine the genetics of social reward by selectively breeding

animals based upon the levels of USV production.

Genetics and Social Reward 4

Using Selective Breeding as a Tool

Selective breeding is an effective tool for developing animal models for human disorders

(Brunelli, 2005). Selective breeding permits modeling an aspect of a disorder that is thought to

depend upon genetic variations in the human population. Certain traits, or phenotypes, can be

selectively bred so that animals display a higher or lower frequency of the desired trait. The idea

is that through several generations of selecting for a particular trait, the genetic information

related to that trait has a higher probability of being expressed; therefore, creating increasing

differences between selected genetic lines (Plomin, DeFries and McClearn, 1991; Snustad,

Simmons, and Jenkins, 1997). One consequence of selective breeding, assuming that the high

and low USV systems are interdependent, is that genetic linkages between the selected systems

and control systems are revealed (DeFries, 1981). Researchers can then dissect the linkages of

these systems which will provide an understanding of the mechanisms controlling the systems in

both genetic lines (Deitrich, 1993).

Selective breeding can provide for more accurate animal models that realistically mimic

behavioral and physiological symptoms of disease. Social and developmental disorders such as

autism spectrum disorder are steadily increasing in the human population and show familial inheritance patterns (Korvatska, Van de Water, Anders, and Gershwin, 2002). Anxiety levels and

sensory sensitivity can be disrupted in children affected with autism and these phenotypes have

been examined in rats by using selective breeding (Hofer, Shair, Masmela, and Brunelli, 2001;

Lavi-Avnon, Shayit, Yadid, Overstreet, and Weller, 2005). Because of the genetic component of

this disorder, selective breeding, which can replicate genetic patterns, is a vital technique that can

be used in researching this disorder. In modeling components of autistic spectrum disorder and Genetics and Social Reward 5

other developmental disorders, we will use selective breeding of USVs to study social behaviors

through different stages of development.

Selective Breeding According to Vocal Output

Previous genetic research demonstrates that vocalizations can be used as a selective

breeding trait (Brunelli, Vinocur, Soo Hoo, and Hofer, 1997). Variations in isolation distress

calls in preweanling rats enable selective screening for these calls. Brunelli and colleagues have

shown that these differences in calls extend throughout the developmental stages (Brunelli,

Keating, Hamilton, and Hofer, 1996) and they have discovered differential developmental markers over time in the rats selected for the high rates of distress vocalizations (Hofer et al.,

2001). Their results show that animals displaying a high frequency of distress calls show greater urination and defecation responses to isolation and more rapid ear canal opening at tens days old.

This research has contributed to the understanding of USVs and development but further research needs to address physiological and functional relationships between these co-selected traits.

Little work has been done on the genetic basis of positive affective states and traits, despite the strong genetic component in humans (Kagan and Snidman, 1999; Lykken and

Tellegen, 1996). Positive emotional phenotypes have been specifically selected in dogs (Scott and Fuller, 1965) and Panksepp and colleagues have discovered a high frequency vocalization or chirp that is emitted during positive emotional states in rats (Panksepp and Burgdorf, 2000). Data strongly support the relationship between affective states and USVs, and also the relationship between social interaction and these 50 kHz chirps (Knutson et al., 2002; Panksepp and

Burgdorf, 1999). Rats that display high rates of 50 kHz vocalizations during tickling demonstrate more play behaviors and are often preferred play partners compared to conspecifics (Panksepp, Genetics and Social Reward 6

Gordon, and Burgdorf, 2002a). Situations involving rewarding stimuli such as food, sexual

partners, play opportunities, drugs of abuse, electrical stimulation of the lateral hypothalamus,

and social contact following acute isolation all elicit 50 kHz vocalizations (Panksepp, Knutson,

and Burgdorf, 2002b; Brudzynski and Pniak, 2002).

Behavioral Tests to Access Socialness of Selectively Bred Lines

Neuroscience has an increasing trend of using animal models to study social behaviors

(Beatty, Dodge, Dodge, White, and Panksepp, 1982; Blanchard et al., 1991; Brudzynski and

Chiu, 1995; Brunelli, 2005; Brunelli, Nie, Whipple, Winiger, Hofer, and Zimmerberg, 2006;

Gerall, 1963; Panksepp, 1998). Valid and reliable measures of social reward are needed to

examine gregariousness and social bonding in animals that express these high levels of

vocalizations. Panksepp and colleagues have been developing these measures over the last 30

years (Panksepp, 1998). Each paradigm or behavioral observation chosen: pup retrieval, maternal

care observations, isolation distress calls, conditioned odor preference, play behavior, and social

investigation selectively measures various social tendencies at different developmental time

points. We intend to use all of these paradigms to study the developmental differences between

animals selected for high rates of 50 kHz vocalizations (High Line) and animals selected for low

rates of 50 kHz vocalizations (Low Line).

In rats and many other organisms, experiences between birth and weaning can have

profound effects on the ability to exhibit normal social behaviors. For example, primate infants

who don’t have early attachment experiences fail to develop normal social behavior and social

problem solving behavior (Kramer, 1992). Handling and maternal separation of rat pups have

been known to effect rough-and-tumble juvenile play behavior (Arnold and Siviy, 2001; Genetics and Social Reward 7

Gonzalez, Lovic, Ward, Wainwright, and Fleming, 2000); therefore, it is important to assess the impact of maternal behaviors for the high and low genetic lines.

Pup Retrieval

One classic paradigm used to assess maternal care has been the pup retrieval task. This task is designed to quantify the maternal motivation to keep the nest in tact. In this paradigm, the nest is disrupted by removing the pups and placing them outside of the nest; then, the dam has the task of restoring the nest by retrieving the pups and placing them back into the nest. This paradigm has been used to assess maternal differences across different strains of rats (Siviy,

Love, DeCicco, Giordano, and Siefert, 2003), it has been used to determine the neural network for maternal care (Gammie, 2005), and it has been used to assess the influence maternal care had on the development of social behaviors, particularly the development of future maternal behaviors, in grown rats (Friedman, Berman, and Overstreet, 2006; Champagne, Francis, Mar, and Meaney, 2003; Gonzalez et al., 2000; Nelson and Panksepp, 1998; Fleming, Kraemer,

Gonzalez, Lovic, Rees, and Melo, 2002). Friedman and colleagues (2006) used this paradigm to show that slower retrieval times are associated with increased immobility during a forced swim test possibly indicating learned helplessness.

In addition to the pup retrieval task, we will investigate the influence of environment, via observations of maternal care, to determine variations between the groups. These data will provide evidence supporting or opposing the notion that the differences in social behaviors are a result of genetic contributions.

Maternal Care Observations

Many other characteristics contribute to maternal care besides pup retrieval. For example, nursing characteristics, anogenital licking, nest building, and crouching over the nest all serve a Genetics and Social Reward 8 purpose in establishing a maternal attachment (Numan and Insel, 2003; Champagne et al., 2003).

Anogenital licking provides stimulation to initiate urination and defecation responses from the pups (Numan and Insel, 2003). Nursing clearly provides nourishment to the litter. Nest building and crouching over the nest serve to protect and regulate body temperature for the young pups that would be unable to do so without the mother and littermates (Numan and Insel, 2003).

Extensive studies have been conducted to determine the impact of these behaviors, particularly licking/grooming, on the pups by using maternal deprivation and observation studies. Infants, both rodent and primate, deprived of maternal care for extended periods of time show dramatic increases in fearfulness, aggressive patterns of social behavior, and delayed cognitive abilities (Champagne et al., 2003). Therefore, differences found between the social behaviors in the genetic lines could be accounted for by variations in rearing. Making systematic evaluations of maternal care and comparing them across genetic lines would determine variations of certain aspects of maternal care between lines and how those variations influence adult prosocial and investigation tendencies. However, if observations of maternal care do not establish differences in rearing, then variations in social behaviors may be more accounted for by genetic contributions.

In order to assess differences in maternal care, we will examine the dams with their litters in their home cage during different phases of activity, the light and dark cycles and on two different postpartum days (Siviy et al., 2003). By examining the maternal care behaviors at postnatal day four and again at postnatal day eight, we have the advantage of comparing maternal behavior in the early to later postpartum periods to examine the impact of learning on the intensity and extent of maternal care.

Genetics and Social Reward 9

Isolation Distress Vocalizations

Previous research has a difficult time obtaining reliable and valid measures for positive affective states in animals, whereas measurements for negative affects states have been more obtainable. The bias towards negative affective state exists because negative affect has greater reliability, an increased external validity, and animals easily learn with negative reinforcement.

Isolation distress vocalizations have been a very effective method of studying affective states in preweanling animals (Brunelli et al., 1997; Winslow et al., 2000) because the consistency in eliciting vocalizations and the intensity of distress vocalizations is reliably high. Isolation distress vocalizations in various species have many neuropharmacological similarities to anxiety- like behaviors in adulthood and anxiety disorders in humans (reviewed in Hofer, 1995, 1996;

Miczek et al., 1991; Newman, 1991; Winslow and Insel, 1991). Previous research has indicated that isolation distress vocalizations are negatively correlated with positive affective states. This has been demonstrated with neuropharmacology in that benzodiazepines decrease isolation distress calls (Vivian, Barros, Manitiu, and Miczek, 1997) and also activity within an elevated- plus maze indicated that animals with a high rate of isolation distress vocalizations as infants were less likely to spend time investigating during this test (Dichter, Brunelli, and Hofer, 1996).

In this isolation paradigm, the animal is briefly removed from the nest and placed individually in an isolation chamber where the pup’s USVs are recorded for two minutes. Upon completing the two minute test, the pup is returned to the nest. By testing for isolation distress calls at an early age, we will obtain a measure for anxiety in rat pups, which could be an indicator for future developmental deficits and provide a window to social and emotional processes.

Genetics and Social Reward 10

Conditioned Odor Preference

Many researchers have studied separation distress or negative affective states (Nitschke,

Bell, Bell, and Zachman, 1975; Brunelli et al., 1997; Winslow et al., 2000), but fewer researchers have attempted to examine the positive affective states associated with attachment in a laboratory setting. Panksepp and colleagues have examined rat pups, young chicks, and young canines in

forming prosocial behaviors and attachment bonding (Nelson and Panksepp, 1998; see Panksepp,

1998 for review). Research has shown that maternal dams are motivated to interact with their

pups (Malenfant, Barry, and Fleming, 1991) but this project aims to examine if the pups are

motivated to interact with their mother. The behavioral paradigm used is the conditioned odor

preference (COP) task which is quite similar to the conditioned place preference paradigm used

to study reward and reinforcement (Bardo and Bevins, 2000; Tzschentke, 1998). Drug

reinforcement and hunger/thirst/reproductive systems have been classic reinforcement and

motivational systems examined with conditioned place preference.

Using the conditioned odor preference paradigm to study social reward has many

advantages compared to other measures of social reward. One major advantage is being able to

test the animals in a reward free state; and therefore, test the animal’s behavior independent of

direct reward exposure. The animal is tested in the absence of the primary rewarding stimuli and

is forced to recall the memory of the reward stimulus during the testing period in order to

demonstrate a spatial preference. Another advantage is that this paradigm allows for

simultaneous testing of reward and locomotor activity. By measuring not only the time spent in

each compartment but the amount of locomotor activity of the animal, we get a measure of

reward preference and also the overall activity level of the animal. Finally, this COP paradigm

can be and has been easily adapted to various other species for testing and is noninvasive. Genetics and Social Reward 11

The COP apparatus has been used to examine the influence of oxytocin on social attachment and learning. Oxytocin mediates many different prosocial behaviors including maternal behavior, sexual behavior, and social memory (Nelson and Panksepp, 1998; Gammie,

2005) and oxytocin’s role in preweanling animals was examined to determine how it could mediate the acquisition of an odor associated with the mother dam. This study by Nelson and

Panksepp (1996) was used as a template for the present research strategy. Preweanling rat pups were conditioned with either oxytocin, an oxytocin antagonist, or saline and were tested in the

COP apparatus for conditioned approach and place preference using olfactory cues. Conditioning the animals involved exposing them to a novel olfactory cue paired with the unconditioned stimulus, which in this case was exposure to their mother. The maternal dam was coated on her ventral surface with the novel olfactory cue, lemon extract. Three 30 minute conditioning sessions were completed in one day with a three-hour maternal deprivation period before and between sessions. Rat pups treated with either saline or oxytocin demonstrated a strong preference for the olfactory cue side of the apparatus whereas the oxytocin antagonist group failed to develop a strong preference for the maternally associated odor. This research provided insightful data regarding the neurochemistry, specifically the role of oxytocin, in infant-maternal attachment and the paradigm allows for investigation of maternal attachment and social learning in young rats. This study sought to elaborate on and potentially integrate the previous research finding.

Play Behavior

Most mammalian species demonstrate a strong urge to play at a young age. This drive for play includes behaviors of play fighting, rough-and-tumble play, and social play (see

Panksepp, 1998 for a review). Several research groups have proposed different functions for this Genetics and Social Reward 12

play behavior and most agree that the role of play is to prepare the animals for future social

encounters (Hofer and Shair, 1992). Many adult organisms retain their playful sequences

expressed during childhood, and use them for interactions later in life with other organisms. Play

behavior functions to maintain and organize social hierarchies and thus, is a very informative

behavioral strategy to measure (van den Berg, Hol, Van Ree, and Spruijt, 1998). There may be

functional homologies between human and animal play (Panksepp, 1998), therefore the study of

animal play provides insights to the biology and chemistry of emotion and social reward for

humans.

Animals deprived of play behaviors during rearing show disturbed social (Meaney and

Stewart, 1979), agonistic (Lore and Flannelly, 1977), and sexual behavior (Gerall, 1963; Gerall,

Ward, and Gerall, 1967; Gruendel and Arnold, 1969; Hard and Larson, 1968). More recent work

by van den Berg et al. (1998) indicates that play is indispensable for development of social

coping. They found that animals deprived of juvenile play did not differ from the socially reared animals in nonsocial tests, i.e. open-field tests, but did differ significantly when tested in social challenges, i.e. sexual interaction tests.

Panksepp and others uncovered several facets of play behaviors including environmental, sensory, gender, and learning factors that effect play (Siviy and Panksepp, 1987; Panksepp,

1980; Panksepp, Burgdorf, Bainfeld, Kroes, and Moskal, 2004). Panksepp and colleagues revealed a developmental time window between 20-40 days postnatally when environmental and social factors can influence the pattern and frequency of play behaviors so much so, that even one day of social isolation can increase peaks in play patterns (Panksepp, 1980). Most studies

using this methodology have indicated that under the proper testing conditions, including Genetics and Social Reward 13 appropriate partners and context, females in various species demonstrate the same levels of rough-and-tumble play as their male counterparts (Panksepp, Siviy, and Normansell, 1984).

Neuroanatomy and neurochemistry of play behaviors have been studied by several research groups in terms of reward brain regions and a variety of neurotransmitters.

Acetylcholine, serotonin, and dopamine have been found to be involved in play behavior production (Knutson and Panksepp, 1997; Beatty et al., 1982). In addition, several neuropeptides in the opioid family influence play (Panksepp, Jalowiec, DeEskinazi, and Bishop, 1985;

Normansell and Panksepp, 1990) as well as recent investigation into the changes in brain cholecystokinin (CCK) levels during rough-and-tumble play behaviors (Burgdorf, Panksepp,

Brudzynski, Kroes, and Moskal, 2005; Panksepp et al., 2004). Neuroanatomical mapping studies have localized regions of the brain that are activated during play behaviors (Gordon, Kollack-

Walker, Akil, and Panksepp, 2002). These include the dorsolateral tectum, inferior colliculus, dorsal periaqueductal gray, ventromedial hypothalamus, dorsal and ventral striatum, and somatosensory cortex.

An understanding of the genetics involved in play behavior will add critical elements as to the functional significance of play. Also, since we will be examining a wide range of testing ages, we hoped to find early indicators of social reward which will provide information about disrupted developmental processes that may lead to abnormal play.

Social Investigation

Social port investigation is a relatively rare paradigm measuring social gregariousness in paired animals. This unique paradigm measures the tendency of an animal to explore a small port associated with social reinforcement or a nonsocial port which indicates general exploration behaviors. This paradigm is very beneficial in testing social behaviors in adults since rough-and- Genetics and Social Reward 14

tumble play tends to wane as the animals age. Previous research using this paradigm has found

control animals displayed a significant preference for the port offering social reinforcement

(Panksepp, Nelson, and Bekkedal, 1997) and this effect can be manipulated by parameters such

as social isolation. Implementation of this paradigm allows for careful analysis of gregariousness

and exploration behaviors across the different genetic lines.

In this paradigm, a pair of animals was placed in separate experimental chambers with the option of interacting in a common port or in an opposing port without social reinforcement.

Photoreceptors placed around the ports automatically record the number of nose pokes the

animals make at each port, as well as the duration of each poke. Animals will only be tested

when weight is greater than 250 grams, usually around postnatal day 90. This standard was set so

animals are unable to escape through the ports; they will only be able to push their noses through

the port openings. Consequently, animals will be tested as adults providing the final test of social

reward along the developmental continuum.

This project is a valuable integration of behavioral genetics into the growing field of

social neuroscience. It has the potential of providing critical information about the production of

USVs in terms of social rewards as well as revealing USVs as indicators of juvenile

temperament. Since previous research has focused more heavily on negative affective states, this

project examines positive affective states and genetics to provide essential missing links in the

current research.

Genetics and Social Reward 15

CHAPTER I: SPECIFIC AIMS

I. Examine relationships between different types of social behaviors expressed at

various developmental stages.

Certain personality traits can remain consistent throughout the lifespan of certain organisms including humans (Kagan and Snidman, 1999; Lykken and Tellegen, 1996). An animal that expresses high levels of prosocial tendencies as a pup could then show facilitated social behaviors throughout its lifespan. We examined the relationship between social tendencies at different developmental time points in the rat to verify if there was a positive relationship between the three prosocial measures (COP, Play Behavior, and Social Port). We expected animals bred in the random line to show a positive relationship between the maternally- associated odor preference, play behavior and social investigation behavior.

II. Examine the impact of selective breeding for USVs on the expression of social

behavior.

Previous research indicates that animals selectively bred for distress vocalizations have fewer prosocial tendencies than control animals (Brunelli et al., 2006). We intended to see if animals selected for high levels of positive affect vocalizations (50 kHz) expressed more social learning and prosocial behaviors than controls. In addition, a longitudinal study was designed to reveal long-term patterns of social tendencies.

We expected high line animals to show reduced isolation distress vocalizations and weaker preference for the conditioned odor when compared to the random and low genetic lines. We expected the high line animals to show enhanced play behavior and social investigation before and after social isolation. Finally, we hypothesized the opposite results for the low genetic line meaning enhanced isolation distress vocalizations and stronger preference for the conditioned Genetics and Social Reward 16 odor, as well as a decrease in play behaviors and social investigation. All comparisons were relative to the random line. Genetics and Social Reward 17

CHAPTER II: METHODS

Subjects

All breeding animals were Long-Evans rats originally purchased from Harlan Animal

Facilities (Indianapolis, Indiana). Animals where transferred to Brock University Animals

Facility (St. Catharine’s, Ontario, Canada) where they were selectively bred for low, random and high genetic lines. At approximately 90 days of age, selectively bred animals were transferred to

Bowling Green State University. Animals were mated at Animal Facilities at Bowling Green

State University (Bowling Green, Ohio) with one male per female. For this study, 270 pups were used from 24 different litters. The animals were housed in clear plastic cages (65 X 24 X 15 cm) with food (Harlan Teklad Rat Chow #8604) and tap water ad libitum. Corn cob chips were provided for bedding. Subjects were maintained on a day-night cycle of 12:12 light/dark cycle

(lights on at 07:00) and room temperature was kept at 22ºC and humidity was controlled at 40-

50%. All procedures were approved by the Institutional Animal Care and Use Committee at

Bowling Green State University.

Random line animals were not intended to be identical to typical Long Evans rats, but were intended to reflect the impact of partial inbreeding. At one point, the random line animals had to be supplemented by bringing in new animals from Harlan Animal Facilities for use in another study. Behavioral testing and tickle-induced 50 kHz USVs from these new supplemental animals compared to the inbred random line animals were statistically indistinguishable (all p’s <

.05; Burgdorf, unpublished observation) indicating that the inbred random line animals are responding as do typical Long Evans rats. Not only does this provide a rationale for using the random line as a control group, but also permits generalizing their social behavioral profile as being typical of Long Evans outbred rats. Genetics and Social Reward 18

Cross Fostering Technique

After two weeks of pair housing (one male and one female per cage), pregnant females were individually housed in cages (65 X 24 X 15 cm) with corn cob bedding. No later than 48 hours after birth, randomly selected high line and low line pups were cross fostered to a dam of the other genetic line. 10 out of the 24 litters were cross fostered with a total of 34 cross fostered pups. High line pups were placed into a litter with a low line dam and low line pups were placed into a litter with a high line dam. Selection was made to equalize total number of pups and to equalize gender; therefore, pups, two or four, of the same gender were selected. Cross fostered pups were permanently tattooed on their forelimb paws to distinguish between pups that were not cross fostered. Animals which were not cross fostered were not tattooed.

Pup Retrieval

Pup retrieval is one of the hallmark behaviors of rodent maternal care. On postnatal day one, dams were tested on a standard pup retrieval task. All pups were removed from the home cage for a period of 2 minutes, while the dam remained in the home cage. After 2 minutes, the pups were returned to the home cage, but were placed outside of the nest. All data were videotaped for offline analysis by a blind experimenter.

Litters were not culled to a particular number and therefore, due to variation in litter size, pup retrievals were only measured for the first four pups retrieved. Retrieval of four pups was chosen because all litters except three had four or more pups. The litters with less than four pups participated in the retrieval task, but these data were not incorporated into the results. One dependent measure was the number of times the dam dropped the pups elsewhere besides the home nest, the pup could be dropped and picked back up several times before it is successfully returned to the nest (Stern, 1996). This measure was used to determine if dam’s pup retrieval is Genetics and Social Reward 19

disorganized and frantic, or if the dam was uninterested and lethargic (Friedman et al., 2006). If

the dam is picking up the pups and dropping them outside the nest, the retrieval behavior is

disorganized, if the dam is not retrieving the pups to the nest nor dropping them outside the nest,

the dam may be lethargic and uninterested. The number of dropped pups is one indicator of a type of maternal behavior. Videotapes were scored to determine the following other characteristics of the retrieval: latency to pick up the first pup and total time to retrieve four different pups to the nest (Gonzalez et al., 2000; Numan and Insel, 2003; Numan, Numan,

Schwarz, Neuner, Flood, and Smith, 2005) Testing session lasted 10 minutes. A behavioral scoring sheet was used and can be seen in Appendix A. Other than nest disturbances on postnatal day one, nests were left undisturbed through postnatal day eight, until habituation was initiated for isolation distress calls on postnatal day nine.

Maternal Care Behaviors

To examine whether maternal care varied between lines, we completed an in-depth examination of maternal care behaviors. The behavior of each dam was assessed at postnatal day four and again on postnatal day eight. Each litter was observed for six different 1 minute periods during the last hour of the light phase and the first hour of the dark phase of the light/dark cycle.

The 1 minute sample periods were randomly chosen from six 10 minute time blocks during each sampled hour, i.e., 1 minute was randomly chosen from minutes 1-10, another minute was randomly chosen between minutes 11-20, etc. Observations made during the dark phase were made under red light illumination. During each 1 minute observation period, the presence or absence of the following maternal behaviors was noted: dam was crouching over the nest in a nursing position, anogenital and/or body licking of the pups, autogrooming, and nest building.

The location of the dam, in or out of the nest, was also noted for each 1 minute observation Genetics and Social Reward 20 period. Behaviors were videotaped and analyzed offline using a behavioral scoring sheet, see

Appendix B.

Isolation Distress Calls

Isolation of rat pups leads to a short bout of distress calls. We wanted to examine if the production of these distress calls varied between the genetic lines. The isolation testing chamber, which was located in a testing room separate from the housing room, consisted of a 500 mL glass beaker with an ultrasonic microphone suspended approximately 25 cm above the base of the beaker. USVs were recorded using a high frequency bat detector, Pettersen D980 ultrasonic detector (Uppsala, Sweden) which digitally recorded at 196 kHz, and USVs were analyzed offline via sonogram (Avisoft Bioacoustics). The pups were habituated for 1 minute to the testing chamber on postnatal day nine. On postnatal day ten, the pups were removed from the colony room and were placed individually in the isolation testing apparatus for 2 minutes with

USVs being recorded. There were no other animals present in the testing room during the testing session. After testing, animals were transported back to the colony room and were returned to their home cage and mother. Animals were tested during the light cycle of a 12:12 light/dark cycle (lights on at 07:00). Data were manually scored offline for total number of 40 kHz distress vocalizations.

Conditioned Odor Preference (COP)

COP measures the affinity of the pup to its mother. The testing apparatus consisted of a

Plexiglas chamber (18 X 5 X 7.6 cm) which was divided lengthwise by visual marker into three equal sections of 6 cm. The testing apparatus was located in a testing room, separate from the colony room. The chamber, which has metal bars across the floor, was suspended 7.6 cm over Genetics and Social Reward 21 two equally spaced glass jars which contained either a cotton ball saturated with 1 mL of pure lemon extract or a cotton ball saturated with 1 mL of distilled water.

Pups were removed from their home cage in the colony room and were habituated to the

COP chamber on postnatal day ten for 1 minute. Conditioning began on postnatal day eleven and animals were separated into six conditioning groups: 1. High lines animals conditioned with a cotton ball 2. High line animals conditioned with the mother dam 3. Random line animals conditioned with a cotton ball 4. Random line animals conditioned with the mother dam. 5. Low line animals conditioned with a cotton ball 6. Low line animals conditioned with the mother dam.

Conditioning was conducted three times during postnatal day eleven with three hours of maternal deprivation proceeding each 30 minute conditioning session. Both conditioning and maternal deprivation time was performed in an experiment room other than the testing room or the colony room. Animals conditioned with the cotton ball were removed from their home cage and were placed in a novel cage (23.5 X 21 X 20.3 cm) with 4 cotton balls in each corner saturated with .25 mL of lemon extract on each cotton ball. The groups conditioned with the mother dam were removed from their home cage and were placed in an identical novel cage with the mother who had been saturated with 1 mL of lemon extract on her ventral surface immediately prior to being placed in the novel conditioning cage. All animals remained in the conditioning cages for 30 minutes. Following the third conditioning session, the mother was bathed with soap and water to completely remove the lemon scent from her ventral surface and she was returned, along with the pups, to the home cage.

Testing began the day immediately following the conditioning day (postnatal day twelve). Testing was preceded by three hours of maternal deprivation with the pups housed in a Genetics and Social Reward 22

group. Each testing session consisted of placing each pup in the middle of the testing apparatus

and giving the pup free access to the place preference apparatus for 5 minutes. The side

containing the lemon scent was counterbalanced across trials. Trials were recorded via

commercially available DVD recorder and camera to be analyzed offline. A trained

experimenter, blind to testing conditions, used a computer-based behavioral scoring system to accurately code compartment entries and time duration via joystick control.

Play Behavior

Juvenile rats, similar to many other organisms, display intense play bouts with conspecifics. Therefore, animals were paired for repeated play sessions with a same sex littermate within 10% of their body weight. The play arena, located in a separate testing room,

was a box (30.5 X 30.5 X 30.5 cm) with three stainless steel sides and one Plexiglas side to allow

for video recording from the side, and a Plexiglas floor. A testing session consisted of removing

the animals from their home cages in the colony room and placing them in the play arena located

in a testing room for 5 minutes with their play partner. Animals were immediately returned to

their home cage post testing. Testing continued for eleven consecutive days with play sessions

occurring every other day, postnatal days 22, 24, 26, 28, 30 and 32. Animals were isolate housed

after weaning and remained isolate housed for the duration of testing days to prevent play bouts

in the home cage during non-testing times and to enhance play behaviors (Panksepp, 1980). All

play behavior was videotaped to permit viewing of behavioral interactions. Videotaped play

behavior was scored by a trained experimenter in real time for two major categories, dorsal

contacts and pins. A pin was scored if one animal was lying on its back with the other animal on

top. A dorsal contact was scored if an animal touches the other with its front paws on the dorsal

surface between the neck and the rump, not including the tail (Panksepp et al., 1984). Behavioral Genetics and Social Reward 23

scoring was performed with computer-assisted software and equipment designed to accurately

tally the number of dorsal contacts and pins, as well as, pin duration. Behavior scoring was conducted by an experimenter blind to all testing conditions.

Social Investigation

The social port investigation paradigm was designed to examine variations in gregariousness between animals and in this case the different genetic lines. The social investigation apparatus consisted of two clear Plexiglas cages (65 X 24 X 15 cm) with two exploration ports measuring 1.58 cm in radius on each end. Cages containing corn cob bedding were placed end to end on a secure table top such that one port was facing the neighboring cage

which was designated the social port. The port on the opposing side of the cage was designated

the nonsocial port and was facing open space. The ports were designed to allow the animals to

place their noses into the ports, but were not able to escape. The social ports were placed

approximately 7 cm apart. Photo beams were located at the each port to count the number of

times an animal poked his/her nose into the port and also to tell the duration of each nose poke.

The photo beams transferred information to a DOS-based custom computer program (Headhov3)

for analysis.

Animals were habituated for 1 minute to the social investigation cages when all animals

in the litter weighed at least 250 grams, approximately postnatal day 90. After the habituation

day, animals were tested the following three consecutive days with their play partner in the other

cage for 30 minutes. After the habituation session, the animals were socially housed with their

play partner for 24 hours prior to the first testing session. Following the first social investigation

test day, the animals were housed in isolation for 24 hours and then retested. The 24-hours of

isolate housing examines the effect of social deprivation on gregariousness. Animals were Genetics and Social Reward 24 returned to pair housing after the second isolation testing day and 24 hours later, the animals were tested for the third and final day. Genetics and Social Reward 25

CHAPTER III: ANALYSIS

Pup Retrieval

Three one-way Analysis of Variance (ANOVAs) were utilized to determine group differences. The between-subject factor was the genetic line (High, Random, and Low) and the dependent variables were the mean latency to begin retrieving in seconds, the mean total duration of retrieving in seconds, and the number of times pups were dropped during the retrieval task. A separate one-way ANOVA was used for each dependent measure and t-tests were used for post hoc comparisons.

Maternal Care Behaviors

One ANOVA was used for each of the maternal behaviors observed (i.e. nursing, allogrooming, autogrooming, nest building, presence in nest) with a between-subject factor of genetic line (High, Random, Low, High Cross Fostered, or Low Cross Fostered). Post hoc t-tests were used to determine group differences.

Isolation Calls

We used two 2-factor ANOVA tests to examine the isolation distress call data. The first

ANOVA had a between-subjects factor for genetic line (High, Random, Low, High Cross

Fostered, or Low Cross Fostered) and the dependent variable was distress calls (40 kHz). The second ANOVA had the same between-subject factor for genetic line, but the dependent variable was higher frequency <50 kHz USVs. We did post hoc t-test comparisons to determine group differences. Pearson correlations were computed between the two types of pup’s calls, distress calls and the higher frequency <50 kHz USVs for each genetic line (High, Random, Low).

Genetics and Social Reward 26

Conditioned Odor Preference

Initially, the number of line crossed into either side of the apparatus was summed to determine the total number of line crosses. This permitted a comparison of locomotor activity

between the genetic lines. A one-way ANOVA was used for this analysis with the dependent

measure being total number of line crosses and a between-subject factor of genetic line (High,

Random, Low, High Cross Fostered, or Low Cross Fostered).

Then, two 4-factor mixed design ANOVA analyses were completed for the different

dependent variables. In the first 4-factor ANOVA, the within-subjects repeated measure was for

the dependent measure of time spent in each place, i.e. lemon or water side of testing apparatus.

Three between-subject factors included: conditioning group (Cotton Ball or Dam exposure),

gender (Male or Female), and genetic line (High, Random, Low, High Cross Fostered, or Low

Cross Fostered). The second 4-factor mixed design ANOVA used had the same between-subject

factors listed above, but the within-subjects repeated measure was for the dependent measure of

the number of entries in either the lemon or water side of the apparatus. T-tests were used for

post hoc comparisons.

A Pearson product moment correlation was completed for each genetic line to examine

the relationship between number of lemon entries, number of water entries, total time spent in

the lemon portion of the testing apparatus and the total time spent in the water portion of the

testing apparatus.

Play Behavior

For number of dorsal contacts, a 3-factor mixed design ANOVA was used. There was a within-subjects repeated measure for testing session (first, second, third, fourth, fifth, and sixth Genetics and Social Reward 27

play day) and two between-subject factors of gender (Male or Female) and genetic line (High,

Random, Low, High Crossed, Low Crossed).

A 3-factor mixed design ANOVA with the same factors as above were used (within-

subject for testing day (first, second, third, fourth, fifth, and sixth play day) and between-subject

factors of gender (Male or Female) and genetic line (High, Random, Low, High Crossed, Low

Crossed)) for the three additional dependent measures: pin number, total pin duration, and the

average pin duration. Post hoc t-tests were used for pairwise comparisons.

In order to determine if the genetic lines were performing pins and dorsal contacts and

the same proportions, a percentage calculation was made to determine the percentage of dorsal

contacts and pins performed out of the total number of play behaviors. The total number of play

behaviors was the summation of the number of dorsal contacts and the number of pins. The percentage of dorsal contacts was determined by taking the number of dorsal contacts divided by the total number of pins and dorsal contacts and then multiplying by 100. The same formula was used for pins across all six testing sessions.

Additionally, for each genetic line, Pearson correlation coefficients were calculated to determine the relationship between number of pins and number of dorsal contacts for each testing day.

Social Investigation

A 4-factor mixed design ANOVA was used to analyze the results of the social investigation test. The number of nose pokes was the dependent measure with two within-subject repeated measure factors which included port type (Social or Nonsocial Port) and housing condition (Pair Housed or Isolate Housed). Two between-subject factors included gender (Male or Female) and genetic line (High, Random, Low, High Cross Fostered, or Low Cross Fostered). Genetics and Social Reward 28

Duration of nose pokes was the second dependent measure for another separate 4-factor mixed

design ANOVA with the port type, housing condition, gender and genetic lines as the other four independent factors.

Pearson correlations were calculated to determine the relationship between number of social and nonsocial nose pokes and the amount of time spent in each port for the isolation day

and the third day of testing, which was the second day of pair housing.

Correlation Analysis

In order to complete the goals of Aim I mentioned earlier, two-tailed Pearson correlations

were computed for each genetic line for the three prosocial measures (Conditioned Odor

Preference, Play Behavior, and Social Port). These correlations were computed to determine if

there is a positive or negative correlation between the side preference for the testing chamber and

number of pins and dorsal contacts, and social port preference. Measures used to determine the

correlation for conditioned odor preference paradigm are the number of entries into the lemon

portion of the testing apparatus and the total amount of time in the lemon portion of the

apparatus. The measures used for the play behavior are the number of pins and dorsal contacts on

play day six, because all animals were showing the most elevated levels of play behaviors during

that play session. Finally, for correlational analysis with the social port testing, number of nose

pokes into both the social and non social ports on the isolate housing day and the second pair-

housing day were used. In addition, the total times spent in both the social and nonsocial port on

the same two days were used. Genetics and Social Reward 29

CHAPTER IV: RESULTS

Pup Retrieval

In the random line, rapid retrieval duration and low drop rates were noted (see Figure 1).

Similar retrieval characteristics were seen in the two genetic lines. Then, three one-way Analysis

of Variances (ANOVAs) were used to determine group differences (High, Random, Low)

between the three dependent measures: 1. latency to begin retrieving pups, 2. duration of

retrieving four pups, 3. number of times the dam dropped the pups outside of the nest. Figure 1

graphically illustrates the results for all three dependent measures. The ANOVA for latency to

begin retrieving pups did not reveal any significant differences between the genetic lines.

Furthermore, the ANOVAs for duration and number of drops also did not reveal significant

differences between the genetic lines. Therefore, our results indicate that there are no differences

in pup retrieval between the various genetic lines.

Maternal Care Analysis

Random line mothers showed no differences in the maternal care between light and dark

cycles or between early and late sample times. Conversely, within groups comparisons indicated

that low line animals showed differences between day and night observations and postnatal day

four and postnatal day eight observations. Low line dams were allogrooming significantly less at

night on postnatal day four than they were during the light cycle on postnatal day four (t(4) =

5.715, p < .01). However, low line mothers showed a significant increase in allogrooming at

night on postnatal day eight compared to postnatal day four (t(4) = -3.162, p < .05).

Comparing between the light and dark cycle, high line dams showed a significant decrease of bouts in the nest at night than in the light cycle on postnatal day four (t (4) = 3.400, p Genetics and Social Reward 30

Figure 1: Pup Retrieval

250 350 9

8 300 200 7 250 6 150 200 5

150 4 100 3 100 2 50 Drops MeanNumberofPup

M ean Latency to R in etrieve Sec 50 a uaino ereiginM ean Sec of Duration Retrieving 1

0 0 0 High n=5 Random n=6 Low n=4 High n=5 Random n=6 Low n=4 High n=5 Random n=6 Low n=4

Pup retrieval was denoted as the dam retrieving four different pups to the home nest. There were no significant differences between the lines for either of the three measures, latency to retrieve, duration of retrieval, and number of pups dropped during retrieval. Variations in litter size were not statistically significant with size ranging from 4 to 17 pups and with a mean of 10.6 pups per litter.

Genetics and Social Reward 31

< .05). High line dams showed significantly fewer bouts of autogrooming at night on postnatal

day eight than on postnatal day four (t(4) = 3.162, p < .05).

While there were no between group differences between the four observations periods, there were slight between group differences when examining the total occurrences cross all time samples. Figure 2 shows significantly fewer occurrences of nest building for the high line dams compared to the random line dams (t(9) = 2.568, p < .05). Additionally, there is a trend (p = .09) for low line dams to have less occurrences of nursing behavior across all observation periods compared to the random line dams (t(9) = 2.070, p = .07; see Figure 3).

While there are a few differences between the dams of each genetic line, there is no significant difference between genetic lines. Overall, the dams in this study did not show robust differences in maternal behaviors or in pup retrieval. It can be provisionally concluded from these results that differences between pups are a result of genetic influence or other epigenetic factor other than maternal care.

Isolation Distress Vocalizations

There was a significant main effect for distress calls (F(1, 215) =257.543, p < .001) which indicated all animals showed significantly more distress (40 kHz) calls than higher frequency calls during isolation testing (see Figure 4). A main effect for genetic line (F (2, 215)

=3.091, p < .05) pointed out that genetic lines had an effect on the number of both types of

USVs. There was also a significant interaction effect between calls and genetic lines (F (2, 215)

=12.786, p < .001). The interaction effect demonstrated the genetic line significantly predicted the most frequent type of USVs the animals would emit. Post hoc analysis indicated that the low line animals showed significantly more distress calls compared to the random line (t(137)=

2.367, p < .05). In addition, both low line and high line animals displayed significantly less high Genetics and Social Reward 32

Figure 2: Total Occurrences of Nest Building during the Light and Dark Cycles of Postnatal

Days Four and Eight

5 *

Total Occurences over all Observation Periods 0 High n=5 Random n=6 Low n=5

* indicates p < .05 Genetics and Social Reward 33

Figure 3: Total Occurrences of Nursing Behaviors during the Light and Dark Cycles on Postnatal

Days Four and Eight

14 # 12

ll Observation Periods ll Observation 10

Total Occurences for a 0 High n=5 Random n=6 Low n=5 # indicates p = .09 Genetics and Social Reward 34

Figure 4: Mean Number of USVs for Each Genetic Line During the Isolation Distress Test

160 High Line n=63 * 140 Random Line n=92 Low Line n=45 120

100

Mean USVs / 2 Min. ** 40 ** 20

0 Distress Calls High Frequency Calls

Notation indicates significant differences when compared to random line. * indicates p < .05, ** indicates p < .01.

Genetics and Social Reward 35

(>50 kHz) USVs than the random line (low line: t (137) =6.194, p < .01; and high line: t (155)

=4.036, p<01).

Figure 5 indicates the results for the cross fostered animals when compared to the random line animals reared by their biological dam. Although the results for the distress calls are

insignificant, there is visible similarity when compared with the distress call data from Figure 4.

Additionally, there are significantly fewer high frequency USVs in the high line compared to the

random line (t (100) =2.589, p < .05), while the low line shows a trend for fewer high frequency

USVs compared to the random line (t (98) =1.926, p < .09). In sum, high line animals reared by a

low line dam were not significantly different from high line animals reared by a high line dam in

terms of distress calls. Similarly, low line animals reared by a high line dam did not differ significantly from low line animals reared by low line dams in rate of distress calls.

There was a significant negative correlation between distress calls and high frequency 50 kHz calls for both the random and low line animals (Random: r = -.375, p < .01, Low: r = -.335, p < .05). For the random and low line animals, the more distress calls the pups emitted, the fewer higher frequency calls the pups made. High line animals did not show a significant correlation between distress call and higher frequency USVs as pups.

Conditioned Odor Preference

COP behaviors were examined in the random line animals to determine a baseline for conditioned odor preference. Random line animals show a slight, but statistically insignificant preference for the lemon side of the testing apparatus when they had been conditioned with the maternal dam (t (53) = 1.90, p = .06). Random line animals conditioned with the lemon scented cotton balls showed no preference for either side of the testing apparatus (see Figures 8 and 9). Genetics and Social Reward 36

Figure 5: Mean Number of USVs for Each Genetic Line After the High and Low Lines Were

Cross Fostered, During the Isolation Distress Test

180

160 High Line n=10 140 Random Line n=92 120 Low Line n=8

100

60 Mean USVs / 2 Min. * # 40

0 Distress Calls High Frequency Calls

Notation indicates significant differences when compared to random line. * indicates p < .05, # indicates p < .09. Genetics and Social Reward 37

Data were examined using both time in each side of testing apparatus and entries into each side as dependent measures.

To determine differences in mobility between the genetic lines, one-way ANOVA analysis was used. The dependent measure was the number of line crosses into both the lemon and water side of the apparatus. The total number of line crosses was examined to determine if either the high or low line animals showed increased locomotor activity. Figures 6 and 7 indicate that the low line animals, regardless of whether they were reared by their biological dam or a cross fostered dam, showed a significant increase in line crosses compared to the random line animals (for low line: t(161) = 5.959, p < .001; for cross fostered low line: t(119) = 2.693, p <

.01). The high line animals failed to show significant variations in line crosses compared to the random line animals.

There were no significant differences related to gender for main or interaction effects. We collapsed the gender variable for all remaining statistical tests. There was a main effect for genetic line (F(2, 260) =6.293, p < .01), which implied genetic lines differed in place preference.

There was an interaction between place preference and genetic line (F(2, 260) = 3.428, p< .05).

The low line animals showed a significant preference to the water side compared to the lemon side of the testing apparatus (t(78) = 3.737, p < .001) and also low line animals showed a significant preference for the water side of the chamber with compared with the random line

(t(179) = 4.078, p < .001). Furthermore, cross fostered low line animals also showed the same significant preference for the water side of the testing chamber as the biologically reared low line

(t(18) = 2.851, p < .05, see Figure 9). Cross fostered low line animals also spent significantly more time in the water side than did the random line animals (t(119) = 3.558, p < .001). Genetics and Social Reward 38

Figure 6: Mean Number of Total Line Crosses During COP

50 High n=73 45 Random n=103 *** Low n=60 40

Mean Number of Total Entries / 5 Min. 5

0 Entries

Individual studentized t-tests analyzed high to random and low to random comparisons. *** indicates p < .001. Genetics and Social Reward 39

Figure 7: Mean Number of Total Line Crosses During COP with the High and Low Line

Animals Having Been Cross Fostered

50 High n=16 45 Random n=103 ** Low n=18 40

10 Mean Number of Entries / 5 Min. 5

0 Entries

Individual studentized t-tests analyzed cross fostered high animals to random and cross fostered low animals to random lines. ** indicates p < .01. Genetics and Social Reward 40

Figure 8: Mean Amount of Time Spent on Each Side of the COP Chamber

180 Lemon Time *** 160 Water Time

140

120

100

40 Average Time in Sec. / 5 Min.

0 High n=73 Random n=103 Low n=60

Individual studentized t-tests analyzed high to random and low to random comparisons for both lemon time and water time. Individual studentized t-tests were used to determine significant differences in lemon versus water time for each genetic line. *** indicates p < .001.

Genetics and Social Reward 41

Figure 9: Mean Amount of Time Spent on Each Side of the COP Chamber for High and Low

Genetic Lines Consisting of Cross Fostered Animals and the Random Line

160 Lemon Time * 140 Water Time

120

100

40 Average Time in Sec. / 5 Min. 20

0 High n=16 Random n=103 Low n=18

Individual studentized t-tests analyzed high cross fostered to random and low cross fostered to random comparisons for both lemon time and water time. Individual studentized t-tests were used to determine significant differences in lemon versus water time for each genetic line. * indicates p < .05. Genetics and Social Reward 42

Therefore, cross fostered low line animals show the same behavioral profile as the low line animals that were reared by their biological dam.

An interaction effect was found for place preference and condition group, i.e., conditioning with the mother or conditioning with the cotton ball, (F(1, 260) = 4.628, p < .05).

Figures 10 and 11 illustrate this interaction. T-tests indicate that low line animals conditioned with the mother show a significantly different preference for the water side than random line animals conditioned with the mother, which show a preference for the lemon side. Cross fostered animals do not differ significantly from the random line animals. Although, interestingly, the high line cross fostered animals show a significant preference for the water side versus the lemon side when they were conditioned with the low line mother (t(7) = 2.545, p < .05, Figure 11). This is opposite of what the high line animals conditioned with the high line mother showed. High line animals conditioned with high line mothers do not show a significant preference to either side of the apparatus (Figure 10), but the high line animals conditioned with a low line mother show a clear preference for the water side of the apparatus (Figure 11). It appears as though high line animals that are exposed to an odor associated with a cross fostered dam show an aversion to that odor during testing. High line animals who were conditioned with a low line mother were the only cross fostered animals to show significant place preference. All other cross fostered animals showed COP profiles similar to their non-cross fostered groups.

For the random line animals, there were significant positive correlations between the amount of time spent in the lemon side of the testing chamber and the number of entries into the lemon side of the chamber (r = .421, p < .01) and also a positive correlations between amount of time spend in the water portion of the testing chamber and the entries into the water portion of the testing chamber (r = .729, p < .01). There was a negative correlation for time spent in the Genetics and Social Reward 43

Figure 10: Mean Amount of Time in Seconds Each Genetic Line, Divided into Conditioning

Group, Spent on Each Side of the COP Chamber

140 Lemon Time * Water Time 120

100

40 Mean Time in Sec. / 5 Min. / 5 in Sec. Time Mean 20

0 High High Random Random Low Low Mother Cotton Mother Cotton Mother Cotton n=38 n=35 n=53 n=50 n=31 n=29

* indicates p < .05. Genetics and Social Reward 44

Figure 11: Mean Amount of Time in Seconds High and Low Line Cross Fostered Animals,

Divided into Conditioning Group, Spent on Each Side of the COP Chamber

180 * 160 Lemon Time Water Time 140

120

100

60 Mean Time / 5 Min. 40

0 High High Random Random Low Low Mother Cotton Mother Cotton Mother Cotton n=7 n=9 n=53 n=50 n=7 n=11

* indicates p < .05.

Genetics and Social Reward 45

lemon side and time spent in the water side of the testing chamber (r = -.632, p < .01; see Table

1). These results were to be expected and the high and low line animals had correlations similar to those found in the random line. For partial correlation matrices see Table 5 for high line animals and Table 6 for low line animals.

Play Behavior

For the random line animal analysis, two two-way ANOVAs were used. The first

ANOVA examined a dependent variable for number of dorsal contacts, a between-subject gender factor, and the within-subject factor for number of testing days (six) and the second ANOVA had the same factor but used number of pins as the dependent measure. For random line animals, there is a main effect for the number of dorsal contacts across the six testing days (F (5, 265) =

9.374, p < .001; see Figure 12, left) with an increasing number after day one and day four, but males and females are not significantly different from each other in number of dorsal contacts encountered during a 5 minute play session. For pins during a play session, there is a main effect across the six testing days (F (5, 135) = 9.586, p < .001; see Figure 12, right). Figure 12 does show the female animals performing more pins on all testing days, but these gender differences are not statistically significant (F (1, 52) = .802, p = .375).

Figure 13 graphically illustrates all four dependent measures (dorsal contact count, pin count, total pin duration, and mean duration for a single pin) used to assess play for each of the genetic lines. A 3-factor (testing day, gender, and genetic line) mixed design ANOVA was used for each dependent measure. For dorsal contacts across the six testing sessions which is represented in Figure 13, top left, there is a within-subject main effect testing session (F(5,

710)=8.766, p < .001) and a between-subject effect for the genetic line (F(2, 142)=3.844, p <

.05). The low line animals show decreased levels of dorsal contacts which is significantly Genetics and Social Reward 46

Figure 12: Random Line Data for Each of the Play Sessions Separated by Gender

70 20

18 60 16

50 14

12 40 10 30 8 Random Males n=22 20 6 Random Males n=22

Random Females n=32 of Pins/ Number Min. 5 Mean 4 10 Random Females n=32 2 Mean Number of Dorsal Contacts / 5 Min. 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Left: Mean number dorsal contacts in a 5 minute play session for each of the six days of testing. Right: Mean number of pins in a 5 minute play session for each of the six testing days.

Genetics and Social Reward 47

Figure 13: Play Behavior for each Genetic Line

70 20 *

65 18 16 60 14 55 12 * 50 10

45 8 High n=28 Random n=54 6 40 High n=28 Low n=36 Mean Dorsal Contacts/ 5 Min.

Random n=54 Min. / 5 of Pins Number Mean 4 35 Low n=36 2 30 0 Day 1Day 2Day 3Day 4Day 5Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

45 4 ** 40 3.5 * ** 35 3 * 30 * 2.5 25 2 20 * 1.5 15 High n=28 1 High n=28 Mean Duration of Single Pin Single of Duration Mean Mean Pin Duration / 5 Min. / 5 Pin Duration Mean 10 Random n=54 Random n=54 Low n=36 0.5 Low n=36 5 0 0 Day 1Day 2Day 3Day 4Day 5Day 6 Day 1Day 2Day 3Day 4Day 5Day 6

Top left: Mean number of pins during a 5 minute play session for all six days of testing for each genetic line. Top Right: Mean number of pins during a 5 minute play session for each of the six days of testing for each genetic line. Bottom left: Mean total duration of pinning time in seconds for each of the 5 minute play sessions on the six testing days for each genetic line. Bottom right: The mean duration for a single pin for each of the genetic lines for each 5 minute play session on each testing day. * indicates p < .05, ** indicates p < .01.

Genetics and Social Reward 48

Table 1: Pearson Correlation Matrix for the Random Line Animals for the COP Paradigm

Water Lemon Water Time Entries Time Lemon Entries 0.072 .421** -0.186 Water Entries .479** .729** Lemon Time -.632** ** indicates p <.01 Genetics and Social Reward 49 different on day six (t (88) = 2.285, p < .05). For dorsal contacts, high and random line animals are not significantly different.

For the number of pins, there is a main effect for testing days (F(5, 710) = 38.287, p <

.001) with pin counts increasing across the testing days (see Figure 13, top right). There is also an interaction effect for genetic line and gender (F(2, 142) = 4.696, p < .05) and an interaction effect for genetic line, gender, and whether the animal was cross fostered (F(1, 142) = 5.065, p <

.05). These interactions will be examined below.

Pin duration is another dependent measure used which is a summation of the seconds an animal was pinned during a 5 minute play session. There was a main effect for testing day (F(5,

710) = 34.226, p < .001, see Figure 13, bottom left) and also a main effect for genetic line (F(2,

142) = 3.764, p < .05) and gender (F(1, 142) = 4.658, p < .05). Post hoc t-tests indicated high line animals spent significantly more time being pinned on play day two when compared to the random line animals on day two (t (80) = 2.099, p < .05, see Figure 13 bottom, left) and again high line animals spend more time than random line animals in the pinning position on day five

(t (80) = 2.329, p < .05). Low line animals also spend more time pinning than random line animals on day five (t (88) = 2.891, p < .01, see Figure 13, bottom left) but did not significantly differ from the random line on any other play days. There was a significant interaction effect for genetic line and gender (F(2, 142) = 4.715, p < .05) and a three-way interaction between genetic line, gender, and whether the animal was cross fostered (F(1, 142) = 10.871, p < .05).

The final dependent measure used to assess levels of play behavior was the average duration of a single pin. These data are represented in Figure 13 bottom right. There was a significant within-subject main effect for testing day (F(5, 710) = 2.456, p < .05). A between- subject main effect was seen for genetic line (F(2, 142) = 3.959, p < .05) and a three-way Genetics and Social Reward 50

interaction effect for genetic line, gender, and whether the animal was cross fostered (F(1, 142) =

4.511, p < .05). The bottom right portion of Figure 13 graphically illustrates the differences

between genetic lines with respect to duration of a single pin across the testing sessions. High

line animals show a significantly longer average pin duration compared to the random line animals on day five (t (80) = 3.106, p < .01) and on day six (t (80) = 2.603, p < .05). Low line animals also show a significantly longer average pin duration compared to the random line animals on day five (t(88) = 2.042, p < .05), but fail to show a significant difference during any other testing day.

Figure 14 shows the gender differences for the high line animals for dorsal contact counts

(left graph) and for pin counts (right graph). High line males and high line females that were reared by their biological dam do not show statistically significant differences in the number of dorsal contacts made during a 5 minute play session (see Figure 14, left panel); however, high line males show significantly more pins on day four (t(26) = 3.698, p < .01) and on day five

(t(26) = 2.478, p < .05) compared to the high line females (see Figure 14, right panel).

For low line animals reared by their biological mothers, there are no significant gender differences for either number of dorsal contacts or number of pins (see Figure 15).

Data for all cross fostered animals are shown in Figure 16 for each of the four dependent measure of play behavior (number of dorsal contacts, number of pins, total duration of pinning, and the average duration of a single pin). The low line animals reared by high line dams, show decreased dorsal contacts on day three (t(72) = 2.770, p < .01) and on day six (t(72) = 2.677, p <

.01) compared to the random line animals (see Figure 16, top left). Contrarily, the high line animals reared by a low line dam show elevated levels of dorsal contacts compared to the random line animals on day four (t(66) = 2.089, p < .05, see Figure 16, top left), but do not show Genetics and Social Reward 51

Figure 14: High Line Animal Data for Each of the Play Sessions Separated by Gender

25 80

70 20

50 15 ** * 40 10 30

20 High Males n=16

Mean Number of Pins / 5 Min. 5 High Females n=12 10 High Males n=16

Mean Number of Dorsal Contacts / 5 Min. / Contacts Mean Number Dorsal of High Females n=12 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Left: Mean number dorsal contacts in a 5 minute play session for each of the six days of testing. Right: Mean number of pins in a 5 minute play session for each of the six testing days. * indicates p < .05, ** indicates p < .01

Genetics and Social Reward 52

Figure 15: Low Line Animal Data for Each of the Play Sessions Separated by Gender

60 25

50 20

40 15 30

10 20 Low Males n=16 Low Females n=20 Low Males n=16

MeanNumber 5 Min. Pins/ of 5 10 Low Females n=20 Mean Number of Dorsal Contacts / 5 Min. 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Left: Mean number dorsal contacts in a 5 minute play session for each of the six days of testing. Right: Mean number of pins in a 5 minute play session for each of the six testing days.

Genetics and Social Reward 53

Figure 16: Play Data for Cross Fostered High Line Animals, Cross Fostered Low Line Animals, and for the Random Line Animals.

70 25 High n=14 Random n=54 60 20 Low n=20 ** 50 15 40 ** ** 30 10 High n=14 20 Random n=54 Low n=20 Min. 5 / Pins of Number Mean 5 10

Min. / 5 Mean NumberDorsal Contacts of 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

3.5 60 * 3 ** * 50 * * 2.5 40 2 * * 30 1.5

20 ** * * High n=14 * * 1 Random n=54 High n=14 Mean Duration of a Single Pin Single a of Duration Mean Mean Pin Duration / 5 Min. 5 / Duration Pin Mean Low n=20 10 Random n=54 0.5 Low n=20 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Top left: Mean number of pins during a 5 minute play session for all six days of testing for the cross fostered high and low genetic lines and the random line. Top Right: Mean number of pins during a 5 minute play session for each of the six days of testing for the cross fostered high and low genetic lines and the random line. Bottom left: Mean total duration of pinning time in seconds for each of the 5 minute play sessions on the six testing days for cross fostered high and low genetic lines and the random line. Bottom right: The mean duration for a single pin for cross fostered high and low genetic lines and the random line for each 5 minute play session on each testing day. * indicates p < .05, ** indicates p < .01. Genetics and Social Reward 54

significant increases on any other day. For pins (Figure 16, top right), the cross fostered animals

do not show significant differences compared to the random line animals. Low line animals who

were cross fostered into a high line litter, show more time spent in the pinning position across all

testing days except day four (day one: t(72) = 2.824, p < .01; day two: t(72) = 2.082, p < .05; day

three: t(72) = 2.287, p < .05; day five: t(72) = 2.378, p < .05; day six: t(72) = 2.317, p < .05; see

Figure 16, bottom left) compared to the random line animals. High line animals cross fostered

into a low line litter showed increased time spent in the pinning position compared to the random

line animals only on testing day three (t(66) = 2.401, p < .05; Figure 16, bottom left). Low line animals cross fostered into a high line litter also showed longer average duration for a single pin compared to the random line animals on day two (t(72) = 2.492, p < .05) and day five (t(72) =

2.388, p < .05; see Figure 16 bottom right). High line animals reared by low line mothers also showed a longer single pin duration on day five (t(66) = 2.441, p < .05) compared to the random line animals.

Cross fostered animals from both the high and low lines failed to show significant gender differences for either number of dorsal contacts or number of pins. Data from high line animals fostered into a low line litters are shown in Figure 17 and data from low line animals fostered into high line litters are shown in Figure 18.

Overall, low line animals, whether cross fostered or reared by their biological mothers, show significant decreases in play measured by number of dorsal contacts in a 5 minute play session, which is statistically significant on several testing sessions. High line animals, regardless or whether they were cross fostered or not, do not differ statistically from the random line animals in number of dorsal contacts and number of pins per play session over the six testing days. High line females reared by their biological dams show statistically significant fewer pins Genetics and Social Reward 55

Figure 17: Cross Fostered High Line Animal Data for Each of the Play Sessions Separated by

Gender

80 25

70 High Males n=6 High Females n=8 20 60

50 15 * 40 30 10

20 High Males n=6

High Females n=8 Mean Number of Pins / 5 Min. 5 Mean Number of Dorsal Contacts 10 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1Day 2Day 3Day 4Day 5Day 6

Left: Mean number dorsal contacts in a 5 minute play session for each of the six days of testing. Right: Mean number of pins in a 5 minute play session for each of the six testing days.

Genetics and Social Reward 56

Figure 18: Cross Fostered Low Line Animal Data for Each of the Play Sessions Separated by

Gender

70 25 Low Male n=12 60 Low Female n=8 20 50

40 15

30 10 Low Male n=12 20 Low Female n=8

Mean Number of Pins / 5 Min. 5 / Pins of Number Mean 5 10 Mean Number of Dorsal Contacts / 5 Min. 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1Day 2Day 3Day 4Day 5Day 6

Left: Mean number dorsal contacts in a 5 minute play session for each of the six days of testing. Right: Mean number of pins in a 5 minute play session for each of the six testing days.

Genetics and Social Reward 57

than their male counterparts. However, if high line females are cross-fostered into a low line

litter, this gender difference dissipates. Number of dorsal contacts does not appear to be a

measure sensitive enough to examine gender differences in play behavior.

In a comparison between the frequency of dorsal contacts and pins, random line animals

spend about 80% of play activity performing dorsal contacts, while spending approximately 20% of the play activity on pinning (see Figure 19). The random line profile suggests a downward

shift in percentage of dorsal contacts from day one to day two, after day two the percentage of

pins and dorsal contacts remains stable with at least a 3:1 (dorsal contacts: pins) ratio. High line

animals show a similar profile to the random line in terms of percentages, also showing a

decrease in dorsal contacts on day two (Figure 20). In fact, high line animals do not differ

statistically from the random line animals on either percentage of pins or percentage of dorsal

contacts for any of the testing sessions. In contrast, low line animals consistently differ from the

random line animals in percentage of pins and dorsal contacts (T-test across all testing sessions;

t(88) = 3.094, p <.01; see Figure 21). Low line animals consistently spend more time pinning

than performing dorsal contacts, with an average dorsal contact percentage of 77.4% and a

pinning average of 22.6%.

There were several positive Pearson correlations present in the random line for the

correlations of pinning and dorsal contacts across the six testing days. Correlation coefficients

for day six, the final testing day, are as follows: Dorsal contacts on day six were positively

correlated with day two dorsal contacts (r = .673, p < .01); day two pins (r = .521, p < .01); day

three dorsal contacts ( r = .441, p < .05); day three pins ( r = .521, p < .01); day four dorsal

contacts ( r = .699, p < .01); day four pins ( r = .477, p < .05); and day five dorsal contacts ( r =

.643, p < .01). Day six pins were positively correlated with the following: day two dorsal Genetics and Social Reward 58

Figure 19: Mean Percentage of Dorsal Contacts and Pins During Play Activity in the Random

Line Animals (N=54)

Pins Dorsal Contacts

100

10 Mean Percentage / 5 Min. Session 0 Day 1Day 2Day 3Day 4Day 5Day 6 Genetics and Social Reward 59

Figure 20: Mean Percentage of Dorsal Contacts and Pins During Play Activity in the High Line

Animals (N=28)

Pins Dorsal Contacts 100

20 Mean Percentage / 5 Min. Session / 5 Mean Percentage Min. 10

0 Day 1Day 2Day 3Day 4Day 5Day 6 Genetics and Social Reward 60

Figure 21: Mean Percentage of Dorsal Contacts and Pins During Play Activity in the Low Line

Animals (N=36)

Pins Dorsal Contacts 100 90 80 70 60 50 40 30 20 10

Mean Percentage / 5 Min. Session 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Genetics and Social Reward 61

contacts ( r = .463, p < .05); day two pins ( r = .582, p < .01); day three pins ( r = .388, p < .05);

day four dorsal contacts ( r = .521, p < .01); day four pins ( r = .702, p < .01); day five dorsal

contacts ( r = .432, p < .05); day five pins ( r = .694, p < .01); day six dorsal contacts (r = .568, p

< .01). The previous five testing day correlations were similar to those of day six and for a

complete correlation matrix for the random line animal play behavior see Table 2.

High and low line correlations the matrices were not considerably different from the

random line. Therefore, see Appendix C for complete high line (Table C1) and low line (Table

C2) correlation matrices for play behavior.

Social Investigation

Behaviors were initially examined in the random line animals to determine a baseline for testing social investigation. The data were examined two ways and are presented in Figure 22.

The first analysis, shown in the top row of graphs in Figure 22, demonstrates how behaviors vary across the 30 minute test session after each of the three days of testing. Also, it shows there was a clear preference for the social port and that preference remains consistent after each test day.

The results indicate there is a clear preference for the social port of the investigation paradigm demonstrated by significantly greater mean time spent in the social port than in the nonsocial

port (Figure 22, top left) and a greater number of nose pokes in the social port compared to the

nonsocial port (Figure 22, top middle). While the number of nose pokes in the social port

differed from the nonsocial port, the mean duration of pokes at either port, did not differ

significantly from one another (Figure 22, top right). Also, Figure 22 fails to show a large

isolation effect on day two after 24 hours of social isolation, with the exception of a modest

increase in the time spent in the social port. Genetics and Social Reward 62

Figure 22: Baseline Investigatory Behavior in Social Port Apparatus

Investigatory behavior expressed in 10 minute blocks across 3 days of repeated testing:

25 120 Social Port 9 Nonsocial Port ) 8 100 20 Sec. ( 7

80 6 15 5 60 4 10 40 3

Mean Time at Port (Sec.) MeanPort Time at 2 5 Social Port 20 Social Port Mean Number Nose of Pokes Nonsocial Port Mean Nose Poke Duration 1 Nonsocial Port 0 0 0 123 123 123 123 123 123 123 123 123 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

Total investigatory behavior in 30 minute test session:

Nonsocial Male 60 350 Nonsocial Male Nonsocial Female 12 Nonsocial Female Social Male Nonsocial Male Social Male ** Nonsocial Female 300 50 Social Female 10 Social Male ** Social Female Social Female ) 250 40 8 Sec. ( 200 *** *** * *** *** * 30 6 * 150 *** ***

Time at Port Port at Time 20 4 100 Mean Number Nose of Pokes

50 10 Mean Nose Poke (Sec.) Duration 2

0 0 0 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

The top panel shows that in a 30 minute test session, the time spent investigating the social port, as well as the number of nose pokes in the social port is consistently higher than those in the nonsocial port. Moreover, no habituation was observed during the three days of testing. In the bottom panel, data are cumulated across the entire 30 minute test session. Although a significant social port preference was observed in both genders, female rats spent significantly more time in the social port for the first two days than the males as well as significantly more time in the nonsocial port compared to the males. Additionally, females consistently show increased poke duration than the males (bottom right). * indicates p < .05, ** indicates p < .01, and *** indicates p < .001

Genetics and Social Reward 63

Table 2: Pearson Correlation Matrix for the Random Line Play Behavior Data Across All Six

Testing Sessions

Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Day 6 Day 6

Pins Dorsal Pins Dorsal Pins Dorsal Pins Dorsal Pins Dorsal Pins Day 1 .662** .577** .064 .356 -.024 .437* .076 .571** .311 .253 .188 Dorsal Day1 .479** .340 .202 .210 .302 .344 .555** .406* .198 .332 Pins Day 2 .534** .554** .309 .595** .252 .774** .524** .673** .463* Dorsal Day 2 .240 .554** .292 .618** .428* .300 .521** .582** Pins Day 3 .548** .319 .203 .410* .251 .441* .125 Dorsal Day 3 .268 .668** .143 .195 .521** .388* Pins Day 4 .527** .739** .525** .699** .521** Dorsal Day 4 .372 .511** .477* .702** Pins Day 5 .529** .643** .432* Dorsal Day 5 .289 .694** Pins Day 6 .568** Dorsal * indicates p < .05 and ** indicates p < .01

Genetics and Social Reward 64

Animals from each group showed a significant preference for the social port (omnibus ANOVA=

F(5, 1015) = 200.018, p < .001). There was a main effect for genetic line (F(2, 203) = 8.659, p <

.001) and an interaction between port preference and genetic line (F(10, 1015) = 3.015, p < .01).

Each genetic line showed a greater mean number of nose pokes in the social port after isolate housing (see Figure 23) however, both the high and low line animals show significantly more social nose pokes after isolate housing compared to the random line (High social poke compared to random social pokes: t(140) = 2.277, p < .05); low line social pokes compared to random line social pokes: t(122) = 2.644, p< .01; see Figure 23, left). Moreover, the high line animals

consistently showed significantly more nonsocial nose pokes compared to the random line (day

one: t(140) = 5.013, p < .001; day two: t(140) = 4.793, p < .001; day three: t(140) = 5.344, p <

.001; see Figure 23, right). The low line animals did show increased nonsocial port nose pokes compared to the random line but only after social isolation (t(122) = 2.932, p < .01, see Figure

23, right).

Cross fostered animals continue to show a significant preference for the social port (see

Figure 24). However, high line animals do not show the clear, significant preference for the nonsocial port that was seen in the noncrossed animals in Figure 23. However, the cross fostered

high line animals, compared to the random line, did show a significant preference for the

nonsocial port after social isolation (t(102)= 2.879, p < .01). For both social and nonsocial ports,

high line animals showed a significant increase in nose pokes compared to the random line

animals (social port after isolation: t(102) = 2.932, p < .01, Figure 24, left; nonsocial port after

isolation: t(102) = 2.879, p < .01, Figure 24, right). The cross fostered low line animals also

showed a significant increase in social port nose pokes after social isolation (t(100) = 2.502, p < Genetics and Social Reward 65

Figure 23: Mean Number of Nose Pokes in the Social and Nonsocial Ports for Each Day

60 ** 55 * High n=54 Random n=88 50 Low n=36

40 *** 35 *** *** ** 30

25 Mean Number of Nose Pokes / 30 Min. Pokes Nose of Number Mean

20 Pair Isolate Pair Pair Isolate Pair Housed Housed Housed Housed Housed Housed

Social Port Nonsocial Port

* indicates p < .05, ** indicates p < .01, *** indicates p < .001 Genetics and Social Reward 66

.05), but did not show significant variation from the random line for any of the nonsocial port nose pokes, Figure 24. For further analysis on differences between genetic lines, gender differences, and differences between different time blocks on different days for all lines, see

Appendix D for complete tables.

In order to determine the relationship between the number of noses pokes in each port and the total time spent in each port, a Pearson correlation was computed. There is almost always a positive correlation between all four measures (pokes into social port, pokes into nonsocial port, time in social port, and time in nonsocial port; see Table 3). For random line animals, more pokes into the social port is correlated with more pokes in the nonsocial port and likewise for time spent in each port. Data for both high and low lines showed similar correlations, see Table 5 for partial high line social port correlations and see Table 6 for partial low line social port correlations.

Correlation Results

To address the issue of the long-term relationship between the social measures, a Pearson correlation analysis was completed for the random line animals (see Table 4). For random line animals, there were only two correlations between testing paradigms. There is a positive correlation between number of pins and the number of nonsocial port pokes after isolate housing

(r = .373, p < .01). Random line animals who engaged in more pins were also more likely to poke the nonsocial port after isolate housing during the social port test. Additionally, random line animals who engaged in more pins, were positively correlated with more time spent in the nonsocial port after pair housing (r = .318, p < .05; see Table 4).

High line animals showed negative correlations with nonsocial port activity and play behavior. For high line animals, those with more pins during play were less likely to nose poke Genetics and Social Reward 67

Figure 24: Mean Number of Nose Pokes in the Social and Nonsocial Ports for Each Day for

Cross Fostered Animals

70 ** * High Crossed n=16 60 Random n=88 Low Crossed n=14 50

40 * ** 30

10 Mean Number of Nose Pokes / 30 Min. 0 Pair Isolate Pair Pair Isolate Pair Housed Housed Housed Housed Housed Housed

Social Port Nonsocial Port

* indicates p < .05, ** indicates p < .01

Genetics and Social Reward 68

Table 3: Pearson Two-Tailed Correlation Matrix for the Random Line from the Social Port

Investigation Paradigm

Isolate Isolate Pair Pair Pair Isolate Pair Housed Housed Housed Housed Housed Housed Housed Social Nonsocial Social Social Nonsocial Nonsocial Nonsocial Port Port Port Port Port Port Time Port Time Time Pokes Pokes Time Pokes

Isolate Housed Social Port Pokes .593** .452** 0.173 .357** .278** .363** .252* Isolate Housed Social Port Time .243* .359** .260* .505** 0.166 .446** Isolate Housed Nonsocial Port Pokes .673** .227* 0.164 .408** .411** Isolate Housed Nonsocial Port Time 0.101 .232* 0.169 .473** Pair Housed Social Port Pokes .567** .421** .251* Pair Housed Social Port Time 0.169 .313** Pair Housed Nonsocial Port Pokes .699** * indicates p <.05 and ** indicates p <.01

Genetics and Social Reward 69

Table 4: Pearson Two-Tailed Correlations between Testing Paradigms for the Random Line Animals

Isolate Isolate Isolate Isolate Pair Pair Pair Pair Lemon Dorsal Social Social Nonsocial Nonsocial Social Social Nonsocial Nonsocial Time Count Pins Pokes Time Pokes Time Pokes Time Pokes Time Lemon Entries .408** -0.065 0.124 -0.011 0.094 -0.081 0.210 0.076 -0.110 0.086 0.065 Lemon Time 0.109 -0.048 0.033 -0.031 -0.031 0.033 -0.032 -0.171 -0.104 -0.180 Dorsal Counts 0.152 -0.181 -0.265 -0.040 -0.010 0.103 -0.067 -0.040 -0.053 Pins 0.032 0.140 .373** 0.204 0.062 0.134 0.264 .318* Isolate Social Pokes .660** .523** .338* .340* .407** .358** .382** Isolate Social Time .448** .569** 0.262 .503** .273* .641** Isolate Nonsocial Pokes .665** .313* .379** .296* .358** Isolate Nonsocial Time 0.246 .420** 0.061 .362** Pair Social Pokes .592** .505** .375** Pair Social Time .272* .463** Pair Nonsocial Pokes .728** * indicates p < .05, ** indicates p < .01 Genetics and Social Reward 70

the nonsocial port after pair housing (r = -.441, p < .05) and also those with more pins were

significantly negatively correlated with time spent in the nonsocial port after pair housing (r = -

.514, p < .05, see Table 5 for full correlation matrix).

Finally, for low line animals, there were no correlations, positive or negative, between

different behavioral paradigms. For low line animals, social tendencies during play did not

correlate with other social tendencies of the COP or the social investigation test. See Table 6 for

complete correlation matrix.

Overall, prosocial measures do not significantly correlate across different developmental

time points. The random line animals engaging in more play behavior, via more pins during a play session, made more investigations of the nonsocial port, but did not correlate with social port activity. Similarly, high line animals engaging in more pinning activity correlated with more

time and nose pokes into the nonsocial ports. It is possible that animals engaging in more rough- and-tumble play as juveniles correlates with more inquisitive adult animals, but furthering testing

would be need to further clarify this correlation with additional measures of seeking and/or

inquisitiveness as adults. Genetics and Social Reward 71

Table 5: Pearson Two-Tailed Correlations between Testing Paradigms for the High Line Animals

Isolate Isolate Isolate Isolate Pair Pair Pair Pair Lemon Dorsal Social Social Nonsocial Nonsocial Social Social Nonsocial Nonsocial Time Count Pins Pokes Time Pokes Time Pokes Time Pokes Time Lemon Entries .502* 0.178 0.256 -0.363 -0.322 -0.093 -0.046 -0.178 -0.344 -0.118 -0.131 Lemon Time 0.134 0.053 -0.038 -0.288 -0.106 -0.272 -0.161 -0.347 0.120 0.037 Dorsal Counts .769** -0.116 -0.284 -0.124 0.018 -0.172 -0.198 -0.088 -0.364 Pins -0.303 -0.261 -0.256 -0.117 -0.390 -0.152 -.441* -.514* Isolate Social .709** 0.233 -0.027 .427* 0.216 0.289 0.342 Pokes Isolate Social Time 0.270 0.113 0.293 0.289 0.190 0.391 Isolate Nonsocial .703** 0.393 0.170 0.392 0.246 Pokes Isolate Nonsocial 0.246 0.343 0.094 0.125 Time Pair Social Pokes .523** 0.394 .430* Pair Social Time -0.121 0.100 Pair Nonsocial .727** Pokes * indicates p < .05, ** indicates p < .01 Genetics and Social Reward 72

Table 6: Pearson Two-Tailed Correlations between Testing Paradigms for the Low Line Animals

Isolate Isolate Isolate Pair Pair Pair Pair Lemon Dorsal Social Isolate Nonsocial Nonsocial Social Social Nonsocial Nonsocial Time Count Pins Pokes Social Time Pokes Time Pokes Time Pokes Time Lemon Entries .724** 0.256 0.005 0.005 0.123 -0.144 -0.063 -0.343 -0.298 0.034 0.125 Lemon Time 0.185 0.058 0.213 -0.063 0.315 0.287 -0.295 -0.373 0.263 0.320 Dorsal Counts .683** 0.054 0.118 -0.202 -0.312 -0.242 -0.334 -0.373 -0.201 Pins -0.154 -0.068 -0.118 -0.204 0.017 0.199 -0.131 0.098 Isolate Social Pokes 0.338 0.325 -0.011 0.219 -0.327 0.381 0.359 Isolate Social Time -0.384 -0.130 -0.268 -0.273 -0.092 0.265 Isolate Nonsocial Pokes .757** .487* 0.119 .632** 0.323 Isolate Nonsocial Time 0.200 0.076 .476* 0.306 Pair Social Pokes .648** .521** 0.200 Pair Social Time 0.307 0.205 Pair Nonsocial Pokes .818** * indicates p < .05, ** indicates p < .01 Genetics and Social Reward 73

CHAPTER V: DISCUSSION

Social Development and Paradigm Comparison

A general trend of social behaviors from infancy to adulthood was examined in this study. The random “control” line animals provided a baseline with which to compare the different selectively bred lines. Overall, the random line dams displayed typical levels of maternal care (Siviy et al., 2003; Champagne et al., 2003; Champagne et al., 2006) and pup retrieval (Li and Fleming, 2003; Friedman et al., 2006). The random line pups when separated from their littermates and dams showed similar rates of isolation distress calls (approximately

115 distress calls/ 2 minutes) compared to other control groups (150 distress call for a longer testing session in the control group for Armstrong et al., (2001) and approximately 100 distress calls per 2 minute test for the random line reported in Brunelli, 2005)) which is assumed to be indicative of anxiety (Hofer, 1996; Miczek et al., 1991; Winslow and Insel, 1991).

While previous research has used various other methods to examine maternal/pup attachment (i.e., nipple attachment latency, contact frequency and duration with the mother, and approach latency; see Nelson and Panksepp, 1996), for the present study a conditioned odor preference paradigm was used to assess the affinity of a pup to its mother. Odor preference was apparent for the maternally associated cue but the linkage was not as strong as previously found in other work (Nelson and Panksepp, 1996; Panksepp, Nelson, and Siviy, 1994). A possible reason for this weaker effect could be improper placement of the lemon scent. Perhaps the scent was placed on, or so close to the nipple that when the pup went to suckle, it ingested some of the lemon extract, found it to be aversive, and then showed an aversion to the lemon odor. Genetics and Social Reward 74

As juveniles, the random line animals showed significant levels of play behavior as

compared with control animals from previous other studies. Our random line animals showed approximately 15 pins/5 minute session compared to approximately 15 pins/5 minute play

session for Panksepp (1980); approximately 13 pins/ 5 minute session for Deak and Panksepp

(2006); 12 pins / 5 minutes session for Siviy et al. (2003). Play behaviors were usually lower on the first day, but behaviors were enhanced after the first session and remained stable throughout the rest of the testing period.

Finally, results from the social investigation testing confirmed a clear preference for the social port by the random line young adults, indicating a normal tendency for gregariousness

(Panksepp et al, 1997; Walker, Diefenbach, Mikulak, and Pert, 2005; Bekkedal, Rossi, and

Panksepp, 1998).

To address the relationship between social behaviors and the stability of those behaviors across a long developmental timeline as discussed in Aim I, a correlation matrix was computed for the three social paradigms (COP, Play and Social Port Investigation). As expected, the prosocial behaviors of rough-and-tumble play and social investigation were positively correlated.

Contrary to our predictions, levels of maternal conditioning were not positively correlated with the levels of play behavior and social port investigation. Results indicate that a pup’s affinity for its mother does not appear to be a predictive factor in juvenile play or adult gregariousness.

Overall, for our random ‘control’ line, rats who engaged in more play behavior tended to be

more inquisitive during their adult testing. A possible reason for the lack of correlation between the COP data and the other prosocial measures could because COP paradigm is not a direct measure of gregariousness and prosocial activity. It is more of an attachment measure and Genetics and Social Reward 75

perhaps a paradigm measuring more social tendencies during that day of age would correlate

with later social tendencies.

Another study examining the stability of social behaviors investigated the effects of trimethylolpropane phosphate (TMPP) on both play and gregariousness (Bekkedal et al., 1998).

Results indicated that rats showing an increase in juvenile play behavior also showed an increase

in social port investigation in adulthood (Bekkedal et al., 1998). Correlational matrices were not

calculated, but their conclusions are consistent with our results. To our knowledge, this is the

only other study attempting to correlate positive social behaviors across an elongated time period

in the rat.

A recent study examined the relationship between negative and positive social

interactions in the rat. Brunelli et al. (2006) published data relating distress vocalizations and

rough-and-tumble play. Their results indicate that animals selected for high levels of infantile

distress vocalizations show deficits in play behavior. Our results are consistent with their data, in that, our low line animals show increased rates of distress vocalizations and lower levels of play behavior. In sum, based on the results from our data and data from other laboratories, selective breeding for USVs, whether infantile distress vocalizations or 50 kHz adult USVs, produce temperamental differences that are expressed during juvenile rough-and-tumble play and continue in adulthood.

One limitation of this study is the lack of measurement for overall levels of arousal.

Researchers have suggested a multidimensional nature of affect which includes a component of arousal present in both positive and negative affect (Green, Goldman, and Salovey, 1993).

Perhaps, within the selection process, the temperament selected for is level of arousal instead of temperamental affect. This study did take into account a measure of locomotor activity during Genetics and Social Reward 76 the COP paradigm by measuring the total number of line crosses into either side of the testing apparatus. We found a significant difference between low line activity and random line activity with low line animals displaying more locomotor activity compared to random line animals. In contrast, the low line animals actually played less which is somewhat contradictory to the hyperactivity found during COP. This effect isn’t all together unexpected based on studies examining the effects of amphetamines and play behavior. Amphetamine injected animals are more aroused and they play less than saline injected animals (Field and Pellis, 1994; Beatty et al., 1982). Actually, being more generally aroused may decrease the time devoted to play because the animals may be exploring or exhibiting general locomotion during the play session.

More testing of arousal and locomotor activity in the future would provide more conclusive data differentiating the levels of arousal between these animals.

Temperament, defined by Zeanah and Fox (2004), refers to the behavioral style exhibited in response to a range of stimuli and contexts. Temperament studies with infants have indicated that infants labeled as being slightly more reactive are more likely than normal to have anxious symptomology later in development, but these data also verifies the influence of environment.

More reactive infants are more likely than normal to be anxious, but not every reactive infant becomes an anxious child (Kagan and Snidman, 1999). Furthermore, social deficits seen in developmental disorders such as autism show a persistent behavioral phenotype in adolescence and in adulthood (Seltzer, Shattuck, Abbeduto, and Greenberg, 2004). These temperamental studies and others have indicated a consistent temperamental characteristic and this is consistent with our animal data indicating a relationship between the juvenile and the adulthood periods in the expression of certain social interactions.

Genetics and Social Reward 77

Major Findings for Selective Breeding

The second aim of this study was to examine the impact of selective breeding for 50 kHz

USVs on the expression of social behavior. Therefore, we selectively bred for a line of animals

with elevated 50 kHz USVs (High line animals) and a line of animals with decreased 50 kHz

USVs (Low line animals) (Burgdorf et al., 2005). Selective breeding for low levels of 50 kHz

USVs appears to have had the larger impact on the expression of social behaviors, but the results

for both lines will be briefly reviewed.

High line animals did not differ significantly from random line animals in most of the

behavioral paradigms. In fact, the first time social behaviors varied significantly between the

high and the random lines was during social investigation testing in early adulthood. The high

line animals consistently showed more investigation of the nonsocial port compared to the

random line animals. Yet, a correlational matrix indicated a similar play/social port correlation as the random line animals. On the other hand, low line animals differed from random line animals in all behavioral paradigms. Low line animals appeared to be in more distress when separated from the mother and littermates, but they did not develop a preference for the maternally associated odor. Low line animals showed significantly more mobility during testing, but showed decreased play activity. The correlation matrix did not show correlations with any of the social

behaviors as seen in the high and random lines.

Based on previous research indicating that animals selectively bred for distress

vocalizations have fewer prosocial tendencies than control animals (Brunelli et al., 2006), we

intended to see if animals selected for high levels of 50 kHz USVs expressed more prosocial

behaviors than controls. And conversely, animals selected for low levels of 50 kHz USVs Genetics and Social Reward 78

expressed fewer social tendencies. The results from this study indicate that selecting for elevated levels of 50 kHz USVs does not enhance social behaviors. However, these results do suggest that selecting for lower levels of 50 kHz USVs has significant and lasting effects on the expression of social behavior.

Influence of Maternal Care: Cross Fostering Technique

For animal researchers, one way to determine the influence of maternal care on the development of a pup is to employ a cross fostering technique. The cross fostering technique involves placing animals from one litter into another litter to compare the impact of maternal care and environment on the expression of various phenotypes. Cross fostering studies have been done with genetic models of many different phenotypes including: hypertension (McCarty,

Cierpial, Murphy, Lee, and Fields-Okotcha, 1992), depression (Friedman et al., 2006), adult aggression (Flandera and Novakova, 1974), ethanol sensitivity, (Sluyter, Hof, Ellenbroek,

Degen, and Cools, 2000) and selectively bred lines for infantile anxiety (Brunelli, Hofer, and

Weller, 2001).

Presently, cross fostering techniques were utilized to determine the role of maternal care on the expression of these social behavioral profiles. High line animals reared by low line dams did show an aversion to the maternally associated odor during the COP test which is contrary to behaviors of the high line animals reared by high line dams. Other than this low affinity for the cross fostered dam, high line animals had a very similar profile to those high line noncrossed animals for the other social paradigms. Low line animals reared by high line dams also showed a similar profile to that of noncrossed low line animals. While not all comparisons between the cross fostered and random lines are statically significant, which may be due to low number of cross fostered animals, the cross fostered animals did share the same tendencies as their Genetics and Social Reward 79

noncrossed conspecifics. A limitation of this study is that the random, high or low line animals

were not cross fostered to another litter of the same genetic line, but considering the few cross

fostered effects, this control seems superfluous.

These results indicate that being reared by a high line dam does not negate the variations in social behaviors seen in the low line animals. Therefore, there are variations between these selectively bred lines that cannot be explained simply by differential maternal care, implicating a genetic component to the differential social tendencies among the lines. Cross fostered high line animals did show a weak affinity for the fostered low line dams indicating that maternal behavior may modify the pup’s affinity for the dam. Future research will need to address the neurodevelopmental changes due to the influence of maternal care and its effect on genetic expression. Additionally, there could be other epigenetic factors that were not examined that could contribute to the behavioral differences observed here, which should be investigated in the future.

Neurobiology of Social Behavior

The methods of selective breeding used in the present study could alter neurobiological mechanisms of emotion and social behaviors. Some of the potential neurobiological mechanisms include oxytocin, endogenous opioids, and catecholamines. For instance, oxytocin, a neuropeptide that is synthesized primarily in the hypothalamus and released into the blood stream and throughout the brain, has been consistently shown to promote maternal/pup bonding, but also has been implemented in various other social behaviors including social cues, social recognition, and social bonding (Lim, Bielsky, and Young, 2005; Nelson and Panksepp, 1998).

The study by Nelson and Panksepp (1996) clearly shows the direct link of oxytocin to maternal/pup affiliation. Results showed that animals, centrally administered with an oxytocin Genetics and Social Reward 80 antagonist, failed to show a strong affinity for their mother, which was seen in the control animals. A similar impairment in maternal conditioning is seen in the low line animals of this study; however, the behavioral characteristics of the low line animals and the oxytocin antagonist treated animals in the above study are not completely congruent since the animals administered with an oxytocin antagonist failed to show any place preference whereas our low line animals showed a clear preference for the unconditioned side. Perhaps the deficits observed in this study are a result of alterations in this neuropeptide system which is inhibiting the mother/pup bonding.

Future studies could collect and measure oxytocin levels in the low line cohort to check for altered amounts.

Additional studies examining the effects of oxytocin and attachment have shown that central administration of oxytocin can reduce the number of distress vocalizations in rat pups

(Insel and Winslow, 1991). Periods of social isolation in rat pups leads to a reduction in oxytocin receptor binding (Noonan et al., 1994). These studies as well as others clearly show the involvement of oxytocin in the formation of maternal attachment.

Endogenous opioids have also been implicated in various social behaviors including maternal attachment, play behavior and social companionship (Panksepp, 1998) and could potentially be involved in the changes seen in these genetic lines. Opioids, just like oxytocin, can also reduce levels of distress vocalizations (Nelson and Panksepp, 1998), but also, low doses of morphine can increase play and opiate antagonists can reduce play (Panksepp, 1998). These data seem slightly contradictory because the same manipulations with opioids can decrease and increase the desire for social companionship and thus gregariousness in many species including rats (Panksepp, Najam, and Soares, 1980) and humans (Kurland, 1978). Panksepp (1998) explains that opiate doses must be kept low to facilitate play because high doses of opiates Genetics and Social Reward 81

reduce all social behaviors and mobility in general. In sum, modest opioid arousal increases play

and play can promote opioid release (Panksepp and Bishop, 1981). While opioids are clearly

involved in modulating play, they are not the only factor mediating social and play interactions

(Panksepp, 1998).

In a study by Vanderschuren, Spruijt, Hol, Niesink, and Van Ree (1996), low doses of

morphine were administered and were shown to increase play. In another study examining the

effects of opioids on play behavior, opioid administration was shown to increase the dominance

of one animal, while opioid antagonist administration increased submission during a play session

(Panksepp, Jalowiec, DeEskinazi, and Bishop, 1985). In a study focusing on the level of

locomotor activity and play, naloxone, an opioid antagonist, was shown to strongly reduce play

behavior, but not locomotor activity (Siegel and Jensen, 1986). The authors concluded that naloxone primarily affects the affective component of play because of the reduced play without diminished activity (Siegel and Jensen, 1986).

Research clearly implicates endogenous opioids in the control of distress vocalizations and social behaviors, particularly in rough-and-tumble play behavior. Analysis of central opioid levels in the low line animals could determine whether endogenous opioids are responsible for the differences in social behavior seen here.

The catecholamine, dopamine, plays a role in the reinforcing effects of certain stimuli and has high concentrations in the lateral hypothalamus, the ventral tegmental area, and nucleus accumbens in the brain. These areas and connections making up what is commonly called the

SEEKING system is the “incentive or appetitive motivational system that mediates wanting”

(Panksepp, 1998, page 125). Dopamine plays an important role in seeking and goads animals to explore and to develop expectancies. Increases in brain dopamine activity may cause young Genetics and Social Reward 82 animals to search for alternative rewards and hence, to diminish play. In genetic lines selectively bred for distress vocalizations, dopamine has been proposed to be elevated in the animals selected for low levels of distress vocalizations, potentially explaining their decreased play behaviors (Brunelli et al., 2006). In a study by Brunelli and Kehoe (2005), measurements were taken of the dopamine metabolic activity in postnatal day ten pups selected for low levels of distress calls. Results showed greater dopamine activity exhibited in the cingulate cortex, hypothalamus, caudate putamen, septum, and periaqueductal gray (Brunelli and Kehoe, 2005).

These data by Brunelli et al. (2006) suggest that the functional deficits in dopamine may be responsible for the differential behavior observed in their selectively bred lines.

Maternal care can also be strongly affected by dopamine. Administration of a dopamine agonist into the nucleus accumbens has been shown to decrease pup retrieval and pup licking

(Keer and Stern, 1999) and lesions of the nucleus accumbens shell also significantly disrupted pup retrieval (Li and Fleming, 2003). Meaney, Brake and Gratton (2002) demonstrated that adult rats that were maternally separated in early life have fewer dopamine reuptake transporters in the ventral striatum. Perhaps, the methods of measuring maternal care in our genetic lines were not sensitive enough to determine the variations in maternal care. We made notations of if a maternal behavior was present or not during a 1 minute observation period. Providing a more accurate measure such as duration or time spent performing a certain aspect of maternal care during an observation period would be a more accurate measure of maternal differences. In the future, our observation methods could be revised and could indicate that there are differences in maternal care among our lines.

Continued research into the effects of oxytocin, opioid, and dopamine should help to clarify the neurochemical differences between our genetic lines. The research summarized here Genetics and Social Reward 83 provides ample evidence that these mechanisms are likely candidates for which brain systems may be affected in these selectively bred lines.

Bipolarity of Positive and Negative Affect

In addition to providing information on the development of social behaviors and disruptions in that development, the results of the study contribute to the ongoing theoretical question about the role of positive and negative emotional in the control of behavior. Many researchers offer persuasive arguments that positive and negative affect are opposing ends of one affect variable (for a review see Russell and Carroll, 1999). For instance, these authors argue that affect is bipolar and that research stating that these concepts are two independent feelings or attitudes are, in fact, the result of inherent errors in measurement and statistics. Russell and

Carroll analyze human survey data with various different statistical techniques and rephrasing of survey questions to provide evidence for the bipolar argument. They go so far as to state,

“Bipolarity provides a parsimonious fit to existing data” (Russell and Carroll, 1999, page 3).

However, this debate is far from being settled and animal research, in particular, continues to offer evidence countering statements made by Russell and colleagues. For example, animal research has indicated separate mechanisms for appetitive and aversive unconditioned reflexes that can vary independently of each other (Konorski, 1967; Mackintosh, 1983; Masterson and

Crawford, 1982).

Animals in this study were selected based on a measure for positive affect, 50 kHz USVs.

In accordance with a bipolarity view, animals selected for low levels of 50 kHz USVs would be expected to be low on positive affect and high on negative affect. One may tend to interpret an increase in distress vocalizations (high negative affect) and a decrease in play behavior (low positive affect) as being in conjunction with a bipolarity theory. However, this conclusion needs Genetics and Social Reward 84

to be verified with more work. For example, an individual with opioid dependence shows low

levels of social interactions, but during periods of elevated opioid levels, reports feelings of euphoria and pleasure (Morrison, 2001). Furthermore, children with autism can fail to engage in many types of social interactions and do have emotional disturbances, but to conclude that they are high on negative affect would be unfounded without further measures of negative affect. In fact, this study was lacking an adequate measure for negative affect during adulthood. Therefore, to state that these low line animals are high on negative affect would be premature at this point.

In sum, these data do not directly provide evidence for or against a bipolar affect model, since this study lacks adequate measures of negative affect at various developmental time points.

In fact, there was only one measure of negative affect, distress vocalizations at postnatal day ten, which we did not significantly correlate with other social behaviors. So we are unable to assert that high negative affect was sustained during subsequent development. Additionally, by

selecting for positive affect vocalizations, we intended to select for high and low positive affect,

not high and low negative affect. Many extensions of this work need to be completed for more

conclusive evidence about the status of positive and negative affect in these animals.

Cacioppo and Berntson (1994) offer an alternative stance on bipolarity than Russell and

Carroll (1999) that not only are positive and negative affective states independent substrates, but also that there are independent biological mechanisms underlying these emotions. Behavioral

testing utilizing measures of anxiety via open-field test, forced swim test, and elevated-plus

maze, would provide a more direct indication of negative affect.

To perform an adequate test on the issue of bipolarity of affect, a behavioral comparison

of animals selected for low positive affect (low 50 kHz USVs) and animals selected for high

negative affect (high isolation distress vocalizations or high 20 kHz USVs) needs to be Genetics and Social Reward 85

completed. A prediction based on bipolarity of emotion is that similar behavioral profiles would

be observed since the selection process would be selecting for the same end on the affect bipolar

continuum. The opposite should also be true: animals selected for high positive affect (high 50

kHz USVs) and low negative affect (low isolation distress vocalizations or low 20 kHz USVs)

would have similar behavioral profiles since they would be selecting for negative affect on a

bipolar continuum. The current study builds a foundation for this future critical test.

Clinical Implications

By selecting for fewer positive affective vocalizations, these selectively bred lines show similar developmental tendencies as those seen is many clinical populations. For example, individuals with autistic spectrum disorder can show widespread disruptions in attachment

(Happé, 1994); also separation anxiety disorder and reactive attachment disorder are also characterized by abnormal attachment (Morrison, 2001). Low line animals in this study also showed an altered affinity for their mother indicated by their nonpreference for a maternally associated odor. The neural network responsible for the difference in high and low line maternal

affinity could provide insights into the neural activity that is disrupted in these developmental

disorders.

Decreased or altered social interaction and social communication are symptoms in many

developmental disorders including: autistic spectrum disorder (Happé, 1994; Baird, Cass, and

Slonims, 2003; Lim et al., 2005), Rett’s disorder, childhood disintegrative disorder, Asperger’s

disorder, and attention deficit/ hyperactivity disorder (ADHD) (Morrison, 2001). Many of these

developmental differences in social behavior are seen in the low line animals including a

different profile for rough-and-tumble play compared to the high and random line animals. It is

possible that the neural mechanisms differentiating the low line animals from the random line Genetics and Social Reward 86 animals are also the neural mechanisms involved in autistic spectrum disorder and other developmental disorders. By identifying the neural mechanisms differentiating social behavior in these animals, we may be able to explore new treatments and/or ways to prevent these diseases.

Clinical trials and studies involving oxytocin, endogenous opioids, and dopamine, the candidates thought to be responsible for the differences in these lines, have been investigated in patients with developmental disorders and in particular autism disorder. A study by Modahl et al.

(1998) demonstrated that children with autism showed significantly lower levels of oxytocin than age-matched control children. Additionally, Hollander et al. (2003) recently reported that repetitive behaviors, which are a symptom of autism, can be reduced by intravenous oxytocin infusion in adults with autism.

Endogenous opioids, because of their effect on social behaviors, have been thought to be elevated in patients with autism. Research on opioid hyperfunction in autistic populations has produced inconsistent results (Lam, Aman, and Arnold, 2006) but research on the effects of opioids on the developing brain continues. Naloxone and naltrexone, opioid antagonists, have been given to autistic populations with some promising effects of alleviating certain autistic symptoms (Lensing et al., 1995; Lam et al., 2006).

There has been considerable interest in dopamine’s involvement in autism since dopamine antagonists (antipsychotics) have been effective in treating hyperactivity, stereotypies, aggression, and self-injury which are all autistic symptoms (Young, Kavanagh, Anderson,

Shaywitz, and Cohen, 1982). Dopamine levels have not been measured centrally in autistic patients, but peripheral dopamine abnormalities do not appear to be present in autism (Anderson and Hoshino, 1997). However, more conclusive studies will be needed to clarify the role of dopamine in autism. Genetics and Social Reward 87

The dopamine system has also been correlated with ADHD (Li, Sham, Owen, and He,

2006) and studies evaluating the efficacy of selective dopamine agonists, like bupropion, hold

promise in the treatment in ADHD (Pityaratstian, 2005; Flindling and Dogin, 1998).

Investigation into the CNS dopamine levels in our selectively bred lines could provide

information regarding dopamine levels and its implication in social behaviors.

Due to the inconsistent data about the neurochemistry and neurophysiology, as well as symptomology observed in patients with autism and other developmental disorders, it is difficult

to create valid and reliable animal models for any of these disorders. Continued research

modeling certain impairments of social behavior, like disruptions of play observed in this study,

can lead to more knowledge regarding aspects of defective social functioning which will enable

understanding of the mechanisms that lead to the disorders.

Conclusions and Future Studies

Results from this study have contributed to the literature on affect and the genetics and

development of social behaviors. Furthermore, it has combined areas of interest including

psychology and genetics and will continue to examine this relationship and their influence on

each other. The selective breeding of genetic lines based on positive affect vocalizations has and

will continue to pave the way to create new models for various emotional disorders that do not

require drug administration or environmental manipulations.

Future studies will need to be implemented to continue the assessment of behavioral

differences, assessment of differences in neurochemistry and neuroanatomy, as well as genetic

testing to determine more precisely the genetic influence on the expression of social behaviors.

Current studies in the laboratories of J. Panksepp, J. Burgdorf, and J. Moskal and studies in our

own laboratory have already been implemented to address and resolve some of the remaining Genetics and Social Reward 88

issues. By continued testing on these selectively bred lines, we will be able to identify the neural differences between these social profiles and be able to make further implications on human populations. Moreover, as causes and characteristics of various developmental disorders like autistic spectrum disorder continue to emerge, more precise animal models will be developed for more accurate testing of pharmacological therapies and behavioral interventions to treat and possibly cure these conditions.

Genetics and Social Reward 89

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APPENDIX A

Total Duration of DOB # of Pups DVD ID Latency to Retrieve retrieving Number of drops 6/21/2005 n=6 A

7/1/2005 n=12 B

7/19/2005 n=4 C

7/23/2005 n=5 D

7/23/2005 n=16 D

7/30/2005 n=4 E

9/15/2005 n=12 F

9/16/2005 n=13 G

9/18/2005 n=10 H

9/22/2005 n= 13 I

9/22/2005 n=14 I

9/23/2005 n=14 J

9/23/2005 n=7 J

9/24/2005 n=17 K

9/24/2005 n=13 K

Genetics and Social Reward 105

APPENDIX B

Litter DOB: Number of Pups: Date: DVD ID: Postnatal Day:______

Light Cycle Time Time Time Minute 1: Minute 2: Minute 3:

Nursing: Nursing: Nursing: Allogrooming: Allogrooming: Allogrooming: Autogrooming: Autogrooming: Autogrooming: Nest Building: Nest Building: Nest Building: In/Out of nest: In/Out of nest: In/Out of nest:

Time Time Time Minute 4: Minute 5: Minute 6:

Time Time Time Minute 1: Minute 2: Minute 3:

Time Time Time Minute 4: Minute 5: Minute 6:

Genetics and Social Reward 106

APPENDIX C

Pearson correlation matrices for play behavior for the high line (Table C1) and the low line (Table C2).

Table C1: Pearson correlation matrix for the high line play behavior data across all six testing sessions. * indicates p < .05 and **

indicates p < .01.

Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Day 6 Day 6

Pins Dorsal Pins Dorsal Pins Dorsal Pins Dorsal Pins Dorsal Pins

Day 1 Dorsal .662** .577** 0.064 0.356 0.024 .437* 0.076 .571** 0.311 0.253 0.188 Day1 Pins .479** 0.340 0.202 0.210 0.302 0.344 .555** .406* 0.198 0.332 Day 2 Dorsal .534** .554** 0.309 .595** 0.252 .774** .524** .673** .463* Day 2 Pins 0.240 .554** 0.292 .618** .428* 0.300 .521** .582** Day 3 Dorsal .548** 0.319 0.203 .410* 0.251 .441* 0.125 Day 3 Pins 0.268 .668** 0.143 0.195 .521** .388* Day 4 Dorsal .527** .739** .525** .699** .521** Day 4 Pins 0.372 .511** .477* .702** Day 5 Dorsal .529** .643** .432* Day 5 Pins 0.289 .694** Day 6 Dorsal .568**

Genetics and Social Reward 107

Table C2: Pearson correlation matrix for the low line play behavior data across all six testing sessions. * indicates p < .05 and **

indicates p < .01.

Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4 Day 5 Day 5 Day 6 Day 6

Pins Dorsal Pins Dorsal Pins Dorsal Pins Dorsal Pins Dorsal Pins

Day 1 Dorsal .544** .556** 0.258 0.327 0.008 .536** 0.112 0.300 -0.024 0.312 -0.029 Day1 Pins 0.107 0.249 0.211 .382* .362* .437** 0.065 0.252 0.145 0.219 Day 2 Dorsal .457** .514** -0.067 .566** 0.134 .507** -0.028 .579** 0.130 Day 2 Pins 0.252 .550** 0.279 .351* 0.145 0.243 0.174 0.204 Day 3 Dorsal 0.187 .662** 0.142 .701** 0.229 .560** 0.165 Day 3 Pins 0.102 .467** 0.083 .608** 0.074 0.298 Day 4 Dorsal .345* .744** 0.215 .694** 0.231 Day 4 Pins 0.044 .557** 0.197 .561** Day 5 Dorsal 0.184 .715** 0.096 Day 5 Pins 0.158 .495** Day 6 Dorsal .535** Genetics and Social Reward 108

APPENDIX D

Social investigation results separated by genetic line, gender, conditioning day, i.e., day one, day two or day three, and separated by 10 minute time block within the 30 minute testing session. * indicates p < .01, ** indicates p < .01, *** indicates p < .001, and # indicates p < .09.

Table D1:

Day 1- Pair Housing Mean Number of Social Pokes Mean Number of Nonsocial Pokes Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Male N=28 18.00 ± .754 12.75 ± .739 10.14 ± .935 13.61 ± .850 *** 7.68 ± .653 7.57 ± .772 #

High Line Female N=26 20.85 ± 1.274 15.65 ± 1.045 12.46 ± 1.239 13.12 ± .676 9.19 ± .927 * 9.50 ± 1.165 ***

Random Line Male N=42 15.88 ± 1.030 12.10 ± 1.068 9.50 ± .923 9.48 ± .557 6.69 ± .601 5.60 ± .533

Random Line Female N=46 21.67 ± .805 15.02 ± .723 10.24 ± .726 11.41 ± .532 7.22 ± .416 5.50 ± .524

Low Line Male N=16 19.00 ± 2.580 14.44 ± 1.895 11.81 ± 2.262 9.56 ± 1.338 7.75 ± 1.174 7.13 ± 1.169

Low Line Female N=20 23.14 ± 1.010 13.95 ± .821 9.86 ± 1.415 11.67 ± .705 6.67 ± .766 4.57 ± .739

Genetics and Social Reward 109

Table D2:

Day 2- Isolate Housing Mean Number of Social Pokes Mean Number of Nonsocial Pokes Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Male N=28 19.64 ± 1.089 17.21 ± 1.362 15.96 ± 1.189 # 11.18 ± .611 9.32 ± .883 * 9.57 ± 1.108 ***

High Line Female N=26 18.46 ± .898 # 17.00 ± 1.169 16.46 ± 1.341 ** 12.38 ± .613 9.85 ± .688 ** 10.62 ± .775 **

Random Line Male N=42 17.24 ± .934 16.69 ± .953 13.00 ± .937 10.00 ± .503 7.36 ± .556 5.38 ± .501

Random Line Female N=46 20.80 ± .611 15.30 ± .687 11.13 ± .812 11.33 ± .496 7.30 ± .455 7.07 ± .593

Low Line Male N=16 24.13 ± 1.43 *** 19.06 ± 1.346 16.75 ± 1.257 * 13.38 ± .836 ** 11.06 ± 1.230 ** 12.13 ± 1.868 **

Low Line Female N=20 20.86 ± .549 16.19 ± .861 10.67 ± .882 10.90 ± .762 8.00 ± .870 5.43 ± .975

Genetics and Social Reward 110

Table D3:

Day 3- Pair Housing Mean Number of Social Pokes Mean Number of Nonsocial Pokes Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Male N=28 16.39 ± .589 13.07 ± 1.031 10.68 ± 1.179 12.93 ± .703* 10.93 ± .703** 9.79 ± 1.118**

High Line Female N=26 19.62 ± 1.338 13.46 ± 1.145 10.19 ± 1.063 14.15 ± .609* 11.69 ± 1.085** 11.65 ± 1.28***

Random Line Male N=42 16.26 ± .963 13.55 ± .912 11.10 ± 1.007 10.60 ± .496 7.26 ± .590 5.90 ± .596

Random Line Female N=46 18.07 ± .637 12.85 ± .829 11.70 ± .716 12.15 ± .656 8.57 ± .603 7.15 ± .571

Low Line Male N=16 18.44 ± 1.646 15.19 ± 1.691 12.31 ± 1.731 15.06 ± 1.28*** 11.00 ± 1.455 9.75 ± 1.614**

Low Line Female N=20 17.38 ± .919 11.33 ± .846 6.57 ± 1.105** 12.67 ± .615 6.95 ± 1.001** 4.48 ± 1.037*

Genetics and Social Reward 111

Table D4:

Day 1- Pair Housing Mean Time in Social Port Mean Time in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Male N=28 62.46 ± 3.734# 72.00 ± 7.465 77.89 ± 10.863 47.09 ± 3.716*** 46.12 ± 5.573* 49.23 ± 6.885*

High Line Female N=26 86.60 ± 6.446 101.4 ± 8.864 98.92 ± 12.798 64.23 ± 6.988*** 69.88 ± 9.570*** 102.5 ± 18.29***

Random Line Male N=42 50.94 ± 4.593 65.08 ± 7.849 60.02 ± 8.745 23.96 ± 1.891 26.77 ± 4.569 23.82 ± 3.258

Random Line Female N=46 81.55 ± 3.573 89.43 ± 7.012 78.93 ± 8.369 42.16 ± 2.645 35.43 ± 3.281 37.93 ± 5.204

Low Line Male N=16 47.76 ± 7.037 56.65 ± 11.300 57.41 ± 13.342 26.17 ± 5.874 22.34 ± 4.821 31.65 ± 7.829

Low Line Female N=20 88.14 ± 5.085 81.07 ± 9.570 75.24 ± 13.767 40.47 ± 3.364 40.02 ± 4.816* 31.20 ± 7.145 Genetics and Social Reward 112

Table D5:

Day 2- Isolate Housing Mean Time in Social Port Mean Time in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Male N=28 67.06 ± 4.299 92.85 ± 9.610 111.4 ± 11.099 42.71 ± 3.420** 56.29 ± 7.413** 65.89 ± 11.122*

High Line Female N=26 105.6 ± 6.098 113.0 ± 9.665# 105.9 ± 8.006# 93.24 ± 8.762*** 82.30 ± 7.724*** 107.42 ± 12.20**

Random Line Male N=42 59.34 ± 4.971 92.60 ± 6.705 92.47 ± 8.445 25.01 ± 1.854 28.82 ± 2.873 33.21 ± 6.391

Random Line Female N=46 108.7 ± 5.276 93.95 ± 6.588 83.02 ± 8.881 48.81 ± 3.399 46.71 ± 5.150 63.59 ± 8.728

Low Line Male N=16 81.79 ± 8.002* 92.59 ± 11.604 99.93 ± 11.377 39.21 ± 5.714* 51.56 ± 6.999* 78.35 ± 15.036**

Low Line Female N=20 116.7 ± 5.817 107.6 ± 5.835 82.52 ± 8.337 50.66 ± 4.618 66.40 ± 10.275* 44.98 ± 9.138 Genetics and Social Reward 113

Table D6:

Day 3- Pair Housing Mean Time in Social Port Mean Time in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Male N=28 65.12 ± 4.638 77.37 ± 9.126 71.80 ± 9.611 59.42 ± 4.611*** 80.93 ± 11.52*** 78.91 ± 13.051**

112.79 ± 95.94 ± 6.723 89.31 ± 8.991 72.66 ± 8.855 93.44 ± 9.001*** 112.1 ± 16.92*** High Line Female N=26 14.30***

Random Line Male N=42 58.81 ± 4.634 85.75 ± 7.355 89.32 ± 11.814 28.89 ± 2.928 30.72 ± 3.381 37.14 ± 8.022

Random Line Female N=46 83.45 ± 5.473 72.86 ± 6.579 79.73 ± 7.383 58.38 ± 5.882 62.66 ± 7.258 49.51 ± 5.621

Low Line Male N=16 59.73 ± 4.354 87.91 ± 15.169 90.56 ± 23.440 49.97 ± 7.181* 59.22 ± 11.935# 80.01 ± 15.637*

Low Line Female N=20 100.82 ± 6.298* 91.74 ± 9.702 62.67 ± 14.313 52.61 ± 3.974 65.04 ± 16.456 37.21 ± 9.324 Genetics and Social Reward 114

Table D7:

Day 1- Pair Housing

Mean Poke Duration in Social Port Mean Poke Duration in Nonsocial Port

Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Male N=28 3.53 ± .197 5.65 ± .514# 7.95 ± 1.021 3.77 ± .379** 5.86 ± .638** 6.03 ± .646*

High Line Female N=26 4.38 ± .371 6.72 ± .572 7.93 ± 1.064 4.85 ± .502** 7.20 ± .769** 10.06 ± 1.615***

Random Line Male N=42 3.13 ± .197 4.54 ± .374 5.55 ± .770 2.57 ± .192 3.53 ± .378 3.64 ± .467

Random Line Female N=46 3.86 ± .120 5.98 ± .436 7.60 ± .621 3.73 ± .179 4.84 ± .401 5.82 ± .590

Low Line Male N=16 2.50 ± .191 3.30 ± .517 4.42 ± 1.45 2.48 ± .339 2.59 ± .438 3.57 ± .588

Low Line Female N=20 3.92 ± .238 5.82 ± .640 7.03 ± 1.458 3.57 ± .255 6.16 ± .522# 5.08 ± .917 Genetics and Social Reward 115

Table D8:

Day 2- Isolate Housing

Mean Poke Duration in Social Port Mean Poke Duration in Nonsocial Port

Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Male N=28 3.50 ± .214 5.35 ± .462 6.71 ± .545 3.72 ± .291* 5.67 ± .625* 6.26 ± .728 High Line Female N=26 5.94 ± .397 6.82 ± .585 7.02 ± .545 8.19 ± 1.101*** 8.95 ± .851** 10.1 ± 1.052

Random Line Male N=42 3.41 ± .228 5.45 ± .314 6.82 ± .503 2.48 ± .165 3.86 ± .267 5.10 ± .638 Random Line Female N=46 5.32 ± .266 6.03 ± .384 6.54 ± .575 4.43 ± .329 6.08 ± .508 8.06 ± .823

Low Line Male N=16 3.40 ± .292 4.89 ± .520 5.96 ± .595 2.85 ± .333 4.65 ± .452 5.79 ± .700

Low Line Female N=20 5.68 ± .339 6.88 ± .394 8.02 ± .933 4.63 ± .242** 7.63 ± 1.000# 6.93 ± 1.068

Genetics and Social Reward 116

Table D9:

Day 3- Pair Housing

Mean Poke Duration in Social Port Mean Poke Duration in Nonsocial Port

Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Male N=28 3.98 ± .257 5.78 ± .487 8.68 ± 1.827 4.60 ± .305*** 6.70 ± .711* 7.89 ± 1.789 High Line Female N=26 5.45 ± .555* 7.23 ± .847 8.30 ± 1.431 6.55 ± .561*** 9.03 ± .981* 10.17 ± 1.269

Random Line Male N=42 3.61 ± .233 6.50 ± .514 8.37 ± 1.240 2.64 ± .189 4.28 ± .309 5.26 ± .900 Random Line Female N=46 4.61 ± .249 5.75 ± .348 6.62 ± .559 4.72 ± .329 7.04 ± .658 6.73 ± .493

Low Line Male N=16 3.39 ± .213 5.70 ± .535 6.80 ± .959 3.27 ± .316 4.96 ± .535 12.54 ± 5.776**

Low Line Female N=20 5.92 ± .309** 8.50 ± 1.022** 7.50 ± 1.227 4.17 ± .252 7.42 ± 1.146 5.33 ± 1.160

Genetics and Social Reward 117

Table D10:

Day 1- Pair Housing Mean Number of Social Pokes Mean Number of Nonsocial Pokes Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Crossed Male N=8 17.83 ± 1.440 14.17 ± 2.063 11.92 ± 1.725 10.92 ± 1.554 9.00 ± 1.651 # 6.58 ± 1.743

High Line Crossed Female N=8 17.86 ± 1.010 14.43 ± 2.057 14.71 ± 3.517 10.57 ± .972 8.00 ± 1.175 7.29 ± 1.796

Random Line Male N=42 15.88 ± 1.030 12.10 ± 1.068 9.50 ± .923 9.48 ± .557 6.69 ± .601 5.60 ± .533

Random Line Female N=46 21.67 ± .805 15.02 ± .723 10.24 ± .726 11.41 ± .532 7.22 ± .416 5.50 ± .524

Low Line Crossed Male N=10 12.75 ± 3.172 12.00 ± 3.342 6.50 ± 4.213 12.25 ± 2.250 7.00 ± .707 4.25 ± 1.601

Low Line Crossed Female N=4 27.25 ± 2.810 15.25 ± 1.702 14.25 ± 3.326 10.75 ± .479 7.00 ± 1.633 7.50 ± 2.784

Genetics and Social Reward 118

Table D11:

Day 2- Isolate Housing Mean Number of Social Pokes Mean Number of Nonsocial Pokes Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Crossed Male N=8 23.08 ± 1.979** 16.25 ± 1.666 17.25 ± 2.975 * 12.83 ± 1.079 * 7.33 ± .847 7.83 ± 1.866

High Line Crossed Female N=8 24.86 ± 2.272 # 21.86 ± 2.405 ** 13.29 ± 1.554 11.43 ± 1.478 11.29 ± 1.539* 10.00 ± 2.478

Random Line Male N=42 17.24 ± .934 16.69 ± .953 13.00 ± .937 10.00 ± .503 7.36 ± .556 5.38 ± .501

Random Line Female N=46 20.80 ± .611 15.30 ± .687 11.13 ± .812 11.33 ± .496 7.30 ± .455 7.07 ± .593

Low Line Crossed Male N=10 17.50 ± 1.848 17.00 ± 5.447 16.00 ± 7.821 12.75 ± 1.315 7.00 ± 2.273 4.50 ± 2.598

Low Line Crossed Female N=4 25.00 ± 2.739 28.25 ± 5.23*** 16.50 ± 1.258 11.00 ± 1.871 9.00 ± 1.472 9.50 ± 2.630

Genetics and Social Reward 119

Table D12:

Day 3- Pair Housing Mean Number of Social Pokes Mean Number of Nonsocial Pokes Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Crossed Male N=8 19.25 ± 1.661 # 19.00 ± 2.209 ** 11.50 ± 2.116 11.67 ± 1.189 10.33 ± 1.539* 7.75 ± 1.088

High Line Crossed Female N=8 18.29 ± 2.212 8.71 ± 1.248 # 7.14 ± 1.738 # 10.57 ± 1.172 9.71 ± 1.796 5.43 ± 2.034

Random Line Male N=42 16.26 ± .963 13.55 ± .912 11.10 ± 1.007 10.60 ± .496 7.26 ± .590 5.90 ± .596

Random Line Female N=46 18.07 ± .637 12.85 ± .829 11.70 ± .716 12.15 ± .656 8.57 ± .603 7.15 ± .571

Low Line Crossed Male N=10 15.25 ± 1.548 6.50 ± 2.255* 5.25 ± 3.092# 9.25 ± 2.394 3.75 ± .854 1.50 ± .866#

Low Line Crossed Female N=4 19.50 ± 1.848 13.50 ± 3.096 5.05 ± 3.703* 11.25 ± 2.658 8.50 ± 3.122 3.25 ± 1.974

Genetics and Social Reward 120

Table D13:

Day 1- Pair Housing Mean Time in Social Port Mean Time in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Crossed Male N=8 53.91 ± 7.504 57.34 ± 9.201 76.03 ± 18.397 30.09 ± 5.500 46.93 ± 12.482 # 46.24 ± 15.304 High Line Crossed N=8 64.96 ± 12.144 67.97 ± 7.059 74.53 ± 11.127 61.31 ± 7.767* 69.34 ± 18.10 ** 62.41 ± 21.614 Female Random Line Male N=42 50.94 ± 4.593 65.08 ± 7.849 60.02 ± 8.745 23.96 ± 1.891 26.77 ± 4.569 23.82 ± 3.258

Random Line Female N=46 81.55 ± 3.573 89.43 ± 7.012 78.93 ± 8.369 42.16 ± 2.645 35.43 ± 3.281 37.93 ± 5.204

Low Line Crossed Male N=10 34.63 ± 7.357 51.59 ± 24.180 106.3 ± 68.899 25.49 ± 5.284 22.27 ± 2.882 16.70 ± 5.289

Low Line Crossed Female N=4 99.05 ± 15.930 87.81 ± 30.381 106.1 ± 32.105 49.80 ± 5.411 46.48 ± 15.569 45.60 ± 22.421 Genetics and Social Reward 121

Table D14:

Day 2- Isolate Housing Mean Time in Social Port Mean Time in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 90.06 ± 116.6 ± 21.352 97.55 ± 20.226* 42.86 ± 5.005 40.43 ± 9.223 50.55 ± 18.135 High Line Crossed Male N=8 12.245** 89.25 ± 101.4 ± 10.452 127.1 ± 18.056 # 73.15 ± 7.439 62.87 ± 7.146 146.0 ± 44.98*** High Line Crossed Female N=8 15.070**

Random Line Male N=42 59.34 ± 4.971 92.60 ± 6.705 92.47 ± 8.445 25.01 ± 1.854 28.82 ± 2.873 33.21 ± 6.391

Random Line Female N=46 108.7 ± 5.276 93.95 ± 6.588 83.02 ± 8.881 48.81 ± 3.399 46.71 ± 5.150 63.59 ± 8.728

Low Line Crossed Male N=10 39.01 ± 8.486 53.48 ± 22.389 105.3 ± 43.668 29.99 ± 5.905 27.56 ± 9.321 22.52 ± 12.570

175.5 ± 22.274 106.7 ± 16.605 109.0 ± 36.236 42.59 ± 3.753 71.71 ± 23.826 70.48 ± 16.363 Low Line Crossed Female N=4 ** Genetics and Social Reward 122

Table D15:

Day 3- Pair Housing Mean Time in Social Port Mean Time in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Crossed Male N=8 80.24 ± 11.472* 112.9 ± 15.816# 122.9 ± 27.225 42.56 ± 11.264 59.82 ± 12.497 46.24 ± 9.984

High Line Crossed Female N=8 92.23 ± 19.246 77.85 ± 20.023 47.59 ± 17.424 76.79 ± 11.746 101.8 ± 19.165 # 79.10 ± 34.157

Random Line Male N=42 58.81 ± 4.634 85.75 ± 7.355 89.32 ± 11.814 28.89 ± 2.928 30.72 ± 3.381 37.14 ± 8.022

Random Line Female N=46 83.45 ± 5.473 72.86 ± 6.579 79.73 ± 7.383 58.38 ± 5.882 62.66 ± 7.258 49.51 ± 5.621

Low Line Crossed Male N=10 41.47 ± 9.057 32.36 ± 8.572* 94.30 ± 64.729 20.64 ± 4.560 13.11 ± 3.759 7.85 ± 3.993

Low Line Crossed Female N=4 91.82 ± 14.151 87.56 ± 11.677 31.19 ± 23.128 39.75 ± 13.357 58.27 ± 24.673 58.03 ± 48.738 Genetics and Social Reward 123

Table D16:

Day 1- Pair Housing

Mean Poke Duration in Social Port Mean Poke Duration in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Crossed Male N=8 3.00 ± .326 4.29 ± .637 5.58 ± .974 2.53 ± .378 4.92 ± 1.044 5.61 ± 1.086

High Line Crossed Female N=8 3.52 ± .444 5.13 ± .815 5.64 ± .554 6.02 ± .820** 7.97 ± 1.203** 7.83 ± 2.451

Random Line Male N=42 3.13 ± .197 4.54 ± .374 5.55 ± .770 2.57 ± .192 3.53 ± .378 3.64 ± .467

Random Line Female N=46 3.86 ± .120 5.98 ± .436 7.60 ± .621 3.73 ± .179 4.84 ± .401 5.82 ± .590

Low Line Crossed Male N=10 2.79 ± .201 3.35 ± 1.432 39.75 ± 36.6*** 2.07 ± .276 3.32 ± .639 5.28 ± 2.048

Low Line Crossed Female N=4 3.73 ± .661 5.36 ± 1.380 6.73 ± 1.702 4.63 ± .488 6.81 ± 1.493 4.20 ± 1.715 Genetics and Social Reward 124

Table D17:

Day 2- Isolate Housing

Mean Poke Duration in Social Port Mean Poke Duration in Nonsocial Port

Block 1 Block 2 Block 3 Block 1 Block 2 Block 3

High Line Crossed Male N=8 3.80 ± .360 7.04 ± 1.032* 4.84 ± .863# 3.28 ± .278 6.31 ± 1.653* 4.71 ± .973 High Line Crossed Female N=8 4.15 ± .405# 5.93 ± .709 5.68 ± .438 5.74 ± .524 7.91 ± 1.077 17.79 ± 5.580***

Low Line Crossed Male N=10 2.20 ± .366 2.82 ± .747* 6.50 ± 2.782 2.55 ± .783 4.27 ± .961 2.93 ± 1.68

Low Line Crossed Female N=4 4.25 ± .374 6.43 ± .406 7.12 ± 2.821 4.49 ± 1.286 7.54 ± 1.668 7.71 ± .809

Genetics and Social Reward 125

Table D18:

Day 3- Pair Housing

Mean Poke Duration in Social Port Mean Poke Duration in Nonsocial Port Block 1 Block 2 Block 3 Block 1 Block 2 Block 3 High Line Crossed Male N=8 4.07 ± .407 6.15 ± .786 10.09 ± 2.629 3.49 ± .604 6.07 ± .849 5.63 ± 1.091

High Line Crossed Female N=8 5.00 ± .743 10.93 ± 4.542** 5.65 ± 1.912 7.44 ± .939** 11.02 ± 2.017* 11.71 ± 4.044

Random Line Male N=42 3.61 ± .233 6.50 ± .514 8.37 ± 1.240 2.64 ± .189 4.28 ± .309 5.26 ± .900

Random Line Female N=46 4.61 ± .249 5.75 ± .348 6.62 ± .559 4.72 ± .329 7.04 ± .658 6.73 ± .493

Low Line Crossed Male N=10 2.62 ± .366 6.05 ± 1.151 10.27 ± 5.360 2.33 ± .180 4.24 ± 1.962 4.30 ± 1.544

Low Line Crossed Female N=4 4.76 ± .802 7.40 ± 1.338 3.78 ± 2.231 3.59 ± 1.104 8.15 ± 2.215 7.80 ± 6.009 Genetics and Social Reward 126