FEAR AND ASSESSMENT OF SAFETY IN RATS SELECTIVELY BRED FOR
DIFFERNTIAL EMISSION OF 50 KHZ ULTRASONIC VOCALIZATIONS
Emily S. Webber
A Thesis
Submitted to the Graduate College of Bowling Green State University in partial
fulfillment for the degree of
Masters of Arts
August 2009
Committee:
Howard Casey Cromwell, Ph.D, Advisor
Verner Bingman, Ph.D.
Laura Dilley, Ph.D.
i
Abstract:
H. Casey Cromwell, Advisor
The goal of this study was to explore fear in rats that were selectively bred for variations in 50 kHz ultrasonic vocalizations (USVs) emission. Animals’ USVs are related to affective states and have been shown to be vital for communication and social interactions. 50 kHz USV emission during a tickle paradigm was used as the selection criterion for choosing breeders. Three animal lines were bred: high, low and random.
The high line animals emit significantly more 50 kHz USVs when compared to the random line animals while the low line animals emit significantly fewer 50 kHz USVs than the random line animals. Random line animals were produced by arbitrarily choosing 2 breeders from two different litters. Prior studies have suggested a variation of affective states in these animals. Behavioral strategies were used as measures of whether or not the animals diverge on traits related to fear and the assessment of safety: 1) social recognition, 2) play suppression, and 3) prepulse inhibition (PPI). The social recognition task requires basic discrimination abilities and examined the ability of these animals to assess safety and familiarity in social situations. The play behavior paradigm investigated instinctual fear responses, conditioning and extinction of play suppression by observing play behavior for several days after exposure to the aversive unconditioned stimulus. The
PPI test was used to assess reflexive fear and sensorimotor gating by measuring the acoustic startle response and the inhibition of this response. Results showed that low line animals had impaired social recognition abilities compared to random and high line animals. During play high line animals demonstrated exaggerated conditioned fear to the play apparatus. Conversely, low line animals failed to show contextual fear conditioning. ii
High and low line animals had deficiencies in PPI. High line animals emitted more 22
kHz USVs than the other lines. Overall these findings suggest that the high line animals
may have exaggerated emotional conditioning abilities and the low line animals may
have deficits in emotional associative learning. This animal model may serve as a useful tool in examining the basic genetics and neurobiology of emotional learning.
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“As soon as there is life, there is danger.”
- Ralph Emerson iv
This manuscript is dedicated to Karl and Lillian Englund, their never ending support and love has helped to educate me, providing me with the knowledge and skills to pursue my
passion. They are forever in my heart, my mind and my determination to succeed. v
ACKNOWLEDGEMENTS
I thank my advisor H. Casey Cromwell for all of guidance and support that he has
given me throughout the course of this project. In addition you have provided me with
essential feedback required to write this manuscript. Thank you for all of your help and
efforts.
I also would like to thank my committee members Verner Bingman and Laura
Dilley for their service on this committee and advice given throughout this process. In
addition, I thank Jaak Panksepp for agreeing to be a special guest at my thesis proposal
meeting; his insight has guided the interpretation and analysis of my results.
I also acknowledge and thank Jeff Burgdorf, for helping me learn the scoring
process of USVs on spectrograms. Another individual who has helped me grasp the prior
research on this topic is graduate student, Kelley Harmon. Thank you for all your help in
the interpretation of both the prior research and the project at hand. Your advice has been
invaluable to me.
I also thank Travis Beckwith and Samantha Peña for all their help in running the experiments, you did an exemplary job. This project was funded by the Hope for
Depression Research Foundation. I also thank the J.P. Scott Center for Neuroscience
Mind and Behavior for providing an atmosphere of collaboration and constructive criticism necessary for my development as a graduate student. Employees of University
Animal Facilities are also in my debt, as they took care of the animals’ daily needs
throughout this project.
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TABLE OF CONTENTS
Page
CHAPTER I. INTRODUCTION ..……………………………………………….... 1
Animal Models of Emotion ………………………………………… 1
Ultrasonic Vocalizations and Rat Behavior ……………………….. 2
Neurobiology of USVs ……………………………………………. 5
Selective Breeding ………………………………………………… 9
Fear as a Key Social Emotion …………………………………….. 13
Behavioral Paradigms Used in this Study …………………………. 16
Social Recognition ……………………………………….... 16
Play Suppression …………………………………………... 19
Prepulse Inhibition …………………………………………. 20
CHAPTER II AIMS OF THIS STUDY …..…….…………………………………22
Aim I: Social Recognition ………………………………………… 22
Aim II. Play Suppression …………………………………………. 23
Aim III: Prepulse Inhibition ………………………………………. 23
CHAPTER III. METHODS ……………………………………………………….. 25
Breeding …………..………………………………………………… 25
Social Recognition .………………………………………………… 25
Play Suppression ….……………………………………………….. 26
Prepulse Inhibition ….……………………………………………… 28
CHAPTER IV. STATISTICAL ANALYSIS ....…………………………………… 30
Social Recognition …………….…………………………………… 30 vii
Play Suppression …………………………………………………... 31
Prepulse Inhibition ………………………………………………… 32
CHAPTER V. RESULTS …………………………………………………………. 32
Social Recognition ………………………………………………… 32
Play Suppression …………………………………………………... 45
Prepulse Inhibition ………………………………………………… 68
CHAPTER VI. DISCUSSION ...………………………………………………….. 79
Summary ……………………………………..……………………. 79
Social Recognition ………………………………………………… 79
Play Suppression …………………………………………………... 82
Prepulse Inhibition ………………………………………………… 84
Previous Research: A Closer Look ………………………………... 85
Alternative Hypothesis: Variations in Associative Learning ..…….. 87
Future Research ……………………………………………………. 91
Clinical Implications ………………………………………………. 92
REFERENCES ……………………………………………………………………. 95 viii
LIST OF FIGURES
Figure Page
1 Percentage of Social Investigation ………………………………………….. 36
2 Percentage of Social Motivation ……………………………………………. 40
3 50 kHz USVs During Social Recognition ………..………………………….. 44
4 Pin Number During Play Suppression …...... 46
5 Pin Duration in seconds During Play Suppression ………………..………… 48
6 Doral Contacts During Play Suppression …………………………………… 50
7 50 kHz USVs on the Worn Cat Collar Day …………………………………. 52
8 ASR for Different Trial Types During PPI ...... 71
9 PPI (%) for the Different Selectively Bred Lines of Animals ………………. 72
10 22 kHz USV emission over different trial types During PPI ……….………. 73
11 22 kHz USV Examination of Trial and Sex Interaction During PPI ….……. 74
12 22 kHz USV Emission of Different Lines of Animals During PPI ……....… 75
13 22 kHz USV Emission Sex Differences During PPI ……………….…….… 76
14 22 kHz USV Emission Over Blocks of Trials During PPI …………….…… 77
15 22 kHz USV Emission Investigating Trial Block and Animal
Line Interaction ……………………………………………………………… 78
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LIST OF TABLES
Table Page
1 Independent Samples t-test for Social Investigation Percentage ……………….. 33
2 Means ± S.E.M Social Investigation Percentage ……………………………….. 34
3 Independent Samples t-test for Social Motivation Percentage …...... 38
4 Means ± S.E.M Social Motivation Percentage ..……………………………….. 39
5 Independent Samples t-test for 50 kHz USVs During
Social Recognition ………...……………………………………………………. 42
6 Means ± S.E.M. for 50 kHz USVS during Social Recognition ………………… 43
7 Independent Samples t-tests Pin Number During
Play Suppression …...……….………………………………………...... 53
8 Paired Samples t-tests of Pin Numbers for Random Line Animals
During Play Suppression …..…………………...... 54
9 Paired Samples t-tests of Pin Numbers for High Line Animals
During Play Suppression ……..………………………………………………... 55
10 Paired Samples t-tests of Pin Numbers for Low Line Animals
During Play Suppression ……..………………………………………………... 56
11 Means ± S.E.M. of Pin Numbers During Play Suppression ………..…………. 57
12 Independent samples t-tests on Pin Duration During
Play Suppression ……..……………………………………………………….. 58
13 Paired Samples t-tests of Pin Duration for Random Line Animals
During Play Suppression …………….………………………………………... 59 x
14 Paired Samples t-tests of Pin Duration for High Line Animals
During Play Suppression …………………………………………………….... 60
15 Paired Samples t-tests of Pin Duration for Low Line Animals
During Play Suppression ……………………………………………………… 61
16 Means ± S.E.M. of Pin Durations During Play Suppression ………….………. 62
17 Independent Samples t-tests Dorsal Contact Number
During Play Suppression ……………………………………………………… 63
18 Paired Samples t-tests of Dorsal Contact Numbers for Random Line
Animals During Play Suppression ……………………………………………. 64
19 Paired Samples t-tests of Dorsal Contact Numbers for High Line
Animals During Play Suppression ……………………………………………. 65
20 Paired Samples t-tests of Dorsal Contact Numbers for Low Line
Animals During Play Suppression ……………………………………………. 66
18 Means ± S.E.M. of Dorsal Contact Numbers During
Play Suppression ……………………………………………………………… 67
1
CHAPTER I: INTRODUCTION
Animal Models of Emotion
Animal models have been essential in making progress within psychology and
other scientific fields. These models allow us to observe and manipulate specific
behaviors and relate these data to the psychological processes like emotion, cognition and
perception. The majority of this research has relied on lesions, micro electrical
stimulation with electrodes and drug injections to induce and measure a desired
emotional state in an animal (Heise et al., 2005; Insel & Hulihan, 1995; Leeman et al.,
2003). While these techniques can be very useful, they lack key developmental and genetic components important for understanding how these affective states may develop.
Selective breeding is a technique that allows researchers to examine the role of genetic predispositions in animal models of emotional development. The current study
investigated how possible genetic predispositions may alter the development of social
behavior.
Results from prior research have demonstrated that genes play a crucial role in the
regulation of emotion (Butcher et al., 2006). Previous work by J.P. Scott used selective breeding of dogs and mice to examine the genetic factors involved in emotional differences with a focus mainly on aggression (Scott, 1949). It is well known that genetics play a role in production of mental illness (Hariri & Weinberger, 2003; Kagan &
Snidman, 1999; Robinson et al., 1992; Smoller et al., 2003). Utilizing an animal model of rats selectively bred based upon differential rates of 50 kHz ultrasonic vocalizations
(USVs) emission (Burgdorf et al., 2005; Harmon et al., 2008; Panksepp & Burgdorf,
2003; Panksepp & Burgdorf, 2000; Panksepp, 2007, Panksepp, 2000) we hope to gain 2 insight about the interaction between potential genetic and well controlled environmental influences on social learning and emotion. Previous research has associated 50 kHz
USVs with positive affective states of rats (Panksepp and Burgdorf, 2000; Burgdorf,
2007; Burgdorf et al., 2000; Knutson et al., 2002). Prior research has revealed key relationships between hormones, neurochemicals and neural circuits related to emotional states using this animal model (Panksepp & Burgdorf, 2003, Burgdorf et al., 2001,
Burgdorf et al., 2007, Burgdorf et al., 2009; Knutson et al., 1999).
This present study was based on previous research using selectively bred lines of animals (Burgdorf et al., 2005). The animals are bred based upon numbers of 50 kHz
USVs emitted during a tickle paradigm (Burgdorf et al., 2005). The current project utilized a set of behavioral paradigms that have been validated as useful in understanding social and affective behaviors (Brunelli et al., 2006; Siviy et al., 2006; Thor & Holloway,
1982; Rosa et al., 2005a, 2005b). This project extends the previous work on these selectively bred animals (Burgdorf et al., 2005; Harmon et al., 2008; Panksepp &
Burgdorf, 2000) by using behavioral paradigms to assess social abilities and fear reactivity. Previous research has found variation in social behavior in these animals; however, the impact of potentially fearful and aversive stimuli on behavior has not been examined. It is important to understand potential variations that exist in these animal lines in reflexive fear reactivity, fear conditioning and extinction. Alterations in these processes may have an impact on the development of social abilities.
Ultrasonic Vocalizations and Rat Behavior
Oral communication between animals is essential in the development of social behavior and dominance hierarchies (Burman et al., 2007; Sales, 1972). This skill is 3
highly adaptive as it can give animals the ability to respond appropriately in diverse social situations. Many animals use oral communication for a variety of reasons. For
example, birds use song to communicate (Beecher & Burt, 2004), while ants use chemical trails to organize their colonies (Watmough & Edelstein-Keshet, 1995). Non- human primate research has also found that vervet monkeys vocalize alarm calls in response to danger (Ribeiro, 2007). USVs are vocalizations that range from 22 kHz to 70 kHz (Panksepp, 2007). They are inaudible to the human ear. These USVs are emitted by some rodents as a tool for communication. USVs in rats initiate play behavior, warn others of possible danger and are important for appropriate mating and aggressive behavior (Burman et al., 2007, Corrigan & Flannelly, 1979; Panksepp, 1998).
Burman and colleagues (2007) designed a study in which USVs were recorded and played back to rats (Burman et al., 2007). The animals in this study were placed into a chamber that was connected to an arena, 22 kHz and 50 kHz USVs were played and emergence behavior into the arena was monitored (Burman et al., 2007). Results showed that playing 22 kHz USVs increased the latency to enter the arena, as well as a decrease in time spent outside the emergence box (Burman et al., 2007). Results for the 50 kHz
USVs did not portray an inverse to this relationship; they had no effect on latency or time spent in the arena (Burman et al., 2007). This lack of effect may be due to flaws in the experimental design. The animals were first exposed to 22 kHz USVs and then tested with 50 kHz USVs. This may have caused the animals to be aversively conditioned to the arena because no extinction procedure was utilized (Burman et al., 2007). Results from the 22 kHz playback study are consistent with previous research findings that 22 kHz USVs are used as an alarm system to warn others about nearby predators (Knutson 4 et al., 2002). Corrigan and Flannelly (1979) found that rats previously defeated by other aggressive males produced 22 kHz vocalizations when re-exposed to the aggressive animals. The 22 kHz vocalizations are much longer than the 50 kHz vocalizations
(Knutson et al., 1999). It is clear that the 22 kHz USVs are important for communicating danger and fear; however, there is substantial data supporting the existence of positive
USVs with positive valence (Burgdorf & Panksepp, 2002; Panksepp, 2007; Panksepp &
Burgdorf, 2000, 2003).
USVs associated with positive stimuli occur at approximately 50 kHz (Burgdorf
& Panksepp, 2002). Previous research has used these 50 kHz USVs as a means of studying the affective states of rats and has associated these vocalizations with “laughter”
(Panksepp, 2007; Panksepp & Burgdorf, 2000, 2003). 50 kHz USVs have been recorded during positive social events such as play, tickling and in anticipation of social and pharmacological rewards (Burgdorf et al., 2000; Burgdorf et al., 2001, Burgdorf et al.,
2007; Knutson et al., 2002). Tickling these rats simulates rough-and-tumble play, and consists of vigorous somatosensory stimulation targeting the dorsal and ventral surfaces.
The animals respond to this tickling by approaching the tickle hand, and will even “play bite” the tickler’s hand to solicit this stimulation in anticipation of its rewarding qualities
(Burgdorf et al., 2000).
While USVs are emitted as a communicative tool, it is also important to note that they have interesting anticipatory qualities (Burgdorf et al., 2000). USVs are emitted in anticipation of reward or punishment (Burgdorf et al., 2000; Burgdorf et al., 2001). This suggests that they are not simply linked to the experience of positive or negative stimuli, but related to affective states. Burgdorf and colleagues (2000) investigated USVs in 5
response to rewarding electrical brain stimulation (ESB) to the ventral tegmental area
(VTA) and lateral hypothalamus (LH) (Burgdorf et al., 2000). Animals were given
rewarding electrical pulses at a fixed interval. As time to the reward decreased, the rate of
50 kHz USV emission increased (Burgdorf et al., 2000). This technique was repeated
and animals were given the opportunity to self-administer, as the reward time was
approached these animals self-stimulated and provided a similar increase in 50 kHz USV
emission (Burgdorf et al., 2000). Importantly, increases in locomotor behavior were not
correlated with increases in 50 kHz USV emission (Burgdorf et al., 2000). Furthermore
the same study found that this anticipatory vocalization behavior generalized to food
reward. This suggests a general relationship to positive affect and not a specific link to any single reward outcome. These results provide compelling evidence that these USVs
display a clear anticipatory relationship. These vocalizations are not just a simple reaction
to an event, but part of an affective memory, formed by expectations that guide
observable behavior (Burgdorf et al., 2000). Observations of behavior and knowledge of
the biological nature of USV emission can give us clues about variations in neural
circuitry that may exist in these selectively bred animals.
Neurobiology of USVs in Rats
USVs are produced through a process called laryngeal braking (Blumberg &
Alberts, 1990). Rats emit these USVs during prolonged expirations from the lungs
(Blumberg & Alberts, 1990). Roberts (1974) found that audible vocalization productions
were not altered in a helium rich environment like USVs, suggesting a difference
mechanism of acoustic production between the two types of vocalizations. He reported
that USVs in a variety of rodent species were a special type of ultrasonic whistle, similar 6 to bird songs (Roberts, 1974). Wetzel and colleagues (1980) reported that the inferior laryngeal nerves were important for USV production, while the superior laryngeal nerves were responsible for the modulation of frequency and intensity of USVs. These laryngeal nerve fibers are part of the polyvagal nerve (Wetzel et al., 1980).
The same study examined the neurobiology of the motor involvement of these nerve fibers (Wetzel et al., 1980). Labeling of the inferior and superior laryngeal nerves revealed that the nucleus ambiguus has projections to these laryngeal structures (Wetzel et al., 1980). The dorsal formation of the nucleus ambiguus has nerve fibers that ascend to the inferior laryngeal nerves (Wetzel et al., 1980). The ventral portion of the nucleus ambiguus has projections to the superior laryngeal nerve structures (Wetzel et al., 1980).
Research has also implicated the periaqueductal gray (PAG) in USV production (Hofer,
1996; Kroes et al., 2007; Leeman et al., 2003; Oka et al., 2008; Sadananda et al., 2008).
Stimulation of the lateral PAG produced 22 kHz USVs in rats (Carrive, 1993).
The PAG has efferent fibers that synapse near the nucleus ambiguus, suggesting that these structures may work together to produce USVs (Hofer, 1996). Using anterograde and retrograde tracing techniques; Oka and colleagues (2008) confirmed that the central nucleus of the amygdala sends inhibitory projections to the PAG. They also found that the PAG has neurons that project to the nucleus ambiguus (Oka et al., 2008).
Specific neurochemicals and brain systems have been shown to be involved in
USV production. Dopamine (DA) is one of the neurotransmitters thought to be critical for
50 kHz USV emission (Burgdorf et al., 2007; Panksepp & Burgdorf, 2003). Dopamine is a neurotransmitter that has been associated with reward circuits and has also been found to be essential for reward-response learning (Alarco et al., 2007). The ventral tegmental 7
area (VTA) is rich in dopamine projection neurons, which extend to various areas of the
basal ganglia associated with reward, including the LH, nucleus accumbens, as well as to
other areas associated with locomotion (Alarco et al., 2007). This is referred to as the
mesolimbic dopamine pathway and is a crucial pathway involved in the neurobiology of
emotion and reward (for review see Wise, 2004).
Positive affect in rats measured by an increase in 50 kHz USV emission has been
produced when even relatively low doses of DA agonists such as cocaine and amphetamine (AMPH) have been administered to limbic brain regions in rats (Panksepp
& Burgdorf, 2003). Research has demonstrated that administration of DA agonists facilitate positive emotional states and increase 50 kHz USVs while reducing 22 kHz
USVs (Knutson et al., 1998). The administration of DA agonists in place preference studies have been used to examine the motivation of the animals to receive rewarding stimulation (Panksepp & Burgdorf, 2003).
Animals in place preference paradigms were exposed to two chambers, one paired with a rewarding drug, while the other paired with a placebo or neutral stimulus
(Panksepp & Burgdorf, 2003). Animals were either given the dopamine agonist AMPH or a neutral substance also referred to as a vehicle (VEH) (Panksepp & Burgdorf, 2003).
After the association of drug and context was learned by these animals, they were allowed to choose between entering or staying in the placebo or drug paired chamber
(Panksepp & Burgdorf, 2003). When the experimental rats were re-exposed to the rewarding chamber they emit higher numbers of 50 kHz USVs than when re-exposed to the neutral chamber (Panksepp & Burgdorf, 2003). This data is consistent with previous findings by Wintink and Brudzynski (2001), who found that DA agonists increased 50 8
kHz USVs, while the DA antagonist haloperidol decreased 50 kHz USVs. All of this
research provides compelling evidence that DA is playing a very important role in USV
emission; however, it may not be the only key neurotransmitter involved.
Opiates have also been associated with reward and positive affective states
(Neslter, 1997). Opiates bind to receptors that are responsive to natural endorphins
produced in the brain (Neslter, 1997). The place preference experiment discussed
previously was repeated with the use of the opiate agonist morphine (MORPH) instead of
AMPH. This provided results similar to findings from dopamine agonist experiment
(Panksepp & Burgdorf, 2003). After the animals were conditioned to the preferred
chamber with MORPH they emitted higher numbers of 50 kHz USVs than when
confined to the non-preferred chamber (Panksepp & Burgdorf, 2003). Research regarding drug addiction has found that administration of opiates increases DA signaling
from the VTA to the nucleus accumbens, suggesting that these two neurotransmitter
systems are functionally related (Laviolette et al., 2002; Nestler, 1997).
While opiate agonists provide increases in USVs, the opposite of this has been
found using drugs that have been shown to have aversive qualities such as naloxone
(NAL) and lithium chloride (LiCl) (Burgdorf et al., 2001). Naloxone is an opioid
antagonist and LiCl has been associated with desensitization of opioid receptors causing hyperalgesia, or increased sensitivity to pain (Johnston & Westbrook, 2004). Rats in this
study were exposed to a similar paradigm, a NAL group, a LiCl group and a VEH control
group. Results indicated that the rats showed place aversion in response to the drugs.
Furthermore when the rats were confined in the previously aversive chamber they emitted
significantly fewer 50 kHz calls and significantly more 22 kHz USVs (Burgdorf et al., 9
2001; Panksepp & Burgdorf, 2003). This is the converse of the other findings regarding
DA, opiates and place preference, providing us with a better understanding of the
neurological mechanisms of USVs (Burgdorf et al., 2001; Panksepp & Burgdorf, 2003;
Wintink & Brudzynski, 2001). This demonstrates that dopamine and endorphins are
involved in USV emission
Overall, administration of drugs that induce positive affective states increase 50
kHz USV and decrease 22 kHz USV emission, while drugs that produce anxious or
negative affect cause the inverse trend in ultrasonic vocalization emission. If the
selectively bred lines of animals are producing differential rates of USVs in response to
stressful situations, it may be due to differences in neurochemical systems in limbic brain
regions. Understanding how selective breeding may be modulating these essential
neurotransmitter systems can tell us more about the genetics underling emotional
processes along with the social consequences of their manipulation.
Selective breeding
The current study utilized selective breeding to manipulate genomic material
related to different phenotypes. This procedure has a long history in psychobiological
research (Brunelli et al., 1997; Brunelli et al., 2001; Brunelli et al., 2005; Brunelli et al.,
2006; Burgdorf et al., 2005; Scott, 1949). Breeding animals based upon measurable behavioral traits has given scientists enormous empirical power when studying the role of genetics in behavior. J.P. Scott utilized selective breeding to study emotion by
monitoring aggressive behaviors of mice and used these results to help explain genetics
behind social evolution in humans (Scott, 1949, 1953). He specifically used a C57 black
strain of mice that were highly inbred, causing almost no genetic or behavioral variability 10
(Scott, 1949, 1953). Using an animal model of inbred mice gave researchers an avenue
to control for genetic confounds and focus on the role that environment has on the
development of social behavior (Scott, 1949, 1953). J.P. Scott used this technique to
examine the effects of prior experience on aggressiveness and fighting abilities in the
mouse model (Scott, 1949, 1953) Other work has utilized selective breeding in rats to
examine genetic mechanisms involved in emotion (Burgdorf et al., 2005; Burgdorf et al.,
2008; Brunelli et al., 1997; Brunelli et al. 2001; Brunelli, 2005; Harmon et al., 2006;
Harmon et al., 2008) and its impact on observable social behavior (Brunelli et al., 2006).
Brunelli and colleagues (2006) studied differences in play behavior in high, low
and random lines of juvenile animals bred for 25 generations with selective pressure applied to 45 kHz infantile USVs. Infantile 45 kHz USVs are emitted in response to maternal and litter separation (Brunelli et al., 2000). Play is an essential part of juvenile behavior, as it allows for development of motor and social abilities (Panksepp, 1998).
Results indicated that when compared to a randomly bred line, both high and low line animals had reduced aspects of their play behavior. The low line had significantly more deficits in this paradigm (Brunelli et al., 2006). Nape contacts (paw touching the other animals’ neck) in the high line animals were reduced, suggesting a slight deficiency in
play initiation. No differences were observed in 50 kHz USVs, pins (one animal pinning the other on its back) and walk-overs (one animal walker over the other animals’ dorsal surface) when compared to the random line (Brunelli et al., 2006). The low line animals
had deficiencies in all areas of play when compared to the random lines, suggesting a
global deficiency in this essential social behavior (Brunelli et al., 2006). Deficiencies in
social behaviors are part of the pathology of a variety of mental illnesses, such as social 11
anxiety disorder, panic disorder, major depression and autism spectrum disorder (Amaral,
2002; Kagan & Snidman, 1999; Kim & Gorman, 2005; Robinson et al., 1992; Smoller et al., 2003).
Local research has conducted selective breeding on Long-Evans rats for approximately 5 years, over 18 generations (Harmon et al., 2006; Harmon et al., 2008).
The animals bred for this study have been bred based upon how many 50 kHz signals were emitted during a “tickle” paradigm. Three lines are created during this process: a high line, a low line and a random line (Burgdorf et al., 2005). The high line rats emit significantly more USVs while the low line rats emit significantly fewer of USVs than the random line animals (Burgdorf et al., 2005). The tickle paradigm occurs at 23 days of age and consists of 2 minutes of robust tickling a day. During the 2 minutes the animals are exposed to alternating trials of tickle and no-tickle lasting 15 seconds each. After 3 days of tickle, they are exposed again to the 2 minute tickle trial and 50 kHz USVs are recorded and scored (Burgdorf et al., 2005). Tickling these rats simulates rough-and- tumble play. During this experience they will follow the tickler’s hand around the tickle box and administer play bites as solicitations for tickle. This “tickle” is administered primarily to the dorsal and ventral surfaces. In order to tickle the animals’ ventral surfaces they are gently flipped onto their dorsal surface and tickled vigorously with the index finger. After just five generations this breeding provided differences in social behavior and vocalizations between the lines (Burgdorf et al., 2005).
Harmon and colleagues (2008) ran studies on the 17th generation of these selectively bred rat pups. Her studies along with other researchers have found some profound differences between the genetic lines (Harmon et al., 2008). In a conditioned 12
odor preference task the low line animals lacked the typical preference for maternal
associated odor cue (Harmon et al., 2008). The low line animals were equally interested in a previously exposed odor regardless of associative history. This behavior may be due to impaired learning, altered stress reactivity or changes in early environment. Another early motivational change found in low line animals is an increase in isolation or 45 kHz calls and locomotor activity during maternal and litter separation, suggesting that low line animals are more anxious than the other lines (Harmon et al., 2008). Low line animals tended to show less social contact compared to random lines during social contact assays
(Burgdorf et al., 2009). The same study also found that low line rats produced more fecal boli during the open field task which is a test designed to measure anxiety (Burgdorf et al., 2009).
The low animals emit more 22 kHz USVs than the high line animals (Burgdorf et
al., 2005). This demonstrates that the behavioral differences between these lines arise
from differences in affective states and not merely because the low line animals are
unable to vocalize (Burgdorf et al., 2005). This provides further support that these
animals are being bred based upon a trait that is related to affect rather than just simply a
part of the basic function involved in USV production. High line animals show more
vocalizations and less avoidance in response to tickle paradigm (Panksepp & Burgdorf,
2000). In addition, rats that produce a greater USV response to “tickling” also show a
significantly higher amount of play solicitations than low lines (Panksepp & Burgdorf,
2003). Monitoring USVs provides us with a window into the emotional states of rats
(Panksepp, 2007). USV emission has provided us with an indicator of whether the rats 13
evaluate the environment as safe or fear-inducing. Fear is an emotional state that influences both USV production and the social behavior of rats.
Fear as a key social emotion
Fear is an emotion that undoubtedly has many adaptive qualities. Without it animals would not survive. It is an essential mechanism hardwired into the mammalian brain. Fear can cause a freezing response, which can help avoid predator detection
(Antoniadis & McDonald, 2001). The physiological changes associated with fear can provide an edge in a fight and influences organisms to avoid situations that previously elicited the fear response. This is accomplished by associating fearful stimuli or events with contextual cues, this is called fear conditioning (Siviy et al., 2006; Walker & Davis,
1997). Fear can be a very powerful and adaptive emotion, but when reactivity to fear is exaggerated or blunted, it can also be very maladaptive (Amaral, 2002; Marcin &
Nemeroff, 2003; Siviy et al., 2006).
Jaak Panksepp has conducted groundbreaking research in affective neuroscience and he proposed that fear can be better understood by separating it into four distinct categories: learned fear with punishment, learned fear without punishment, spontaneous fear with punishment and spontaneous fear without punishment (Panksepp, 1998). The paradigms in this study were used to assess some of these categories of fear.
Learned fear with punishment is a form of conditioned fear. This type of fear has been measured by the acoustic startle response in a fear enhanced startle paradigm
(Panksepp, 1998; Walker & Davis, 1997). This is accomplished by pairing a light
(conditioned stimulus) to a foot shock (unconditioned stimulus), and then administering the light as a cue before a loud noise causing an increase in the acoustic startle response 14
(Panksepp, 1998; Walker & Davis, 1997). Learned fear with punishment can also be associated to contextual stimuli, rather to just a cue. This is achieved by using the conditioned place aversion test. An animal can be exposed to two arenas connected to each other. The animal is confined in one arena paired with the administration of an aversive drug and then confined while in the other chamber paired with the administration of a neutral drug (VEH). When the animal is allowed to move about the two arenas freely, they will avoid the chamber paired with the aversive drug (Burgdorf et al., 2001). This is contextual fear conditioning. Learned fear without punishment has been modeled only in the partial reinforcement extinction effect (PREE), which causes animals to produce a high response rate during extinction training (Panksepp, 1998).
This is thought to be due to the anxiety or frustration of non-reward (Panksepp, 1998).
Spontaneous fear with punishment can be achieved by direct stimulation of the amygdala in animals. Mild stimulation can elicit freezing while stronger stimulation may cause the animals to attempt escape (Panksepp, 1998). The amygdala is a portion of the limbic system that has been implicated in fear and emotion (Kim & Gorman, 2005;
Kluver & Bucy, 1937; Knight et al., 2005; Panksepp 1998). The limbic system is a cluster of brain nuclei involved in emotional processing and memory (Kim & Gorman,
2005). The current project investigated this by using an experiment to examine pre-pulse inhibition (PPI), which requires the animals to inhibit the startle response to a loud noise by the presentation of a preceding softer tone. PPI is not a learned phenomenon, it occurs on the first pre-pulse pulse trial and is reflexive (Braff et al., 2001; Koch, 1999).
Spontaneous fear without punishment is a form of unconditioned fear (Panksepp,
1998). For example, the open field task, assesses anxiety/fear of an animal by how long 15 it stays nears walls or corners of an apparatus versus exploring the center (Panksepp,
1998). This type of fear does not require learning. Spontaneous fear without punishment was assessed in the present study by using a predatory odor and a social recognition paradigm which involves general assessment of safety. The predatory odor component will be accomplished by introducing a worn cat collar into the play chamber; cat scent has proven itself to be useful as a fearful and stressful stimulus in rat research (Blanchard et al., 2001, Panksepp, 1998; Siviy et al, 2006).
Prior research has found that specific neural regions are more involved in different types of fear reactions. Walker and Davis (1997) report a distinct functional and neurological double dissociation between the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). Results demonstrate that while lesions to the basolatoral amygdala disrupts both conditioned and unconditioned fear, more thorough investigations revealed that the CeA is necessary for conditioned fear and the
BNST necessary for unconditioned fear (Walker & Davis, 1997). This double dissociation in structure and function suggests that fear reactivity has dynamic mechanisms that can produce different behavioral responses to a variety of fearful stimuli.
An exaggerated fear response can inhibit normal behavior and social interaction in unnecessary situations. Research has found that exaggerated fear reactivity plays a role in a variety of mental illnesses including post traumatic stress disorder (PTSD), phobias, behavioral inhibition, social anxiety disorder and obsessive compulsive disorder (Kim &
Gorman, 2005). Emotional affect has been found to play a crucial role in fear reactivity
(Marcin & Nemeroff, 2003). Other research has suggested through non-human primate 16
studies that social anxiety may be a result of hyperactivity of amygdala assessments of
fearful stimuli (Amaral, 2002).
All of this research provides indisputable evidence that fear has an essential role
in basic survival and may both facilitate and hinder social learning (Amaral, 2002;
Marcin & Nemeroff, 2003). Hyperactivity or exaggeration of these fear responses have
been found to cause deficits in social behavior (Amaral, 2002; Kagan & Snidman, 1999;
Kim & Gorman, 2005; Robinson et al., 1992; Smoller et al., 2003). Behavioral
differences found between the selectively bred lines may be attributed to variations in the
fear response. Through selective breeding of different extremes of affect we can better
understand how these affective states lead to abnormal fear responses and influence basic social abilities.
Behavioral Paradigms Used in this Study
Social Recognition
Animals need to be able to determine a relationship between themselves and
another organism. It is necessary for their survival to stay with their littermates and avoid
other forms of life that could be harmful. The social recognition task is a test that
measures social memory in animals (Paz-Y-Miño C, 2002; Thor & Holloway, 1982;
Ferguson et al., 2001). Thor and Holloway (1982) used a social recognition test in adult
male and juvenile male Long-Evans rats. Results of testing revealed different levels of
social investigatory behavior of adult males in response to the exposure of familiar and
unfamiliar juveniles. Adult rats were exposed and re-exposed to the same juveniles with
varying intervals between trials. The longer the interval between exposures, the more the
investigatory behavior increased; however, after a 10 minute interval it was found that 17
adult male investigation of the juvenile male actually decreased (Thor & Holloway,
1982). This suggests memories for social partners last for short time periods (Thor &
Holloway, 1982). Males were exposed and allowed to investigate a juvenile male for five
minutes and after a 10 minute interval the experimenters introduced a novel juvenile.
This was done to examine whether or not the previous decrease in investigatory behavior reflects fatigue or habituation rather than recognition (Thor & Holloway, 1982). Results showed that male rats increased rather than decreased their investigation when exposed to
the novel juvenile. The decline in investigation to the familiar male in the first experiment is not explained simply by fatigue, if that were the case the exposure to the novel juvenile after the same ten minute interval would not have elicited such an increase
in investigatory response (Thor & Holloway, 1982).
Thor and Holloway extended their study by using this paradigm to study juvenile
social memory (Thor & Holloway, 1982). Juveniles were exposed to the same procedure
previously discussed as the adult males, with a 10 minute interval between exposures
(Thor & Holloway, 1982). Results showed that juvenile rats do not show a decrease in
investigatory behavior after a 10 minute break when exposed to a familiar animal (Thor
& Holloway, 1982). Further experiments were conducted with inter-exposure intervals
increasing minute by minute to investigate exactly how long these juveniles can remember each other (Thor & Holloway, 1982). Results indicated a decrease in investigatory behavior up to four minutes after initial exposure. Juveniles require shorter
intervals between exposures suggesting that their social recognition abilities may not yet be fully developed. 18
This paradigm has been used at Bowling Green State University to study the
influence of environmental toxins and social learning (Jolous-Jamshidi et al., 2007). The current research used the same apparatus as this previous work; however, the paradigm was modified in the hopes of gaining a stronger effect. The previous paradigm consisted
of three five minute socialization periods: The first two time blocks included the same littermate and the last a different littermate (Jolous-Jamshidi, 2007). The new paradigm contained three time blocks with the same stimulus animal and a fourth with the different stimulus animal. USVs were also monitored during this experiment. It was expected that an additional exposure to the same stimulus animal would lead to enhanced recognition and greater investigatory behavior in response to the different stimulus animal.
The social recognition task is a method used to test social memory. Testing the different selectively bred lines could reveal a difference in this ability. We predicted that low line animals would show deficits in social recognition revealed by a lack of increase in time spent investigating the different stimulus animal. In addition, it was predicted that there would be no dramatic rise in social investigation upon introduction of the different stimulus animal in the low line animals. This finding will help explain previous data and extend our knowledge in regards to the social and emotional abilities of these animals.
Previous work by Harmon and colleagues (2006) found variations in play behavior demonstrated by the low line animals. The present study aims to examine whether the deficit seen in social behavior could be due to an inability to recognize a littermate. It is possible that enhanced fear may cause deficits in social recognition and alter socially directed behavior. Play studies have shown that rats deprived of direct social contact exhibit more playful behavior after isolation (Holloway & Sutter, 2004; 19
Panksepp & Burgdorf, 2000). Research has also found that rodents who were socially housed emitted significantly less vocalizations and demonstrated significantly less play behavior compared to those who were isolated (Panksepp & Burgdorf, 2000). This suggests that the USV emission and play behavior are dependent on prior social experiences. Research has also provided evidence that any type of deprivation of direct social contact will increase later play (Holloway & Suter, 2004). Animals housed in cages with wire mesh dividing them, that could see and smell each other without physical contact exhibited the same tendency of rats housed in isolated conditions (Holloway and
Suter, 2004). Furthermore rats, when given the choice, prefer to spend their time with other rats that emit more 50 kHz vocalizations (Panksepp, 2007). Social housing seems to be able to have an effect on the degree and type of interactions between animals. The current study kept the juveniles isolated during the social recognition and play behavior paradigms. The variations found in social behavior in these selectively bred lines of animals may be due to deficiencies in social recognition abilities, specifically causing altered juvenile play behavior (Burgdorf et al., 2009; Harmon et al., 2006; Harmon et al.,
2008).
Play Suppression
The play suppression experiment will utilize cat scent to examine instinctual fear responses in these selectively bred lines. Prior research has used cat hair to elicit this response due to its naturalistic fear eliciting properties in rats (Panksepp, 1998). This has provided interesting insights into instinctual fear response and its effects on social behavior. Results showed that play behavior in juvenile rats was severely inhibited by the presence of cat hair, and this effect persisted over several days (Panskepp, 1998). Napes 20 and pins (indicators of play) were significantly decreased during the extinction of this behavior due to the cat hair (Panksepp, 1998). These results have been replicated by other researchers with worn cat collars (Siviy et al., 2006). This suggests that juvenile rats are sensitive to the smell of predators and that predatory odor suppresses social behavior such as play.
Previous research has provided a paradigm for play including the introduction of cat hair; however, other researchers have utilized the introduction of both a worn and unworn cat collar (Siviy et al., 2006). The cat collar makes for a better stimulus because it is more practical than using tufts of cat fur. It can be more easily stored, acquired and implemented in experiments. Both these worn and unworn cat collars can be re-used. In addition, utilizing cat collars offers a practical avenue to provide a reliable control for this experiment with an unworn cat collar. Baseline play data was examined in the animals before exposed to an unworn and then a worn cat collar (Siviy et al., 2006). Play was suppressed and remained significantly below baseline for 6 days (Siviy et al., 2006).
This thesis project used the same paradigm and monitored additional USVs during the worn cat collar testing day to compare differences between the selectively bred lines of animals’. These data gave us insight on the effects of selective breeding on the innate fear response to predator odor and social behavior; however, another measure of reflexive fear reactivity will also be examined.
Prepulse Inhibition
Research investigating the reflexive fear response has utilized a prepulse inhibition (PPI) procedure (Braff et al., 2001; Rosa et al., 2005a, 2005b). PPI is a well established paradigm to examine reflexive startle and the inhibition of startle reflexes 21
(Braff et al., 2001; Rosa et al., 2005a, 2005b). It is a procedure involving two tones, a
prepulse or cue followed by a loud and aversive tone (Koch, 1999; Braff et al., 2001).
By following this procedure with the selectively bred animals, we can examine any differences in the reflexive fear response between the animal lines.
To understand PPI it is wise to first understand the how it is measured. This experiment examines the acoustic startle response (ASR) of an animal. Startle has been
speculated to be a response or preparation to predator attack or a blow; it consists of a fast twitch of body and muscles (Koch, 1999). Startle is not just an auditory phenomenon; it can occur at the other senses as well and is influenced by genetics, background noise, loudness, inter-trial intervals, and drugs (Braff et al., 2001; Koch, 1999). Inhibition of an
ASR to a loud tone based upon the presentation of a weaker prepulse is how PPI is assessed (Braff et al., 2001; Koch, 1999). PPI is used to measure a reflexive process called sensorimotor gating.
Sensorimotor gating is a process by which the central nervous system alters a motor response to an intense stimulus (loud tone) due to the presentation of a weaker stimulus (prepulse). This process occurs on the first prepulse-pulse trial, suggesting that this it is reflexive and does not require learning (Braff et al., 2001; Koch, 1999). PPI abilities increase with the intensity and length of the prepulse, which provides better inhibition of the startle (Braff et al., 2001).
Human research has found that a reduction in PPI abilities is associated with a variety of mental illnesses including schizophrenia, panic disorder and obsessive
compulsive disorder (Braff & Geyer, 1990; Braff et al., 2001; Geyer & Swerdlow, 1990;
Ludewig et al., 2002; Hoenig et al., 2005). Prior studies have found that the 22
dopaminergic systems play a key role in PPI, providing an avenue to test efficacy of
neuroleptic drugs (Geyer et al., 2001; Mansbach et al., 1988; Braff et al., 2001). Research
has also found interesting genetic components to PPI. Family members of patients
diagnosed with schizophrenia also demonstrate some deficiencies in PPI (Cadenhead et
al., 2000). Swerdlow and colleagues (2001) investigated the role of genetics and
environment of sensory motor gating, they concluded that key genetic components exist
which require further investigation. Furthermore, scientists have successfully carried
out selective breeding in rats for PPI deficits (Schawbe et al., 2007). This evidence
suggests that sensorimotor gating has a role in a variety of mental illnesses, involves the
dopaminergic pathway, and carries genetic components that can be selected for.
CHAPTER II: AIMS
The specific aims of this study were to examine whether or not there are
significant differences between selectively bred lines in both USV emission and
behavioral responses using three experiments:
1. Social recognition abilities in juvenile animals.
2. Play suppression in response to introductions of a natural predatory
odor.
3. PPI as a reflexive measure of anxiety in situations of unpredictable
stressors.
Aim 1: Social Recognition
The specific aim of this experiment was to examine how the selectively bred line animals may show variations in basic social recognition abilities. In order for an animal to recognize a potential play mate, they first must be sure that the environment is safe. 23
Prior studies have found that fearful environments suppress play in juvenile rats and cause freezing behavior (Siviy et al., 2006; Blanchard et al., 2001). An anxiogenic phenotype may impede on social behavior in safe situations, causing deficiencies in the development of essential social abilities such as social recognition. Alterations in social recognition in these selectively bred lines of animals may be causing variations seen previously in play behavior (Harmon et al., 2006).
Aim II: Play Suppression
The goal of utilizing the play suppression paradigm was to gain a better understanding of how selective breeding affects both the response to natural fear, and the extent to which this fearful reaction influences social behavior. When rats are afraid they exhibit a number of behaviors: freezing, avoidance, aversive calls, and risk assessment
(Takahashi et al., 2005; Panksepp, 2007). Studies have shown that rats exposed to the smell of a predator such as a cat show dramatic increases in fear behavior that remain elevated for up to 7 days after re-exposure to the environment (Takahashi et al., 2005).
Using this data to examine the extinction of suppressed play behavior due to fear conditioning should provide interesting insights on differences in coping abilities that these animals may possess. We predicted that the high line animals would demonstrate an accelerated fear extinction process while the low line animals would produce a slower fear extinction process. Results have implications on the consequences of selective breeding for 50 kHz USV emission on the instinctual fear response in juvenile social behaviors.
Aim III: Prepulse Inhibition 24
This experiment explored differences between the lines in reflexive behavior to
unpredictable aversive stimuli. Differential responding to unpredictable fearful stimuli
assessed the animals’ baseline fear reactivity. These results have ramifications on the
genetic elements of reflexive fear behavior and sensorimotor gating processes. It was
predicted that the low line animals may have a deficiency in PPI and an exaggerated ASR
than controls, as prior studies have suggested that they are more anxious than the random line animals (Burgdorf et al., 2009; Harmon et al., 2006; Harmon et al., 2008).
25
CHAPTER III: METHODS
Breeding Methods:
Long-Evans rats were bred at Bowling Green State University. Breeders were at least 70 days of age. Six to eight litters were bred per line creating approximately 180 total animals. One male was bred with two females for each of the three lines of animals: high, random and low. The colony room was kept at around 22 degrees Celsius with humidity at around 40-50%, and subjects were housed on a 12:12 light/dark cycle. The lights turned on at 7:00 AM and off at 7:00 PM. The random line animals are designed to represent the impact of partial inbreeding. Animals tested of the same age from another study were also used as controls for PPI. Burgdorf and colleagues (2009) reported that typical Long-Evans rats provided statistically indistinguishable responding when compared to the selectively bred random lines. Each litter was weaned and isolated at 21 days of age. They were housed in 65 X 24 X 15 cm clear plastic cages with corn cob bedding. They were given ad libitum access to food (Harlan Teklad Rat Chow #8604) and tap water. This project was approved by the Institutional Animal Care and Use
Committee at Bowling Green State University.
Social Recognition Methods:
26
Long-Evans rats from the selectively bred lines were tested at 22 and 23 days of
age. Half of each litter was tested at PND 22 and the other half was tested on PND 23.
Splitting the testing over two days ensured that the animals were tested around the same time of day. The testing animal was placed into a tube that connects two test chambers.
The two testing chambers have dimensions of 25 x 20x 13 cm. The tube has a length of
16.5 cm and a diameter of 4 cm. The direction the experimental animals were placed facing was counterbalanced between littermates over the two days, with the first half of animals (PND 22) facing the social box on the first trial. The direction that the animals faced in each of the following 3 trials was alternated. Two males and two females from each litter were chosen as stimulus animals. One animal served as the same stimulus animal and the other as the different stimulus animal for each gender.
The stimulus rats were placed behind a wire grate so that the test subjects could
smell but not touch them. The experimental rats were placed into the tube of the
apparatus and the blockades were removed. This allowed the rats to go to either the
chamber with their conspecific (social chamber) or an empty chamber (non-social
chamber). Four testing sessions lasting 5 minutes were conducted with a three minute
break in between. During the three minute inter-trial interval (ITI) the test rat was placed
into its’ home cage. During the first 3 sessions the experimental rats were exposed to the
same stimulus rat, and in the last session they were exposed to the different stimulus rat
(Trial 1: TS1; Trial 2: TS2; Trial 3: TS3; Trial 4: TD). Each session was recorded onto a
DVD. USVs were also monitored during testing.
Play Suppression Methods 27
At twenty four days of age same sex littermates were paired based upon similar
weights, and then placed in a 30 X 30 X 30 cm Plexiglas play chamber with corn cob
bedding. The bedding in the play chamber was changed between gender and litters. The
play chambers have black paper taped to three of the sides. Two chambers were utilized
in this paradigm, one for the worn cat collar and the other for the rest of the testing days.
The rats were exposed to three days of acclimation during which one of the animals in
each pair was marked with a Crayola magic marker. The same animals were marked
throughout the play testing. Each play bout lasted 5 minutes.
Recording did not take place during the acclimation phase. On the fourth day the
animals were video recorded during their play bouts onto a DVD (DVD format:
Panasonic DVD recorder and camcorder) using an aerial view. This served as the
baseline measure of play behavior for the comparison of extinction testing sessions. On the fifth day four 2 cm strips of unworn cat collar were warmed up in 55 degrees Celsius water for 10 minutes. This was done to warm up the collars as they are stored in a freezer to preserve freshness of the cat scent. They were then placed into each corner of the play chamber and buried in the corn cob bedding, this served as a control for the worn cat collar. On the sixth day, four 2 cm pieces of warmed worn cat collar were placed in each corner of the worn cat collar play chamber in the same manner. A separate play chamber was used for the worn cat collar to ensure that the smell did not linger during the extinction phase. Eight consecutive days of play without any collars were conducted after the worn cat collar exposure. This was done to investigate how long it took for the rats play behavior to return to baseline, this is the extinction phase. USVs were recorded on the worn cat collar day (Pettersen D980 ultrasonic detector, Uppsala, Sweden). 28
Prepulse Inhibition Methods
:
At 50 and 51 days of age rats were placed into a Startle Response chamber and
exposed to a variable interval (VI) set of tones. The litters were counterbalanced by
gender between the two days. The animals were tested between PND 50 and 80. USVs
were recorded in half of the test animals due to limitations of resources. The variable
session consisted of random exposure to 3 trial types: 1. prepulse alone 2. pulse alone 3.
combination of prepulse and pulse together. The third trial measured pre-pulse inhibition
while the other two trials provided information about how these animals responded to the pulse and prepulse in isolation of each other. Initially there was a 5 minute acclimation period to the apparatus with continuous white noise at 70 dB. The prepulse occurs at about 80 dB and lasts 20 ms while the pulse alone occurs at around 118 dB and also lasts for 20 ms. The interval between the prepulse and the pulse in the third trial is 30 ms. The animals were exposed to 60 trials (20 of each) in a pseudorandom order with an inter-trial interval average of 15 seconds, the same random session was used for every animal. 29
The startle chambers were calibrated according to the directions written in the
manual on every day that testing took place (San Diego Instruments, CA). The program
was then set up and made ready for the animals. Animals were in the room when the
program was entered into the computer. The subjects were then placed into the tube of
the startle response chamber and restrained by two plastic blockades. The tube had a
diameter of 9 cm and a length of 20 cm. One of the startle chambers had a device to record ultrasonic vocalizations during testing (Pettersen D980 ultrasonic detector;
Uppsala, Sweden). After the session was complete each animal was returned to their
home cage and the tubes were cleaned out. USV data was saved onto a computer and
scored on a spectrogram using the computer software Avisoft Pro (Germany). Both 50
kHz and 22 kHz USVs were counted manually. 30
CHAPTER IV: STATISTICAL ANALYSIS
Social Recognition Analysis:
Recorded sessions (DVD format: Panasonic DVD recorder and camcorder) were
scored using behavioral scoring software (ODLOG) in real time. Behaviors in the social
box were divided into 3 categories based on degree of social interaction. Division of these behaviors was used to gain a better sensitivity of social behavioral differences between groups.
1. Social investigation: the experimental animal physically interacting with the
grate, this includes biting the grate, poking the nose through the holes, and
climbing on the grate. Also the experimental animal facing and approaching the
stimulus animal at an angle less than 90 degrees
2. General behaviors: grooming, sleeping, non-investigatory behavior while in
the social box.
3. Tube time: the amount of time the animal spent in the tube.
4. Non-social box time: the amount of time the animal spent in the nonsocial box.
The first two behaviors described previously were summed as “total social box duration”. The social investigation duration was taken as a percentage of total time in the social box. This controlled for the amount of time the animals spent in either the social or non-social box. This will be referred to as a percentage of social investigation (SI).
(Social investigation duration/total time in social box duration) x 100.
A motivational percentage (SM) was also calculated, this was accomplished by adding the total time in the social box and dividing it by total time in the apparatus.
(Time spent in social box/ total time in apparatus) x 100. 31
The time the animal spends in the tube and nonsocial box was also coded for.
USVs were recorded (Pettersen D980 ultrasonic detector, Uppsala, Sweden) and counted manually on a spectrogram using AviSoft Pro (Germany). A mixed design ANOVAs were used to analyze social behavior. The between subjects variables were sex and line and the within subjects factors were the 4 different trials. Percentage of SI and SM as calculated by the equations described previously were used as the dependent variables. A series of t-tests were used to examine differences between trials, the Bonferroni correction was used for each set of t-tests. The alpha value was set at .1 and divided by the number of t-tests used in each analysis. This value was used as the criterion.
Play Suppression Analysis
Play behavior (DVD format) was scored in real time using behavioral scoring software (ODLOG). Two behaviors were scored: dorsal contacts; which consists of paw touches to the dorsal surface of the other testing animal and pins. Pins are defined as one animal pinning the other on their back with at least 3 paws in the air (Panksepp, 1998).
USV data was counted manually on a spectrogram using AviSoft Pro.
Mixed design ANOVAs were used to analyze play behavior. The between subjects variables were animal sex and line. The within subject variables were day of exposure to play apparatus. Dependent variables were dorsal contact number, pin number and pin duration. Independent variables were animal line and sex. A series of t- tests were used to examine line differences between days, the Bonferroni correction was used for each set of t-tests. The alpha value was set at .1 and divided by the number of t- tests used in each analysis. This was the criterion used.
Prepulse Inhibition Analysis 32
ASR was measured by the startle chambers in units of 1.22 mV. A ratio
consistent with the literature was used to transform the animals ASR into a percentage of
PPI (Braff et al., 2001).
100- [(prepulse + pulse/ pulse alone) x 100]
Mixed design ANOVAs were conducted on the PPI ratio, USV emission and
ASR. Animal weight was used as a covariate for the ASR analysis, as weight has direct bearing on startle amplitude. The Greenhouse-Geisser was used for within subject variables. USVs were transformed by dividing the number of USVs emitted by the number of seconds in each trial. Between subject measures included sex and animal line. Within subjects measures examined differences in the dependent variables: ASR,
PPI and USV emission. Both 22 kHz and 50 kHz USVs were converted into a ratio of
USVs/sec in order to control for the length of the trials. Two of the trials in the VI condition were removed from the analysis. Only animals that had ASR, PPI, USV and weight data were used in the analysis.
CHAPTER V: RESULTS
Social Recognition
Results were obtained from individual animals Random line n= 17 (9 females, 8 males, 3 litters); low line n = 31 (16 males, 15 females, 5 litters); high line n = 22 (5 females, 17 males, 6 litters). There was a main effect for trial type for percentage of SI
(F(2.624, 177.027) = 11.786, p = .000). The Greenhouse-Geisser correction was used for a violation of sphericity. No other main effects or interactions were found between trial types for each group. 33
Paired samples t-tests were used to investigate the main effect of trial on each
animal line (table 1 & 2). The random line animals demonstrated a significant decrease
from TS1 to TS3 and TS2 to TS3 (t(16) = 5.304, p = .000; t(16) = 3.908, p = .000; figure
1). They produced a non-significant increase from TS3 to TD (t(16) = -2.114, p = .051).
There was not a significant difference in percentage of SI between the TS1 and crucial
TD trial. This observation suggests that SI was reinstated in the final trial when the
different stimulus rat was placed in the apparatus in the final trial.
High line animals produced a non-significant decrease from TS1 to TS2 and TS1
to TS3; suggesting that these animals demonstrate social recognition abilities to the same
stimulus animal (figure 1). The high line animals also produced a non-significant decrease in percentage of SI from TS1 to TD (figure 1). While TS1 and TD are
significantly different, there is a non-significant increase from TS3 to TD. This implies
that these animals may have slight deficiencies in social recognition abilities. The high
line animals investigation did not significantly differ from the random line during the TD
trial, the lack of significance found between TS3 and TD in the high line animals is due
to a less dramatic decrease in SI over the first three trials.
The low line animals produced a significant decrease from TS1 to TD (t(30) =
3.401, p = .002; figure 1). The low line animals are showing a decrease in SI over the
first three trials suggests that they possess recognition abilities; however, because there
was a decrease rather than an increase during exposure to the different stimulus animal
this may reflect habituation or frustration of non-rewarding social interaction rather than
social recognition. 34
Table 1: SI Percentage, paired samples t-tests investigating within subjects effects over trials.
TS1 vs. Trial 2 TS2 vs. TS3 TS3 vs. TD TS1 vs. TS3 TS2 vs. TD TS1 vs. TD
Random t(16) = 1.308, t(16) = 3.908, t(16) = -2.114, t(16) = 5.304, t(16) = 0.422, t(16) = 1.451, p = .209 p = .001 p = .051 p = .000 p = .679 p = .166 * * High t(21) = 2.495, t(21) = 0.758, t(21) = -0.482, t(21) = 2.162, t(21) = 0.231, t(21) = 2.225, p = .021 p = .457 p = .635 p =.042 p = .819 p =.037
Low t(30) = 2.386, t(30) = 0.895, t(30) = 1.356, t(30) = 2.777, t(30) = 2.367, t(30) = 3.401, p = .024 p = .185 p = .378 p = .009 p = .025 p = .002 **
Bonferoni correction: p = .1/18 = .005. 35
Table 2: SI Percentage Means ± S.E.M.
TS1 TS2 TS3 TD
Random 67.68 ± 2.85 62.04 ± 2.14 46.88 ± 5.00 59.63 ± 5.08
High 67.53 ± 1.89 60.61 ± 3.36 56.66 ± 4.66 59.56 ± 3.99
Low 69.29 ± 2.21 63.42 ± 1.82 60.38 ± 2.30 57.64 ± 2.33
All Animals 68.34 ± 1.31 62.20 ± 1.65 55.93 ± 2.17 58.72 ± 2.01
36
Figure 1: Means ± S.E.M. for percentage of SI.
Random 80 High 70 Low
60
50
40
30 20 Social Investigation (%) 10
0 TS1 TS2 TS3 TD4 Trial Type
37
The same analysis on percentage of SM found a main effect of trial type (F(2.571,
172.270) = 26.702, p = .000). The GGC was used again to correct for a violation of sphericity. No other main effects or interactions were found between trials and groups.
Paired samples t-tests were used to investigate the main effect of trial on each animal line
(table 3 & 4). The random line animals demonstrated a significant increase in SM from
TS1 to TD (t(16) = -4.463 p = .000) and TS2 to TD (T(16) = -4.230, p = .001; Figure 2).
The random line animals did not show a significant increase from TS2 to TS3, showing that their motivation to investigate reflected their ability to recognize the animal as the same as the previous exposures. There was a dramatic increase from TS3 to TD, suggesting that the increase in investigation co-occurring with an increased motivation to explore the new stimulus animal. The high line animals demonstrated a significant increase from TS1 to TD (t(21) = -4.735, p = .001; Figure 2). The high line animals also failed to show an increase in SM from trials TS2 to TS3, suggesting again that their decrease in investigation of the same stimulus animal reflected recognition of the conspecific. The high line animals showed a non-significant increase in SM from TS3 to
TD, consistent with their non-significant increase in SI. Low line animals produced a significant decrease from TS1 to TS2 (t(30) = -4.554, p = .000), TS1 to TS3 (t(30) =
-5.200, p = .000) and TS1 to TD (t(30) = -4.634, p = .000; Figure 2). They did not show a significant increase from TS2 to TS3, reflecting their non-significant decrease from
TS2 to TS3. All of the animal lines failed to show a significant increase in social
38
Table 3: SM paired samples t-tests comparing wthin subjects effects across trials.
TS1 vs. TS2 TS2 vs. TS3 TS3 vs. TD TS1 vs. TS3 TS2 vs. TD TS1 vs.TD
Random t(16) = -2.60, t(16) = -1.063 t(16) = -1.808, t(16) = -2.292, t(16) = -4.230, t(16) = -4.463, p =.038 p = .303 p =.090 p =.036 p =.001 p =.000 * * High t(21) = -2.173, t(21) = -0.318, t(21) = -1.272, t(21) = -2.604, t(21) = -1.589, t(21) = -4.735, p =.041 p = .754 p =.217 p =.017 p = .127 p =.000 * * Low t(30) = -4.554, t(30) = -1.706, t(30) = 0.165, t(30) = -5.200, t(30) = -1.667, t(30) = -4.634, p =.000 p = .098 p = .870 p = .000 p = .106 p =.000 * * *
Bonferroni correction criterion: p = .1/18 = .005. 39
Table 4: SM: Means ± S.E.M.
TS1 TS2 TS3 TD
Random 56.38 ± 6.04 72.39 ± 2.91 78.59 ± 5.66 87.89 ± 3.05
High 55.13 ± 3.31 67.04 ± 3.71 68.55 ± 3.73 75.68 ± 3.83
Low 55.7 ± 5.23 73.02 ± 1.86 77.85 ± 3.16 77.29 ± 2.69
All Animals 55.69 ± 2.06 70.99 ± 1.72 75.51 ± 2.31 79.36 ± 1.96
40
Figure 2: Means ± S.E.M. for percentage of SM.
Random High 100 Low 90 80 70 60 50 40 30 Social Motivation (%) 20 10 0 TS1 TS2 TS3 TD4 Trial Type
41
motivation from TS2 to TS3, suggesting that their recognition abilities caused
investigation not to be motivating. The high line animals showed an increase in percentage of SM from TS3 to TD reflective of their increases in SI. The low line animals showed a slight decrease between these two trials, suggesting that the different stimulus animal in TD did not motivate social approach by the low line animals. USV emission during social recognition can give an indicator of how rewarding these animals found each trial.
Results from the data analysis of 50 kHz USV emission revealed a main effect of trial type (F(2.579, 172.809) = 6.431, p = .000). No other main effects or interactions of
50 kHz USVs on trial type were found. Paired samples t-tests were used to investigate the main effect of trial on each animal line (table 5 & 6). Random line animals produced non- significant decrease from TS1 to TS3; TS1 to TD; TS2 to TS3; TS3 to TD (Figure 3).
The high line animals demonstrated no significant differences in 50 kHz USV emission across trials. The low line animals showed non-significant decreases from TS1 to TS3 and TS1 to TD in 50 kHz USV emission (Figure 3). Due to the low amount of 22 kHz
USVs emitted during this experiment they were removed from the analysis. 42
Table 5: 50 kHz USVs during the social recognition experiment.
TS1 vs. TS2 TS2 vs. TS3 TS3 vs. TD TS1 vs. TS3 TS2 vs. TD TS1 vs. TD
Random t(16) = 1.93, t(16) = 2.456, t(16) = -2.017, t(16) = -2.897, t(16) = 0.696, t(16) = 2.897, p = .072 p = .06 p = .061 p = .011 p = .496 p = .015
High t(21) = 1.401, t(21) = -0.158, t(21) = -1.226, t(21) = 1.414, t(21) = -1.320, t(21) = -0.012, p = .176 p = .05 p = .05 p = .05 p = .05 p = .05
Low t(30)= 1.162, t(30) = 1.219, t(30) = 0.350, t(30) = 2.623, t(30) = 1.284, t(30) = 2.559, p = .254 p = .232 p = .729 p = .014 p = .209 p = .016
Bonferoni correction: p = .1/18 = .005. 43
Table 6: Means ± S.E.M for 50 kHz USVs during the social recognition experiment
TS1 TS2 TS3 TD
Random 41.41 ± 12.09 23.18 ± 6.30 10.24 ± 5.29 20.29 ± 5.32
High 16.23 ± 8.37 5.55 ± 1.57 5.73 ± 1.91 16.86 ± 13.85
Low 28.48 ± 7.59 22.48 ± 9.50 13.06 ± 5.41 11.39 ± 3.58
All Animals 27.77 ± 5.26 17.33 ± 4.56 10.07 ± 2.63 15.11 ± 3.51
44
Figure 3: Means ± S.E.M. for 50 kHz USVs emitted during the social recognition paradigm.
Random 60 High Low 50
40
30
20
50 kHz USV Emission 10
0 TS1 TS2 TS3 TD4 Trial Type
45
Play Suppression
Results were obtained from individual animals. Random line n = 42 (22 females,
20 males, 5 litters); high line n = 50 (18 females, 32 males, 7 litters), low line n = 58 (22
females, 36 males, 9 litters). A 2x3x11 mixed design ANOVA was used to examine
effects of line and sex on pin number. There was a main effect of day of play (F(6.152,
885.897) = 39.162, p = .000). There was also a day of play and line interaction
(F(12.304, 885.897) = 8.036, p = .000; figure 4). The Greenhouse-Geisser correction was
used for a violation of sphericity. There were no sex effects. There was a main effect of line (F(2, 144) = 5.956, p = .003). Post-hoc Bonferroni comparisons revealed that the low line animals overall produced more pins than the high line (p = .006) and the random line
(p = .024) animals.
Independent samples t-tests were used to investigate differences in pin number
between groups (table 7). Low line animals produced more pins than the random line on
extinction days 1 - 3. The high line produced significantly more pins than the random on
the first extinction day; however, the high line produced significantly less pins than the
random line on extinction day 7. Low line animals made significantly more pins than the
high line animals on extinction days 1, 3 and 6 – 8.
A series of paired samples t-tests investigated the within subject effect on trial for
each group, all three lines demonstrated significant suppression of pin number on the
worn cat collar day (tables 8- 11). The random line animals return to baseline levels of
pins on extinction day 7 (table 8). High line animals never return to baseline number of
pins (table 9). Low line animals return on the first extinction day (table 10). 46
Figure 4: Means ± S.E.M for pin number during play suppression.
Random 25 High † Low
20 * ф † 15 † ▲ †
10 Pin Number Pin 5
0
rn 2 3 4 8 ine o xt1 xt xt xt xt5 xt6 xt7 xt W E E E E E E E E Basel Unworn Day of Play
The symbol that indicates each different line is placed above where significant differences were found in the results (Random = ф; High = *; Low = †).
Bonferroni: 1/33 = p = .003 47
A 2x3x11 mixed design ANOVA was used to examine the effects of line and sex on pin duration. A main effect of line was found (F(7.062, 1016.976) = 28.82, p = .000; figure 5). There was also a day of play and line interaction on pin duration (F(14.125,
1016.976) = 7.011, p = .000. There were no between subjects effects of line or sex on pin duration.
Independent samples t-tests were used to investigate any differences of pin duration between the lines on each day of play (table 12). Low line animals produced longer pin durations on extinction day 1 than high line animals. High line animals produced significantly longer pin durations on the baseline day of play and unworn cat collar day when compared to low line animals. Low line animals produced significantly longer pins than the high line animals on extinction day 8. Random line animals produced more pins than the high line on extinction day 7.
Paired samples t-tests were conducted to investigate the within subjects effects on day of play on pin duration, all three lines demonstrated significant suppression of pin duration on the worn cat collar day (tables 13 – 16). Random line animals return to their baseline pin duration on extinction day 3 (table 13). High line animals briefly return to 48
Figure 5: Means ± S.E.M. for pin duration during the play suppression experiment.
Random 35 High * Low 30 * † 25 ф ф * 20 15 10 5 0 Pin Duration in Seconds in Duration Pin
e n 5 7 n xt2 xt4 li Ext1 E Ext3 E Ext Ext6 Ext Ext8 se Wor a Unworn B Day of Play
The symbol that indicates each different line is placed above where significant differences were found in the results (Random = ф; High = *; Low = †).
Bonferroni: .1/33 = p = .003 49 baseline on extinction day 3, but fall below on extinction day 6 never return to original levels of pin duration (table 14). Low line animals return to baseline levels of pin duration on the first extinction day (table 15).
A 2x3x11 mixed design ANOVA was conducted investigating the effects of sex and animal line on dorsal contacts on day of play. There was a between subjects effect of sex on dorsal contacts (F(1, 144) = 3.900, p = .05). Pairwise comparisons revealed that males (66.365 ± 3.426) produced more dorsal contacts than females (56.010 ± 3.970). A main effect of day of play on dorsal contact number was found (F(7.374, 106.881) =
61.583, p = .000). There was also a day of play*line interaction (F(14.748, 106.881) =
6.565, p = .000; figure 6).
Independent samples t-tests were used to investigate any between subjects differences of dorsal contact on each day of play (table 17). Low line animals produced more dorsal contacts than the random line on the worn cat collar day and extinction day
1. High line animals produced significantly more dorsal contacts when compared to the random line on extinction day 1. The random line produced more dorsal contacts than the high line on extinction day 7. Low line animals produced more dorsal contacts than the high line on extinction day 7.
Paired samples t-tests were conducted to examine the main effect of day of play for each animal line, all three lines showed significant suppression of dorsal contacts on the worn cat collar day (tables 18-21). Random line dorsal contact number returned to baseline on extinction day 4 (table 18). High line animal dorsal contact frequency returned to baseline on the third extinction day (table 19). Low line animals dorsal contact number returned to baseline on the second extinction day (table 20). 50
Figure 6: Means ± S.E.M. for number of dorsal contacts during play suppression.
ф Random High 120 † * † Low 100 * 80 † 60 † 40 Pin Number Pin 20 0
e 2 3 4 5 6 7 8 rn xt xt xt xt xt xt xt elin Wo Ext1 E E E E E E E as B Unworn Day of Play
The symbol that indicates each different line is placed above where significant differences were found in the results (Random = ф; High = *; Low = †).
Bonferroni: .1/33 = p = .003 51
A one-way ANOVA was conducted on both 50 and 22 kHz USV emission on the
worn cat collar day. There were no between subjects effects of line on 22 kHz USV
emission. A main effect of 50 kHz USV emission on line was found (F(2, 147) = 3.734, p = .026. Post-hoc Bonferonni comparisons revealed that the high line emitted fewer 50 kHz USVs during exposure to the worn cat collar than the random line (p = .021; figure
7). 52
Figure 7: Means ± S.E.M. 50 kHz USVs during the worn cat collar day.
300
250 * 200
150
100 50 kHz USVs 50
0 Random Low High Animal Line
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 < ***).
53
Table 7: Independent samples t-tests pin number during play suppression.
Baseline Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 High vs. t(83.309) t(82.993) t(65.161) t(64.570) t(90) = t(90) = t(90) = t(90) = t(90) = t(72.251) t(50.507) Random = 1.558, = 1.590, = 0.658, = 3.407, 1.135, -0.357, 0.490, 1.022, -2.690, = -4.353, = 2.851, p = .123 p = .166 p = .513 p = .001 p = .259 p = .722 p = .626 p = .310 p = .009 p = .000 p = .006 * * Low vs. t(97.973) t(95.669) t(62.981) t(60.067) t(81.585) t(83.823) t(94.393) t(97.069) t(98) = t(97.947) t(90.633) Random = -0.653, = -0.070, = 2.733, p = 5.697, = 3.893, = 3.226, = 2.572, = 2.390, 0.346, = 0.360, = 2.965, p = .535 p = .944 = ..008 p = .000 * p = .000 p = .002 p = .012 p = .05 p = .019 p = .218 p = .719 * * Low vs. t(106) = t(106) = t(87.690) t(74.709) t(101.879) t(88.898) t(90.508) t(104.589) t(92.907) t(90.886) t(66.979) High -2.017, -1.459, = 2.109, = 3.980, = 2.618, = 3.411, = 2.314, = 1.424, = 3.670, = 4.479, = 5.223, p = .046 p = .148 p = .046 p = .000 p = .028 p = .001 p = .023 p = .157 p = .000 p = .000 p = .000 * * * * *
Bonferroni: .1/33 = p = .003 54
Table 8: Paired samples t-tests on pin number during play suppression for random line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(42) = 0.495, t(42) = t(42) = t(42) = 8.124, t(42) = 7.811, t(42) =4.251, t(42) = 4.492, t(42) = 3.411, t(42) = 1.622, t(42) = 3.374, p = .624 13.968, 12.810, p = .000 * p = .000 * p = .000 * p = .000 * p = .001 * p = .113 p = .002 p = .000 * p = .000 * Unworn ------t(42) = t(42) = t(42) = 8.174, t(42) = 7.232, t(42) = 4.093, t(42) = 3.926, t(42) = 3.060, t(42) = 1.181, t(42) = 2.731, 13.073, 12.127, p = .000 * p = .000 * p = .000 * p = .000 * p = .004 p = .244 p = .009 p = .000 * p = .000 * Worn ------t(42) = .253, t(42) = t(42) = t(42)= t(42) = t(42) = t(42) = t(42) = p = .801 -4.053, -6.119, -4.819, -5.756, -7.182, -8.222, -5.581, p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * Ext 1 ------t(42) = t(42) = t(42) = t(42) = t(42) = t(42) = t(42) = -3.824, -6.847, -5.118, -5.129, -7.239, -7.931, -5.880, p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * Ext 2 ------t(42) = t(42) = t(42) = t(42) = t(42) = t(42) = -1.867, -3.355, -3.189, -4.346, -5.647, -3.072, p = .069 p = .002 p = .003 p = .000 * p = .000 * p = .004 Ext 3 ------t(42) = t(42) = t(42) = t(42) = t(42) = -1.716, -2.516, -3.743, p = -5.029, -2.649, p = .094 p = .016 .001 * p = .000 * p = .011 Ext 4 ------t(42) = t(42) = t(42) = t(42) = -0.691, -1.901, -3.114, -1.084, p = .493 p = .064 p = .003 p = .285 Ext 5 ------t(42) = t(42) = t(42) = -1.482, -3.628, -0.783, p = .146 p = .001 * p = .438 Ext 6 ------t(42) = t(42) = 0.298, -2.448, p = .676 p = .019 Ext 7 ------t42) = 1.860, p = .07
Bonferroni correction: 1/55 = p = .001 55
Table 9: Paired samples t-tests of pin number during play suppression on high line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(49) = 0.130, t (49) = t (49) = 8.446, T (49) = 5.791, t (49) = 8.041, t (49) = 5.483, t (49) = 4.021, t (49) = 7.126, t (49) = 7.414, t (49) = 7.746, p = .897 10.815, p = .000 p = .000 p = .000 p = .000 p = .000 p= .000 p = .000 p = .000 p = .000 * * * * * * * * * Unworn ------t (49) = 9.738, t (49) = 8.073, T (49) = 5.948, t (49) = 7.787, t (49) = 5.556, t (49) = 4.016, t (49) = 7.510, t (49) = 7.157, t (49) = 7.374, p = .000 p = .000 p = .000 p = .000 p = .000 p = .000 p = .000 p = .000 p = .000 * * * * * * * * * Worn ------t(49) = -.3.268, T (49) = - t (49) = -4.085, t (49) = -6.087, t (49) = -6.545, t (49) = -4.282, t (49) = -3.569, t (49) = -4.606, p = .002 * 3.792, p = .000 * p = .000 * p = .000 p = .000 p = .001 p = .000 p = .000 * * * * * Ext 1 ------T(49) = -2.230, t(49) = -1.740, t(49) = -5.132, t(49) = -6.585, t(49) = -1.752, t(49) = -1.090, t(49) = -0.780, p = .03 p = .088 p = .000 * p = .000 p = .086 p = .281 p = .439 * Ext 2 ------t(49) =.953, t(49) =-2.171, t(49) = -3.141, t(49) = 0.496, t(49) = 0.914, t(49) = 1.329, p = .345 P = .035 p = .003 p = .622 p = .365 p = .190
Ext 3 ------t(49) = -3.129, t(49) = -4.292, t(49) = -0.312, t(49) = 0.395, t(49) = 0.788, p = .003 p = .000 * p = .756 p = .694 p = .435
Ext 4 ------t(49) = -2.378, t(49) =2.474, t(49) = 2.991, t(49) = 3.285, p = .021 p = .017 p = .004 p = .002
Ext 5 ------t(49) =3.525, t(49) = 4.187, t(49) = 4.409, p = .001 * p = .000 * p = .000 *
Ext 6 ------t(49) = 0.775, t(49) = 1.137, p = .442 p = .061
Ext 7 ------t(49) = 0.342, p = .734
Bonferroni correction: 1/55 = p = .001 56
Table 10: Paired samples t-tests of pin number during play suppression on low line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(58) = T(58) = t(58) = 0.494, t(58) = 0.883, t(58) = 1.268, t(58) = t(58) = t(58) = 1.095, t(58) = 0.289, t(58)= -1.768, -0.585, 7.870, p = .623 p = .381 p = .21 -0.261, -0.189, p = .278 p = .774 p = .082 p = .0561 p = .000 p = .795 p = .850 * Unworn ------T(58) =7.269, t(58) =0.836, t(58) = 1.265, t(58) = 1.852, t(58) = 0.100, t(58) = 0.140, t(58) = 1.441, t(58) = 0.641, t(58)= -1.559, p = .000 * p = .407 p = .211 p = .069 p = .992 p = .889 p = .115 p = .524 p = .125
Worn ------t(58)= -5.460, t(58)= -5.975, t(58)= -6.033, t(58)= -5.949, t(58)= -6.992, t(58)= -6.321, t(58)= -6.190, t(58)= -5.923, p = .000 * p = .000 * p = .000 p = .000 p = .000 p = .000 p = .000 p = .000 * * * * * * Ext 1 ------t(58) = 0.520, t(58) = 0.758, t(58)= -0.731, t(58) = t(58) = 0.473, t(58) = t(58)= -2.042, p = .605 p = .452 p = .468 -0.937, p = .638 -0.200, p = .046 p = .353 p = .842 Ext 2 ------t(58) = .435, t(58)= -1.244, t(58)= -1.482, t(58) = 0.269, t(58) = t(58)= -2.438, p = .665 p = .219 p = .144 p = .789 -0.601, p = .018 p = .551 Ext 3 ------t(58) =-1.716, t(58) =-2.313, t(58) = -.027, t(58)= -1.288, t(58)= -2.783, p = .092 p = .024 * p = .978 p = .203 p = .007
Ext 4 ------t(58) = 0.120, t(58) = 1.455, t(58) = 0.690, t(58)= -1.741, p = .905 p = .154 p = .493 p = .087
Ext 5 ------t(58) = 1.453 t(58) = 0.568, t(58)= -1.982, p = .152 p = .572 p = .052
Ext 6 ------t(58)= -1.008, t(58)= -2.752, p = .318 p = .008
Ext 7 ------t(58)= -2.695, p = .009
Bonferroni correction: 1/55 = p = .001 57
Table 11: Selectively Bred Lines Pin Number, means and S.E.M.
Baseline Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Random 14.62 ± 14.17 ± 0.86 ± 0.76 ± 4.33 ± 5.81 ± 9.3 ± 7.57 ± 9.81 ± 12.38 ± 9.45 ± 1.03 1.11 0.2 0.34 0.91 0.88 2.25 1.42 1.32 1.44 1.56 High 17.48 ± 17.30 ± 1.2 ± 3.86 ± 6.30 ± 5.34 ± 8.48 ± 10.54 ± 5.62 ± 4.88 ± 4.52 ± 1.52 1.64 0.48 0.84 1.39 0.95 1.21 1.48 0.90 0.94 0.75 Low 13.57 ± 14.03 ± 3.29 ± 12.83 ± 11.92 ± 12.34 ± 14.02 ± 13.84 ± 11.95 ± 13.16 ± 18.22 ± 1.23 1.53 0.87 2.09 1.67 1.84 2.06 1.79 1.47 1.59 2.51 All 15.17 ± 15.16± 1.91 ± 6.46 ± 8.09 ± 8.01 ± 10.37 ± 11.23 ± 9.24 ± 10.18 ± 11.20 ± Animals 0.76 0.86 4.71 0.96 0.93 0.80 1.00 0.94 0.77 0.85 4.58
58
Table 12: Independent samples t-tests of pin duration during play suppression.
Baseline Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8
High vs. t(84.824) t(80.094) t(55.568) t(50.970) t(90) = t(90) = t(90) = t(90) = t(90) = T(81.205) = t(73.275) Random = 1.887 = 2.737, = 1.156 = 3.680, 0.718, 0.718, 0.184, 1.521, -2.044, -3.080, = -2.408, p = .063 p = .008 p = .253 p = .001 p = .464 p = .850 p = .168 p = .132 p = .044 p = .002 p = .015 * * Low vs. t(98) = t(98) = t(59.566) t(59.849) t(96.226) t(93.251) t(97.013) t(98) = t(98) = T(98) = t(98) = Random -1.432, -1.606, = 2.770, = 5.378, = 1.972, = 1.103, = 1.751, 1.311, p -0.333, -0.029, 1.121, p = .155 p = .112 p = .007 p = .000 p = .051 p = .273 p = .083 = .193 p = .74 p = .997 p = .265 * Low vs. t(106) = t(89.416) t(89.415) t(92.285) t(106) = t(106) = t(106) = t(106) = t(106) = T(101.102) t(96.202) High -3.104, = -3.937, = 1.835, = 2.680, 1.180, 0.834, 0.260, -0.369, 1.611, = 2.931, = 3.749, p = .002 p = .000 p = .070 p = .009 p = .234 p = .399 p = .795 p = .713 p = .110 p = .004 p = .000 * * *
Bonferroni correction: 1/33 = p = .003 59
Table 13: Paired samples t-tests of pin duration during play suppression on random line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(42) = t(42) = 11.471, t(42) = t(42)= 5.472, t(42)= 3.897, t(42)= 3.377, T(42)= 2.313, t(42)= 0.772, t(42)= -.244, t(42)= 1.217, 0.472, p = .000 * 11.250, p = .000 * p = .000 * p = .002 p = .026 * p = .455 p = .808 p = .231 p = .640 p = .000 * Unworn ------t(42)= 11.053, t(42)= t(42)= 5.894, t(42)= 3.771, t(42)= 3.738, T(42)= 2.023, t(42)= 0.518, t(42)= t(42)= 0.927, p = .000 * 10.708, p = .000 * p = .001 * p6 = .001 * p = .05 * p = .607 -0.540, p = .359 p = .000 * p = .592
Worn ------t(42 )= 0.493, t(42) = t(42) = t(42) = t(42) = t(42) = t(42) = t(42) = p = .625 -4.312, -6.035, -5.347, -5.025, -6.643, -7.555, -5.871, p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * P = .000 * p = .000 * Ext 1 ------t(42)= -4.355, t(42)= -6.239, t(42)= - t(42)= -5.055, t(42)= -6.780, t(42)= -7.535, t(42)= -6.157, p = .000 * p = .000 * 5.511, p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * Ext 2 ------t(42)= t(42)= t(42)= t(42)= t(42)= t(42)= -1.941, -2.204, -2.266, -3.578, -4.842, -3.008, p = .059 p = .033 p = .029 p = .001 * P = .000 * p = .004 Ext 3 ------t(42)= t(42)= t(42)= t(42)= t(42)= -0.218, -0.975, -2.283, -3.923, -2.207, p = .829 p = .335 p = .028 P = .002 p = .033
Ext 4 ------t(42)= -.907, t(42)= t(42)= t(42)= p = .370 -3.032, -3.697, -1.695, p = .004 P = .001 * p = .098
Ext 5 ------t(42)= t(42)= t(42)= -2.113, -2.936, -0.943, p = .041 p = .005 p = .351
Ext 6 ------t(42)= t(42)= -1.374, 0.400, p = .177 p = .692
Ext 7 ------t(42)= 1.290, p = .204
Bonferroni correction: 1/55 = p = .001 60
Table 14: Paired samples t-tests of pin duration during play suppression on the high line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(49) = - t(49) = t(49) = 7.369, t(49) = 5.321, t(49) = 4.742, t(49) = 2.847, t(49) = 1.642, t(49) = 5.035, t(49) = 5.142, t(49) = 6.155, 1.009, 10.028, p = .000 * p = .000 * p = .000 * p = .006 p = .107 p = .000 * p = .000 * p = .000 * p = .318 p = .000 * Unworn ------t(49) = 9.061, t(49) = 7.446, t(49) = 5.756, t(49) = 5.157, t(49) = 3.403, t(49) = 2.156, t(49) = 5.661, t(49) = 5.374, t(49) = 5.961, p = .000 * p = .000 * p = .000 * p = .000 * p = .001 * p = .036 p = .000 * p = .000 * p = .000 *
Worn ------t(49) = -3.313, t(49) = -3.810, t(49) = -3.953, t(49) = -4.964, t(49) = -5.905, t(49) = -4.644, t(49) = -3.580, t(49) = -4.754, p = .002 p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .001 * p = .000 *
Ext 1 ------t(49) = -1.883, t(49) = -2.522, t(49) = -4.698, t(49) = -6.503, t(49) = -2.695, t(49) = -2.041 t(49) = -1.479, p = .066 p = .015 p = .000 * p = .000 * p = .01 p = .047 p = .145
Ext 2 ------t(49) = -0.779, t(49) = -2.517, t(49) = -3.461, t(49) = -0.755, t(49) = -0.125, t(49) = -0.390, p = .439 p = .015 p = .001 * p = .454 p = .901 p = .698
Ext 3 ------t(49) = -1.737, t(49) = -2.652, t(49) = 0.095, t(49) = 0.555, t(49) = 1.107, p = .089 p = .011 p = .925 p = .581 p = .274
Ext 4 ------t(49) = -1.809, t(49) = 1.944, t(49) = 2.664, t(49) = 2.632, p = .077 p = .058 p = .01 p = .011
Ext 5 ------t(49) = 2.824, t(49) = 3.505, t(49) = 3.513, p = .007 p = .001 * p = .001 *
Ext 6 ------t(49) = 0.544, t(49) = 1.222, p = .0589 p = .228
Ext 7 ------t(49) = 0.611, p = .544
Bonferroni correction: 1/55 = p = .001 61
Table 15: Paired samples t-tests of pin duration during play suppression on the low line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(58) = 1.153, t(58) = 5.072, t(58) = .848, t(58) = 1.310, t(58) = 0.663, t(58) = -0.553, t(58) = -0.950, t(58) = -0.151, t(58) = -1.536, t(58) = -1.681, p = .254 p = .000 * p = .04 p = .195 p = .51 p = .583 p = .356 p = .881 p = .130 p = .098
Unworn ------t(58) = 4.176, t(58) = 0.174, t(58) = 0.773, t(58) = .086, t(58) = -1.403, t(58) = -1.969, t(58) = -0.843, t(58) = -2.223, t(58) = -2.381, p = .000 * p = .863 p = .466 p = .932 p = .166 p = .054 p = .402 p = .03 p = .021
Worn ------t(58) = -3.875, t(58) = -3.949, t(58) = -3.855, t(58) = -4.828, t(58) = -4.929, t(58) = -3.971, t(58) = -5.010, t(58) = -5.357, p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 *
Ext 1 ------t(58) = 0.584, t(58) = -0.056, t(58) = -1.396, t(58) = -2.241, t(58) = -0.812, t(58) = -2.211, t(58) = -2.455, p = .0562 p = .956 p = .168 p = .029 p = .42 p = .031 p = .017
Ext 2 ------t(58) = -0.769, t(58) = -2.647, t(58) = -3.503, t(58) = -1.703, t(58) = -3.273, t(58) = -3.565, p = .455 p = .01 p = .001 * p = .089 p = .002 p = .001 *
Ext 3 ------t(58) = -1.750, t(58) = -2.259, t(58) = -1.136, t(58) = -2.970, t(58) = 3.445, p = .085 p = .028 p = .261 p =..004 p = .001 * Ext 4 ------t(58)= -0.564, t(58)= 0.408, t(58)= -1.400, t(58)= 1.514, p = .575 p = .685 p = .167 p = .136
Ext 5 ------t(58)= 1.013, t(58)= -0.992, t(58)= -1.390, p = .316 p = .326 p = .17
Ext 6 ------t(58)= -2.025, t(58)= -2.417, p = .048 p = .179
Ext 7 ------t(58)= -0.416, p = .469
Bonferroni correction: 1/55 = p = .001 62
Table 16: Selectively Bred Lines Pin Duration, means and S.E.M.
Baseline Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Random 19.09 ± 18.32 ± 0.78 ± 0.92 ± 7.52 ± 10.60 ± 11.05 ± 12.97 ± 17.00 ± 19.74 ± 15.95 ± 1.64 1.68 0.26 0.37 1.61 1.67 1.93 2.48 2.47 2.56 2.58 High 24.46 ± 27.02 ± 1.95 ± 6.14 ± 9.38 ± 11.12 ± 15.66 ± 18.98 ± 10.91 ± 9.71 ± 8.48 ± 2.32 2.7 0.98 1.49 1.94 2.17 2.69 2.96 1.77 2.01 1.72 Low 15.47 ± 14.12 ± 5.55 ± 14.41 ± 13.82 ± 13.94 ± 16.62 ± 17.59 ± 15.83 ± 19.63 ± 20.17 ± 1.79 1.86 1.7 4.32 2.45 2.52 2.53 2.40 2.39 2.72 2.60 All 19.49 ± 19.59 ± 3.01 ± 7.56 ± 10.22 ± 12.03 ± 14.74 ± 16.76 ± 14.52 ± 16.35 ± 15.09 ± Animals 1.17 1.32 0.75 1.15 1.16 1.30 1.44 1.52 1.31 1.48 1.42
63
Table 17: Independent samples t-tests on dorsal contact number during play suppression.
Baseline Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8
High vs. t(90) = t(90) = t(61.872) t(72.025) = t(90) = t(90) = t(90) = t(90) = t(90) = t(90) = T(90) = Random 1.181, 0.960, = 2.005, p 3.160, p = 0.090, 0.084, 0.235, -0.153, -1.149, -4.696, -1.771, p = .963 p = .340 = .049 .002 p = .928 p = .933 p = .815 p = .879 p = .254 p = .000 p = .080 * * Low vs. t(97.835) t(97.094) t(62.904) t(75.198) = t(97.651) = t(96.653) t(98) = t(98) = t(98) = t(98) = T(98) = Random = -1.515, = -0.923, = 3.297, p 4.364, p = 0.831, = 0.537, 0.201 -1.107 -1.782 -1.629 0.014 p = .133 p = .359 = .002 .000 p = .408 p = .593 p = .841 p = .271 p = .078 p = .105 p = .989 * * Low vs. t(106) = t(106) = t(93.325 ) t(104.241) t(105.259) t(106) = t(106) = t(106) = t(106) = t(106) = T(106) = High -1.386, -1.705, = 1.745, = 1.430, = 0.728, 0.417, -0.046, -0.967, -0.476, 3.075, 2.080, p = .169 p = .086 p = .084 p = .156 p = .468 p = .677 p = .963 p = .336 p = .635 p = .003 p = .04 *
Bonferroni: .1/33 = p = .003 64
Table 18: Paired samples t-tests on dorsal contact number during play suppression of random line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(42) = 3.42, t(42) = t(42) = t(42) = 5.177, t(42) = 3.352, t(42) = 1.853, t(42) = 0.681, t(42) = 0.554, t(42) = -1.855, t(42) = 0.588, p = .001 * 16.210, 12.146, p = .000 p = .000 p = .071 p = .500 p = .590 p = .071 p = .560 p = .000 p = .000 * * * * Unworn ------t(42) = t(42) = t(42) = 3.167, t(42) = 1.510, t(42) = -.047, t(42) = -1.577, t(42) = -1.732, t(42) = -3.546, t(42) = -1.106, 14.540, 10.525, p = .003 p = .139 p = .962 p = .123 p = .091 p = .001 * p = .275 p = .000 p = .000 * * Worn ------t(42) = -5.479, t(42) = -7.584, t(42) = -8.730, t(42) = -8.018, t(42) = t(42) = t(42) = t(42) = -8.596, p = .000 p = .000 p = .000 p = .000 -10.809, -12.764, -11.855, p = .000 * * * * p = .000 * p = .000 * p = .000 * * Ext 1 ------t(42) = -5.750, t(42) = -6.995, t(42) = -7.419, t(42) = -8.900, t(42) = - t(42) = t(42) = -7.112, p = .000 * p = .000 * p = .000 * p = .000 10.539, -10.285, p = .000 * p = .000 p = .000 * * * Ext 2 ------t(42) = -2.420, t(42) = -3.551, t(42) = -5.736, t(42) = -4.863, t(42) = -7.349, t(42) = -3.447, p = .02 p = .001 p = .000 p = .000 p = .000 p = .01 * * * * Ext 3 ------t(42) = -1.377, t(42) = -3.616, t(42) = -3.367, t(42) = -4.838, t(42)= -2.201, p = .176 p = .001 p = .002 p = .000 p = .033 * * Ext 4 ------t(42) = -1.410, t(42) = -1.601, t(42) = -4.741, t 42) = -1.307, p = .166 p = .117 p = .000 p = .199 * Ext 5 ------t(42) = -0.212, t(42) = -2.675, t 42) = .035, p = .883 p = .011 p = .973
Ext 6 ------t(42) = -3.103, t(42) = 0.219, p = .003 p = .828
Ext 7 ------t(42) = 2.654, p = .011
Bonferroni correction: 1/55 = p = .001 65
Table 19: Paired samples t-tests on dorsal contact number during play suppression of the high line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(49) = t(49) = t(49) = 6.171, t(49) = 4.092, t(49) = 3.087, t(49) = 1.392, t(49) = 0.839, t(49) = 1.801, t(49) = 4.537, t(49) = 2.944, 1.716, 10.956, p = .000 * p = .000 * p = .003 p = .170 p = .405 p = .078 p = .000 * p = .005 p = .092 p = .000 *
Unworn ------t(49) = t(49) = 5.894, t(49) = 3.429, t(49) = 1.872, t(49) = 0.454, t(49) = -0.161, t(49) = 0.967, t(49) = 3.925, t(49) = 2.320, 11.058, p = .000 * p = .001 * p = .067 p = .652 p = .873 p = .338 p = .000 * p = .025 p = .000 * Worn ------t(49) = -5.217, t(49) = -6.147, t(49) = -6.552, t(49) = -7.567, t(49) = -9.657, t(49) = -7.894, t(49) = -6.181, t(49) = -6.600, p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 *
Ext 1 ------t(49) = -2.656, t(49) = -3.116, t(49) = -3.989, t(49) = -5.481, t(49) = -4.293, t(49) = -1.444, t(49) = -2.719, p = .011 p = .003 p = .000 * p = .000 * p = .000 * p = .105 p = .009
Ext 2 ------t(49) = -1.337, t(49) = -2.482, t(49) = -3.878, t(49) = -2.000, t(49) = -0.285, t(49) = p = .187 p = .017 p = .000 * p = .051 p = .777 -0 .802, p = .427
Ext 3 ------t(49) = -1.590, t(49) = -2.427, t(49) = -0.901, t(49) = 1.388, t(49) = 0.387, p = .118 p = .019 p = .372 p = .171 p = .700
Ext 4 ------t(49) = -0.737, t(49) = 0.397, t(49) = 2.289, t(49) = 1.463, p = .465 p = .693 p = .021 p = .150
Ext 5 ------t(49) = 1.123, t(49) = 3.096, t(49) = 2.562, p = .267 p = .003 p = .014
Ext 6 ------t(49) = 2.345, t(49) = 1.520, p = .023 p = .135
Ext 7 ------t(49) = -1.153, p = .254
Bonferroni correction: 1/55 = p = .001 66
Table 20: Paired samples t-tests on dorsal contact numbers during play suppression of the low line animals.
Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Baseline t(58) = t(58) = t(58) = 6.131, t(58) = 2.384, t(58) = 1.426, t(58) = -0.044, t(58) = -0.994, t(58) = 1.455, t(58) = -1.404, t(58) = -1.523, 3.522, 11.155, p = .000 * p = .02 p = .159 p = .965 p = .325 p = .151 p = .166 p = .133 p = .001 * p = .000 * Unworn ------t(58) = 8.640, t(58) = 2.483, t(58) = 0.395, t(58) = -0.920, t(58) = -1.948, t(58) = -1.252, t(58) = -0.411, t(58) = -2.979, t(58) = -3.183, p = .000 * p = .016 p = .695 p = .361 p = .056 p = .216 p = .613 p = .004 p = .002
Worn ------t(58) = -6.305, t(58) = -7.287, t(58) = -8.175, t(58) = -8.682, t(58) = -8.785, t(58) = -7.668, t(58) = -9.310, t(58) = -9.465, p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 * p = .000 *
Ext 1 ------t(58) = -1.836, t(58) = -3.869, t(58) = -4.818, t(58) = -3.892, t(58) = -2.050, t(58) = -5.162, t(58) = -5.136, p = .072 p = .000 * p = .000 * p = .000 * p = .055 p = .000 * p = .000 *
Ext 2 ------t(58) = -1.347, t(58) = -2.618, t(58) = -1.788, t(58) = -0.732, t(58) = -3.489, t(58) = -3.211, p = .183 p = .011 p = .079 p = .467 p = .001 * p = .002
Ext 3 ------t(58) = -1.660, t(58) = -0.445, t(58) = 0.364, t(58) = -2.600, t(58) = -2.607, p = .102 p = .658 p = .717 p = .012 p = .012
Ext 4 ------t(58) = 1.224, t(58) = 1.599, t(58) = -1.547, t(58) = -1.507, p = .226 p = .115 p = .128 p = .137
Ext 5 ------t(58) = 0.846, t(58) = -2.612, t(58) = -3.098, p = .401 p = .011 p = .003
Ext 6 ------t(58) = -2.875, t(58) = -3.020, p = .006 p = .004
Ext 7 ------t(58) = 0.076, p = .939
Bonferroni correction: 1/55 = p = .001 67
Table 21: Dorsal contact number during play suppression; means and S.E.M.
Baseline Unworn Worn Ext 1 Ext 2 Ext 3 Ext 4 Ext 5 Ext 6 Ext 7 Ext 8 Random 80.45 ± 68.36 ± 11.83 ± 53.02 ± 53.02 ± 61.21 ± 68.67 ± 76.57 ± 77.52 ± 91.79 ± 76.37 ± 4.38 4.95 1.11 5.65 5.65 5.74 7.08 6.09 5.49 6.93 7.64 High 80.14 ± 74.22 ± 18.26 ± 42.90 ± 53.78 ± 61.96 ± 71.02 ± 75.24 ± 67.88 ± 51.72 ± 59.10 ± 4.88 4.51 3.01 5.04 5.63 6.57 7.00 6.11 6.16 5.22 6.21 Low 70.43 ± 62.38 ± 28.20 ± 54.33 ± 60.55 ± 65.67 ± 70.59 ± 67.05 ± 64.14 ± 76.72 ± 76.48 ± 4.96 1.54 4.84 6.20 7.08 6.01 6.28 5.84 5.00 6.06 5.62 All 76.47 ± 68.00 ± 20.31 ± 42.31 ± 56.19 ± 63.19 ± 70.19 ± 72.45 ± 69.13 ± 72.61 ± 70.65 ± Animals 2.81 2.74 2.20 3.15 3.73 3.55 3.88 3.48 3.22 3.72 3.72
68
Prepulse Inhibition
Results were obtained from individual animals. Random line n = 12 (4 females, 8
males, 3 litters); low line n = 13 (8 females, 5 males, 3 litters); high line n = 17 (8
females, 9 males, 4 litters). A 2x3x3 (sex, line, trial) mixed design ANOVA revealed that
there were no significant main effects or interactions of between or within subjects results of ASR on animal line or sex; however, post-hoc Bonferroni tests revealed that the animals overall produced a greater ASR to the PA trials when compared to the PP and
PPI trials (p = .000; p = .000; figure 8). This suggests that the animal lines do not have differences in reflexive fear reactivity to aversive tones.
A one-way ANOVA was conducted on animal line and PPI(%). Results indicated a main effect (F(2, 38) = 3.722, p = .033). Post-hoc Bonferroni tests revealed that the random line animals demonstrated greater PPI(%) than high and low line animals, these results were marginally significant or not significant (p = .052; p = .083; figure 9). This suggests that the high line animals may have deficits in sensorimotor gating processes.
There were no sex effects on PPI(%).
A 2x3x3 (sex, line, trial) mixed design ANOVA revealed a main effect of trial type on 22 kHz USV emission (F(1.233, 43.169) = 8.588, p = .003; figure 10). Post-hoc
Bonferroni tests revealed that the animals more 22 kHz USVS during the PA trials when compared to the PP (p = .04) They also emitted more 22 kHz USVs during the PPI trials when compared to the PA and PP trials (p = .011). There was a trial and line interaction
(F( 2.467, 43.169) = 6.097, p = .002; figure 11). Independent samples t-tests revealed that the high line emitted significantly more 22 kHz USVS than the random line during the
PP, PA and PPI trials (t(17.131) = 2.717, p = .009; t(16.762) = 2.862, p = .003; t(18.608) 69
= 2.924, p = .000). High line animals also emitted significantly more 22 kHz USVs than
low line animals during the PP, PA and PPI trials (t(16) = 2.995, p = .009; t(16) = 3.121,
p = .007; t(16) = 3.437, p = .003). Between subjects results revealed a main effect of
animal line (F(2, 35) = 8.035, p = .001). Post-hoc Bonferroni tests revealed that the high
line produced significantly more 22 kHz USVs than the random and low line animals (p
= .01; p = .003; figure 12). An animal line and sex interaction was also reported (F(2, 35)
= 4.740, p = .015). Males emitted a marginally significant 22 kHz USVs than females (p
= .078; figure 13). There were no significant differences of 50 kHz USV emission during
the PPI experiment.
22 kHz USV emission during the PPI experiment was organized over time into
three blocks of trials, 1-19, 20-39, 40-60. There was a significant main effect of trial
block on 22 kHz USV emission (F(1.057, 37.005) = 9.232, p = .004; figure 14). Post-hoc
Bonferroni tests revealed that the animals emitted significantly more 22 kHz USVs
during the first trial block when compared to the second and third (p = .011; p = .013).
There was a marginally significant difference between the second and third trial block (p
= .056). There was also a trial block and animal line interaction (F(2.115, 37.005) =
6.211, p = .004; figure 15). Independent samples t-tests revealed that the high line
animals emitted significantly more 22 kHz USVs than the random line in the first trial
block (t(20.555) = 2.581, p = .018). High line animals also emitted more 22 kHz USVs than the low line animals during the first and second trial blocks (t(16) = 3.251, p = .005; t(16) = 2.302, p = .035). A between subjects main effect of line was also found on 22 kHz USV emission over the trial blocks (F(2, 35) = 6.154. p = .005). Post-hoc
Bonferroni tests revealed that high line animals emitted significantly more 22 kHz USVs 70 than the random and low line animals (p = .037; p = .009). 71
Figure 8: Means ± S.E.M. ASR for different trial types during PPI
35 *** 30 25 20 15 10 ASR (1.22 mV)ASR (1.22 5 0 PP PA PPI Trial type
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 <
***). 72
Figure 9: Means ± S.E.M of PPI(%) for the different selectively bred lines of animals.
100 90 80 * 70 60 50
PPI(%) 40 30 20 10 0 Random Low High Animal Line
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 <
***).
73
Figure 10: Means ± S.E.M of 22 kHz USV emission over different trial types.
0.9 * 0.8 0.7 * 0.6 0.5 0.4 0.3
22 kHz USVs/sec 0.2 0.1 0 PP PA PPI Trial Type
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 <
***). 74
Figure 11: Means ± S.E.M of 22 kHz USV examining the trial and sex interaction.
0.16 0.14 0.12 0.1 0.08 0.06 * 0.04 0.02 22 kHz22 USVs/sec 0 -0.02 PP PA PPI PP PA PPI
Female Male Trial Type and Sex
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 <
***). 75
Figure 12: Means ± S.E.M of 22 kHz USV emission between animal lines.
1.6 ** 1.4 1.2 1 0.8 0.6 0.4 0.2
22 kHz22 USVs/sec 0 -0.2 Random Low High -0.4 Animal Line
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 <
***). 76
Figure 13: Means ± S.E.M of 22 kHz USV emission between sex.
1.2 * 1 0.8
0.6 0.4
0.2 22 kHz22 USVs/sec 0 Female Male -0.2 Sex
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 <
***). 77
Figure 14: Means ± S.E.M of 22 kHz USV emission over blocks of trials.
0.12 * 0.1 0.08
0.06 0.04
0.02 22 kHz22 USVs/sec 0 Block 1 Block 2 Block 3 -0.02 Trial Type
The symbols (*) provide a level of significance (p = .05 = *; p = .01 = *; p = .001 = *). 78
Figure 15: Means ± S.E.M of 22 kHz USV emission investigating trial block and animal line interaction.
0.25 ** 0.2 0.15
0.1 * 0.05
22 kHz22 USVs/sec 0 Block Block Block Block Block Block Block Block Block -0.05 1 2 3 1 2 3 1 2 3
Random Low High Trial Block and Animal Line
The symbols (*) provide a level of significance (p < .05 < *; p < .01 < **; p < .001 <
***). 79
CHAPTER VI. DISCUSSION
Summary
Results from these experiments suggest that the high and low line animals have alterations in social recognition. The high line animals’ lack of significance is due to a
lack of significant increases or decreases in investigatory behavior. The low line animals
showed significant decreases over the four trials. The play suppression experiment
provided the most striking results. The high line animals show an exaggerated fear
conditioning response while the low line animals did not demonstrate fear conditioning.
The low line animals showed less suppression of dorsal contacts in response to predator
odor; however, the selectively bred animals did not differ in ASR. This suggests there
may be variations in conditioned versus unconditioned fear in these selectively bred
animals; and that they may be modality specific. High and low line animals had deficits
in sensorimotor gating when compared to random line controls. High line animals
produced significantly more 22 kHz USVs during PPI.
Social Recognition
The random and high line animals are showing a significant decrease in
investigatory behavior over the first three trials. The ran8dom line animals then show a
marginally significant increase in investigatory behavior from the TS3 to TD trial. The
random line animals’ investigatory behaviors return to baseline in the TD trial. This
suggests that our random line serves as an adequate control for this paradigm; as they
behave like non-selectively bred Long-Evans rats during this experiment (Thor &
Holloway, 1982). The high line animals show an increase from the third to fourth trial as
well. This is not a significant increase, suggesting that the high line animals may have 80
slight reductions in their social recognition abilities. High line animals show a non-
significant trend of social recognition abilities, the lack of significant could be due to the
lack of a dramatic decrease over the first three trials.
Interestingly, the low line animals show significant decreases over all four trials.
Rather than increasing their SI during the TD trial, they demonstrate another decrease.
The amount of SI the low lines demonstrated in the TD animal trial was significantly lower than baseline. This suggests that they lack basic social recognition abilities.
These variations in SI may be better explained by the percentage of SM data.
Overall the animals demonstrated an increase in motivation from the TS1 to the TD trials.
All three animal lines showed a significant increase from TS1 to TS2; TS1 to TS3 and
TS1 to TD. In addition, all three lines did not show a significant increase in SM from
TS2 to TS3 and TS3 to TD. This suggests that their increasing motivation to spend time
with the stimulus animals becomes less dramatic over the four trials. The low line
animals showed a slight decrease in SM from TS3 to TD, along with a decrease in SI
while inside the social chamber.
Overall the animals also showed a significant decrease in 50 kHz USVs over the
all four trials. The random line animals 50 kHz USV emission rates decreased over the
first three trials, and then a non-significant increase occurred from the TS3 to TD trial,
similar to their investigatory behavior; however, this non-significant increase did not
return their USV emission to baseline rates. There were no significant differences in high
line 50 kHz USV emission data, which was surprising, as prior studies have found these
animals to be highly vocal in social situations (Burgdorf et al., 2005). The low line
animals demonstrated a significant decrease in 50 kHz USV emission over the first three 81 trials, and then another non-significant decrease from TS3 to TD, similar to their investigatory behavior. While there was a lack of significant effects found in this data, the
50 kHz USV emission trends follow the line animals’ patterns of investigatory behavior.
The 50 kHz USV results are difficult to interpret, as they were recorded from the social side of the apparatus, and USV emission on the other side was unlikely to be recorded. In addition, it is impossible to tell whether the stimulus or experimental animal is emitting these calls. It is interesting that the 50 kHz USV emission for each line followed similar patterns of behavior with regard to SI.
This data suggests that the low line animals have deficiencies in social recognition abilities. This may be because the low line animals may have impaired associative learning capabilities. The task not only requires the assessment of a safe environment, but it also requires the animal to make associations between contextual cues and social potential social reward. Differences in social recognition can also arise from alterations in hormone systems. Previous work on social recognition has implicated oxytocin as a key neuro-modulator involved in social recognition abilities (Ferguson, et al., 2006).
Oxytocin is a neuropeptide that is implicated in social bonding (Carter, 1998).
Prior studies have found that oxytocin knock out (OTKO) mice also show deficits in the social recognition paradigm (Ferguson et al., 2006). These deficits were reversed by the administration of oxytocin. Low line animals may have a reduction in oxytocin when compared to the random line animals. Ferguson and colleagues (2006) found that the OTKO mice had a dramatic decrease in gene expression in the medial amygdala
(MeA) when compared to controls. A lack of gene expression in this brain area may account for an inability to recognize a conspecific. The OTKO mice alternatively 82 showed dramatic increases in gene expression in the somatosensory cortex and hippocampus in comparison to control mice (Ferguson et al., 2006). This suggests that animals deficient in oxytocin utilize different strategies when investigating conspecifics.
Future research should examine oxytocin levels in the low line animals. Deficiencies in this neuropeptide may explain variations found in their social behavior (Harmon et al.,
2006).
Play suppression
The findings for the play behavior observations in these selectively bred line animals demonstrated are interesting and surprising. There were no differences in play on the baseline day of play or the unworn cat collar day. High line animals produced more pins than the random line on the first extinction day. The random line produced more than the high line on the 7th extinction day. The low line produced more pins than the random line on extinction days 1-3 and more than the high line on extinction days 1, 3, and 6-8.
The random line’s pin frequency returned to baseline on the 7th extinction day, consistent with the previous research (Siviy et al., 2006). High line animals never returned to baseline levels of pinning. Most strikingly, the low line animals immediately returned to baseline levels of play, completely failing to show contextual fear conditioning. This is not what one might expect from animals that carry an anxiogenic phenotype.
These trends were also found with regard to pin duration. High line animals showed longer pin durations on the baseline day of play and the unworn cat collar day than the low line animals. High line animals produced longer pins than the random line 83
on the first extinction day, while the random line animals produced longer pins on the 7th
extinction day. Low line animals produced longer pins than the high lines on the 8th extinction day.
Random line animals returned to their baseline levels of pin duration on the 6th extinction day, demonstrating that they are a reliable control group for this experiment
(Siviy et al., 2006). High line animals briefly return to baseline on the fifth day of extinction, but fall significantly below from extinction days 6-8. The low line animals return to baseline levels of pin duration on the first extinction day, again failing to show fear conditioning.
Dorsal contact results are less dramatic than the results found with regards to pinning. Males overall produced more dorsal contacts than females. This is contrary to previous research (Harmon et al., 2006), who did not find any sex effects. This could be due to the design of the study, prior work has exposed isolated juveniles to play every other day (Harmon et al., 2006). More consistent exposures to play could be causing a sex effect. There was no line and sex interaction, suggesting this trend is present in all the selectively bred lines. There were no differences in number of dorsal contacts on the baseline and unworn cat collar day. Low line animals produced more dorsal contacts than the random line on the worn cat collar day and extinction day 1. High line animals produced more dorsal contacts than the random line on the first extinction day. The random line animals produced more dorsal contacts than the high line on the 7th extinction day. Low line animals demonstrated more dorsal contacts during the 7th day of
extinction when compared to the high line animals. 84
Random line animals returned to baseline on extinction day 4, consistent with the
previous study (Siviy et al., 2006). The high line animals returned to baseline on
extinction day 3. The low line animals returned to baseline dorsal contact levels on the
second extinction day. Again, the low line animals seem to be showing less contextual
fear conditioning than the other two selectively bred lines.
The introduction of the worn cat collar has also provided results that are
inconsistent with previous research on these line animals and play behavior. Most
strikingly, the low line animals immediately extinguish their play suppression to the worn
cat collar, while the high line animals remain below baseline. This is the opposite of
what was hypothesized. Previous research has demonstrated that the low line animals
display an anxious phenotype, while the high line animals carry an anxiolytic phenotype
(Burgdorf et al., 2009; Harmon et al., 2008). The low line animals are not showing conditioned fear to the play chamber in response to predator order. The random line animals’ extinguishment of play suppression is consistent with research regarding non- selectively bred Long-Evans rats (Siviy et. al, 2006).
Prepulse Inhibition
Overall the animals in this experiment produced a greater ASR during the two more aversive PA trials when compared to the PP and PPI trials. These results were expected and suggest that the experiment was designed and conducted properly. There were significant differences between line or sex on ASR, suggesting that these animals do not differ in their reflexive fear response to aversive tones. 85
Both the high and low line animals demonstrated less PPI (%) than random line controls. This suggests that they have deficits in sensorimotor gating abilities to unpredictable stimuli.
Overall the animals produced greater 22 kHz USVs in response to the PPI trials when compared to the PP and PA trials. There were also more 22 kHz USVs emitted during the PA trials when compared to the PP trials. High line animals emitted significantly more 22 kHz USVs than the random and low line animals during all three trial types. Males also emitted significantly more 22 kHz USVs than females during the
PA trials.
USV emission was monitored over time during the PPI procedure. Overall the animals produced significantly more 22 kHz USVs during the first trial block when compared to the second and third block of trials. This suggests that the animals are either habituating to the procedure, or simply getting tired of emitting USVs. High line animals produced more 22 kHz USVs during the first trial block than the random and low line animals, and more than the low line during the second trial block. This data suggests that the high and low line animals deficits in sensorimotor gating abilities. The high line animals are emitting more 22 kHz USVs. In order to explain these results, previous research needs to be examined more closely.
Previous Research: A closer look
Previous research has suggested that the high line animals are more resilient to anxiety than their controls (Burgdorf et al., 2009; Harmon et al., 2008). They spend more time in the center of an open field apparatus (Burgdorf et al., 2009) which is reinforcing to this hypothesis. High line animals also produced more dorsal contacts than 86
the random line animals during juvenile play (Harmon et al., 2006). They also demonstrate more social contact than the random line animals (Burgdorf et al., 2009).
Prior studies have examined sucrose preference in these line animals; the high line animals’ condition to the sucrose bottle more so than the random line (Burgdorf et al.,
2009). High line animals also emit more 50 kHz USVs in response to conditioned tickle reward than the random and low line animals (Burgdorf et al., 2005).
Harmon and colleagues (2008) found that the low line animals fail to condition to a maternal associated odor cue. Low line animals did not differ in locomotor activity in the open field paradigm when compared to the random line, but produced more fecal boli
(Burgdorf et al., 2009). Low line animals also participated in less social contact than the random line in a social contact paradigm (Burgdorf et al., 2009). The low line animals emit more 45 kHz USVs in response to maternal and litter separation than the random line animals (Harmon et al., 2008). During the porsolt forced swim test the low line animals produced more swimming, fecal boli and less floating than the random line animals (Burgdorf et al., 2009).
This prior research at face value suggests that the previous hypothesis made by
Burgdorf and colleagues (2009) is correct; however, there are some inconsistencies in these results and the implications it has on the fear reactivity of the selectively bred lines of animals. For example, the high line animals display behavior of socially gregarious animals that are more resilient to anxiety in tests of unconditioned fear. The low line animals seem to be much less socially motivated than controls, and provide a greater unconditioned fear response. These results suggest that the low line animals may serve as an animal model for anxiety; however, new research has found that the high line 87 animals have a larger stress response than controls measured by glucocorticoids.
Brudzynksi and colleagues (2008) found that the high line animals have a dramatically larger glucocorticoid response to 30 and 60 minute restraint stress than random and low line animals. Baseline levels of glucocorticods did not differ, suggesting that this phenotype is situational.
Glucocorticoids administration facilitates emotional conditioning processes
(Abrari et al., 2009). This includes conditioned reward and conditioned fear (Abrari et al., 2009). Prior studies have found that oxytocin is essential for social bonding (Insel &
Hullihan, 1995; Piazza & Le Moal, 1997). Differences in oxytocin expression may explain the variations in SM, while differences in fear responses and emotional conditioning may be reflective of alterations in HPA-axis development.
Alternative Hypothesis: Variations in Associative Learning
The data from the current study may be explained by an alternative hypothesis of these animals variation in associative learning. Selectively breeding for USVs reflecting positive affect during a conditioned tickle reward may have caused a co-selection for conditioning processes. Previous studies have shown successful selective breeding for conditioned fear; however, these animals were not tested for conditioned reward (Ponder et. al, 2007). These researchers used selective breeding on high and low fear responses during a conditioned place aversion paradigm and successfully created lines of animals that differed in contextual fear conditioning (Ponder et al., 2007). These lines started to diverge on the third generation (Ponder et al., 2007). They found that the highly fearful line of animals froze significantly more to a context previously paired with foot shock than their non-shocked controls for 7 days (Ponder et al., 2007). They also demonstrated 88
greater fear potentiated startle then their non-shocked controls (Ponder et al., 2007). By
utilizing 50 kHz USVs as a selection criterion for conditioned tickle reward, there may
have been a co-selection for basic and innate associative learning processes. Conditioned
reward and conditioned fear have a shared neural circuitry (Gallagher et al., 1990;
Kruzich & See, 2001; Robledo et al., 1996; Walker & Davis, 1997).
The process of reward conditioning involves various parts of the limbic system.
The most notable brain area involved in this process is the amygdala (Murray, 2007).
Previous studies have shown that rats with bilateral amygdala lesions do not show a conditioned preference to sucrose flavored water when compared to sham lesions (Gilbert
et al., 2003). While these amygdala lesioned animals do not condition to a solution
previous associated with sucrose, they show no difference in their ability to discriminate
and prefer sweetened water (Gilbert et al., 2003). It is thought that this portion of the
limbic system is necessary for the association of stimulus and reward (Hitchcott et al.,
1997; Murray, 2007; Salinas & McGaugh, 1996).
Lesions to the central nucleus of the amgydala (CeA) causes deficiencies in both
conditioned reward and conditioned fear (Gallagher et al., 1990; Kruzich & See, 2001;
Robledo et al., 1996; Walker & Davis, 1997). Takahashi and colleagues (2009) revealed that the MeA is essential for olfactory fear conditioning. Temporary inactivation of the
MeA causes deficits in retrieval of conditioned fear to predatory odor (Takahashi et al.,
2009). The study discussed previously about the OTKO mice also implicated this structure in social recognition processes (Ferguson et al.¸ 2001).
Previous work by Burgdorf and Colleagues (2005) found that the high line animals condition to tickle reward more so than the random and low line animals. It was 89 also found that the high line animals conditioned to a water bottle containing sucrose more so than the random line (Burgdorf et al., 2008). High line animals also show no variation in conditioning to a previously associated maternal odor cue (Harmon et al.,
2008). Low line animals demonstrate no preference for a previously associated maternal odor cue (Harmon et al., 2008) and show a decrease in initiatory play behavior
(Harmon et al., 2006).
The current study found that high line animals demonstrated more baseline play behavior in this experiment, suggesting that they may condition to the rewarding contextual cues of the chamber more so than the low line animals. They also showed a non-significant trend in social recognition. Their non-significance is most likely due to the lack of decrease in investigatory behavior from the TS1 to TS3 trial. This suggests that these animals may have a greater ability to associate the potential social reward than the other lines of animals, reflected as a greater amount of social gregariousness. The low line animal produced steady decreases in investigatory behavior, suggesting that they may have deficits associating potential social reward with contextual cues. The high line animals in this experiment showed exaggerated fear conditioning to the context of the play chamber when compared to the low and high line animals. Most strikingly, the low line failed to demonstrate this innate learning mechanism at all.
Brudzynski and colleagues (2008) found that the high line animals had a greater hormonal stress response to restraint, implicating that these animals may be more fearful than the random and low line animals. Glucorticoids facilitate both conditioned fear and conditioned reward (Abrari et al., 2009). During the selection process to conditioned tickle reward we may have co-selected for these emotional conditioning processes, 90
causing us to have animals that have higher levels of stress hormones and more astute
associative learning abilities.
These animals differ in conditioned fear; however, they may not show variations
in unconditioned fear. Walker and Davis (1997) found a structural and functional double
dissociation between conditioned and unconditioned fear. Results demonstrated that the
CeA was important for the acquisition and expression of conditioned fear (Walker &
Davis, 1997). The bed nucleus of the stria terminalis (BNST) was implicated in expression of unconditioned fear (Walker & Davis, 1997). This double dissociation suggests that it is possible for animals to differ in gene expression with regard to these neurological structures. Low line animals demonstrate more fear responses in the open field and porsolt forced swim test, both examples of unconditioned fear (Burgdorf et al.,
2009). In the current study they showed a diminished fear response to predatory odor.
Research has found contradictory data regarding unconditioned fear in these animals during exposure fearful stimuli. There also seems to be variations in anxious behavior across development, possibly due to alterations in stress hormones and oxytocin.
It is also possible that a co-selection for gene expression of oxytocin occurred.
The tickle procedure mimics rough-and-tumble play, by selecting for rats that emit low numbers of 50 kHz USVs during this paradigm, we may have also selected for rats that lack capabilities in social bonding. Prior studies have found that the low line animals produce less social contact in social contact assays, variations in play behavior and an inability to condition to a conditioned maternal odor cue (Burgdorf et al., 2009; Harmon
et al., 2006; Harmon et al., 2008). High line animals demonstrate more social contact in
social contact assays, produce more dorsal contacts than controls and show no variation 91
in the ability to associate a maternal odor cue to controls (Burgdorf et al., 2009; Harmon
et al., 2006; Harmon et al., 2008). Nelson and Panksepp (1996) found that oxytocin is
essential for the conditioning of pups (PND 15) to a maternally associated odor cue.
These results are consistent with our social recognition data and baseline play behavior
data. The low line animals show a lower amount of SM and inability to discriminate the
different stimulus animal. Their baseline play behavior is lower than that of the high and
random line animals. Prior studies have implicated oxytocin as essential for social
recognition processes (Ferguson et al., 2001), further suggesting that this specific
neuropeptide may be causing the low line animals to have alterations in social bonding;
however, this does not explain the USV data found in the PPI paradigm and the isolation distress call data researched previously (Harmon et al., 2008).
In infancy (PND 10-14) the low line animals show an increase in 45 kHz distress calls when compared to controls, providing initially very strong evidence of a more anxious animal (Harmon et al., 2008). As adults, these low line animals produced dramatically fewer of 22 kHz USVs than the random and high line animals during the
PPI experiment. During the isolation distress call test the animals were in a hypo- responsive period in their HPA-axis activity, a normal part of rat development (Lee et al.,
2007). The increases in 45 kHz USVs found in the isolation study can be explained by a deficiency in oxytocin (Lee et al., 2007). Prenatal stressed rats also emit more of these distress calls during infancy; this is reversed by administration of oxytocin (Lee et al.,
2007). The low line animals in adulthood do not emit many 22 kHz USVs in response to loud tones; this may be because it is not socially relevant stimuli and reflect a hypo- responsive HPA-axis and amygdala activity. The relationship between 45 kHz USVs, 92
oxytocin and glucocorticoids has not yet been fully elucidated; however, new research is
investigating these subtleties in hormones and behavior.
Future Research
Low line animals may model a hypo-active amygdala, while the high line animals
may model a hyper-active amygdala. Oxytocin levels may also be altered in these line
animals. Low line animals’ inabilities to recognize conspecifics and variations in other
tests of social bonding may be due to low levels of oxytocin (Ferguson et al., 2001).
Animals with out any circulating oxytocin fail to show social recognition and have
altered gene expression in the somatosensory cortex and hippocampus in comparison to
controls (Ferguson et al., 2001). A combination of lower levels of oxytocin and blunted
conditioning processes may explain the variation in social behavior found in the line
animals. Gene expression in the BNST should also be examined in the line animals, as it
has an important role in unconditioned fear (Walker & Davis, 1997).
The selection process for these line animals may be better suited to examine the
role of emotional conditioning and oxytocin in social behavior. There has been very little
research regarding selective breeding and conditioning processes (Ponder et al., 2007).
This could be a new frontier for researchers interested in genetic influences of mental
illness. Future research should examine more traditional conditioning paradigms in these
line animals. It is essential to examine whether the variations in conditioning processes occur across modalities. Prior studies on these animals have been primarily social and olfactory in nature. Traditional paradigms examining contextual versus cue conditioning should also be examined. 93
This research examined how these different selectively bred lines of animals
interact with animals from their own litters and lines; it would also be interesting to examine how they interact with each other. Another limitation of this study is the
numbers of litters used. Future work should breed more litters and investigate possible
litter effects that could be confounding the results. Previous research on these animals has
not examined the possible role of litter effects and the resources to do so where not
available for the current study.
Clinical Implications
Clinical implications of this animal model are wide. First and foremost, the high
and low line animals demonstrate deficiencies in sensorimotor gating processes, which
are symptoms of a wide variety of mental illnesses (Braff & Geyer, 1990; Braff et al.,
2001; Geyer & Swerdlow, 1990; Ludewig et al., 2002; Hoenig et al., 2005). This
includes panic disorder, schizophrenia and obsessive compulsive disorder (Braff &
Geyer, 1990; Braff et al., 2001; Geyer & Swerdlow, 1990; Ludewig et al., 2002; Hoenig
et al., 2005). This provides evidence that both the high and low line animals may be a
useful animal model of mental illness in humans.
This alternative hypothesis needs to be investigated. If it rings true then these
animals can provide researchers with an animal model of exaggerated and blunted
emotional conditioning. This could be useful in the examination of the role of genetics in
mental illnesses. For example, if these high line animals in fact are a model of hyper-
conditioning, it would be useful in the study of predispositions to drug addiction. Studies
have found that elevated cortisol secretion in human subjects is correlated with a greater
DA response to amphetamine (Oswald et al., 2005). People with higher levels of stress 94
hormones that have been found to facilitate reward conditioning also have a more robust
physiological response to addictive dopamine agonists (Oswald et al., 2005). High line animals may also serve as a model for those who are more susceptible to post traumatic stress disorder (PTSD), as they show an exaggerated contextual conditioned fear response.
Low line animals fail to condition to fearful contextual cues and show deficiencies in social bonding in comparison to the random line animals. These animals may better represent a model of flat affect. Their deficiencies in social behavior found in prior studies (Burgdorf et al., 2009; Harmon et al., 2006, 2008) were extended in the current study. The low line animals may serve as an animal model of social dysfunction.
These animals may provide research with a genetic-based model of hypo-active amygdala, diminished oxytocin levels and depressed HPA-axis. 95
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