Does Early Manipulation of Oxytocin Influence Serotonin

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DOES EARLY MANIPULATION OF OXYTOCIN INFLUENCE SEROTONIN

INNERVATION WITHIN THE HIPPOCAMPUS?

A thesis submitted

To Kent State University in partial

Fulfillment of the requirement for the

Degree of Master of Arts

Emma N Janosik

August 2020

Except for previously published materials

Thesis Written by

Emma N Janosik

B.A., The University of Toledo, 2017

M.A., Kent State University, 2020

Approved by

______,Dr. Mary Ann Raghanti Advisor,

______,Dr. Mary Ann Raghanti Chair, Department of Anthropology

______,Dr. Mandy Munro-Stasiuk Interim Dean, College of Arts and Sciences

TABLE OF CONTENTS

TABLE OF CONTENTS………………………………………………………………….....…...iii

LIST OF FIGURES……………………………………………………………………………….iv

ACKNOWLEDGEMENTS……………………………………………………………...………..v

I. CHAPTER ONE: INTRODUCTION………………………...……………...……1

Social Recognition………………………………………………………………...2

Oxytocin…………………………………………………………………...………3

Neurochemistry of Social Recognition…………………………………………….5

Neuroanatomy of Social Recognition………………………………………..……6

Serotonin………………………………………………………………………..…9

Interaction Between Oxytocin and Serotonin…………………………………….10

Synthetic Oxytocin and Oxytocin Antagonist……………………………………12

Current Study and Hypothesis……………………………………………………13

II. CHAPTER TWO: METHODS………………………………….………………15

Specimens……………………………………………………………………..…15

Treatment ………………………………………………………………..………15

Tissue Collection…………………………………………………………………15

Immunohistochemistry………………………………………………………..…16

Regions of Interest………………………………………………………….……16

Data Collection ………………………………………………………………..…17

Statistical Analysis…………………………………………………….…………17

III. CHAPTER THREE: RESULTS……………………………………………...…18

iii IV. CHAPTER FOUR: DISCUSSION………………………………………………21

Future Directions …………………………………………………………...……23

V. REFERENCES…………………………………………………………………..25

LIST OF FIGURES

Figure 1 Rodent Olfactory System……………………………………………………………..…7

Figure 2 Mouse Hippocampus……………………………………………………..…………...…9

Figure 3 5HT Staining in prairie vole brain……...…………….………………………………...11

Figure 4 Hippocampal regions of interest……………………………………………………...... 16

Figure 5 Immunohistochemical staining of prairie vole hippocampus………………….…….....18

Figure 6 One-way repeated-measures ANOVA table…………………………………………...19

Figure 7 Bar Graph showing 5HT axon length density in hippocampal regions across treatment groups…………………………………………………………………………………………….19

ACKNOWLEDGMENTS

To my family, thank you for your constant motivation and encouragement. I appreciate all that you have done for me. To my advisor, Dr. Mary Ann Raghanti, thank you for your guidance and support; your understanding, reassurance, and optimism have been influential in my work for which I am incredibly grateful. Thank you for introducing meditation into my life, it has had such a positive impact on me and those around me. To Dr. Spurlock, thank you for making the trip to Scotland possible; I will remember it forever. Thank you to Dr. Meindl for the dedication you show to your students. To my other professors, I have enjoyed learning from you, and I have valued our time together. To my office mate Sarah, I am so glad we shared an office. I will fondly remember our Starbucks trips and the discussions we have had. Finally, thank you to

Karen and Georgia of My Favorite Murder, Emma and Julie of Comments by Celebs, and

Andrew and Cole of Podcast but Outside. Your work has provided me with tears, laughter, and an escape from schoolwork when needed.

CHAPTER ONE: INTRODUCTION

All social behavior, including finding a mate and determining relatedness, involves some form of social recognition (Gabor, Anna Phan, Clipperton-Allen, Kavaliers, and Choleris, 2012;

Wacker and Ludwig, 2012). Organisms rely on both emission and detection of olfactory signals for social recognition (Sanchez-Andrade and Kendrick, 2008). Chemical senses are the most ancient of the senses, and pheromones mediate more interactions than any other cue (Wyatt,

2007). Thus, olfaction serves to mediate behaviors for many animals, one of these being the influence of female fertility on male mating behaviors (Miller and Maner, 2010).

Compared to most other mammals, primates have a decreased reliance on olfaction and an increased visual acuity. Both olfaction and visual cues are essential for food detection and selection as well as for social and sexual communication among human and nonhuman primates

(Nevo and Heymann, 2015; Heymann, 2006). In humans, for example, the body and face of a female not only signals information about her putative reproductive health, these visual cues also convey information about phenotypic and genetic quality when it comes to survival, resistance to disease, and developmental health (Thornhill and Grammer, 1999). Additionally, every individual possesses a unique body odor. Lobmaier, Fischbacker, Wirthmüller, and Knoch

(2018) set out to determine if some women smell more attractive than others and if scent is related to levels of circulating reproductive hormones. Researchers collected the scent of women at peak fertility (i.e., ovulation) and discovered that men were in agreement on how attractive a woman was based on scent; women with higher estradiol and lower progesterone, the hormonal profile associated with ovulation, were rated as more attractive. There is also evidence that

1 shows that a mother’s breast odors are attractive to infants (Lübke and Pause, 2014). Not only that, but mothers have to the ability to identify the scent of their own infants, even as early as a few days after birth. Given the choice of two garments, mothers were able to successfully identify the one belonging to their offspring (Porter, Cernoch, and McLaughlin, 1983). Mice, however, have approximately 2.7 times the number of olfactory receptors as humans, and rodents are therefore able to identify and discriminate odors more easily than humans (Godfrey, Malnic, and Buck, 2003).

The main olfactory system is critical for the processing of both social and nonsocial information (Sanchez-Andrade and Kendrick, 2008). The development and function of this system is dependent upon various hormonal and neurochemical cues. One hormone that influences the olfactory system is oxytocin (OT), a chemical messenger that plays a major role in social behaviors. Oettl et al. (2016) determined that a function of OT is to bring together multiple levels of sensory, motor, and emotion regulating systems for social interaction. OT works in concert with various neurotransmitters, including serotonin, within the olfactory system as well as in other brain regions involved in social recognition and behavior.

The following sections will discuss the neuroanatomy and neurochemistry of these systems.

SOCIAL RECOGNITION

Social recognition includes many nuanced judgments about an individual such as familiarity, social status, reproductive state, health, and kinship, which allow subjects to categorize the general characteristics of an individual (Gheusi et al., 1994; Gabor et al., 2012). In order for any type of social relationship to form, social recognition and social memory must be maintained as they provide the foundation on which social relationships are built (Ferguson,

Aldag, Insel, and Young, 2001; Nelson and Kriegsfeld, 2017). For example, even if a male cannot distinguish between two females, he might exhibit more interest towards the female in the sexually receptive state (Gabor et al., 2012). Not only is social recognition an essential skill for all social species, but the recognition of a familiar individual allows for an appropriate response.

These responses can vary from investigative and cooperative to aggressive or avoidance (Gabor et al., 2012). Recognition of individuals is critical for animals to understand social and reproductive status, health, and kinship of others (Nelson and Kriegsfeld, 2017). In laboratory mice, for instance, a pregnant female might absorb or abort her fetus if she is exposed to a male that was not the sire. This allows her to begin her estrous cycle again (Nelson and Kriegsfeld,

2017).

OXYTOCIN

Oxytocin (OT) is a small neuropeptide consisting of nine amino acids. The primary sources of OT include the magnocellular neurons of the hypothalamic paraventricular and the supraoptic nuclei (PVN and SON), and the parvocellular neurons of the PVN (Gabor et al.,

2012). OT is transported into the posterior pituitary gland and is released to general circulation, acting as the neurohormone that is responsible for regulating uterine contractions as well as milk ejection in mothers (Gabor et al., 2012; Nishimori et al., 1996; Bosch and Neumann, 2012;

Nelson and Kriegsfeld, 2017). OT is also involved in aspects of sexual behavior, a role that is conserved in invertebrates and vertebrates (Carter, 1992). Additionally, OT is released as a neuromodulator in other brain areas, and OT receptors are found in brain regions known to be directly or indirectly involved in social behavior. OT has been correlated to a variety of other prosocial behaviors such as pair bonding, social recognition memory, and trust in a wide variety

3 of species (Gabor et al., 2012; Shamay-Tsoory et al., 2009; Carter, Williams, Witt, and Insel,

1992; Yoshihara, Numan, and Kuroda, 2018).

Because of its role in nursing behaviors, OT plays a critical role in mother-infant bonding. In fact, human studies have shown that mothers with the highest concentrations of OT show the most affectionate contact and social gaze in a 10-minute test with their 4-to-5-month infants as compared to mothers with lower concentrations (Nelson and Kriegsfeld, 2017).

Moreover, women who have the largest increases in OT as their pregnancies progress exhibit the highest levels of maternal bonding when compared to mothers with less of an OT increase

(Nelson and Kriegsfeld, 2017). However, it is not only in mothers where OT has been shown to influence parental bonding. Naber, Van IJzendoorn, Deschamps, van Engeland, and Bakermans-

Kranenburg (2010) concluded that there was an improvement in human father-child interactions after OT intranasal administration and that fathers exhibited more patience, less hostility, and did not show displeasure towards their child. OT treated fathers were also more responsive towards their child during play. Further, Gordon Zagoory-Sharon, Leckman, and Feldman (2010) determined that OT concentrations increase during the first six months of fatherhood.

There has been ongoing research investigating the effects of OT exposure in rodents.

Bales, Pfeifer, and Carter (2004) set out to study if early OT manipulation to male and female prairie voles affects alloparental behavior when tested with novel pups. Alloparental care is the display of parental-like behavior without going through the actual changes associated with pregnancy. In this study, males treated with OT antagonist (OTA) showed reductions in alloparenting behavior and more frequent attacks towards pups. In contrast, males treated with

OT exhibited high levels of spontaneous parental behavior and rare attacks on infants. For both

OT- and OTA-treated females, alloparental behavior declined between the first and second infant

4 exposure and attack patterns were comparable to that seen in males. This suggests that prairie voles are not always more parental after a second exposure to an infant. In a similar study,

Yamamoto et al. (2004) designed an experiment to test the hypothesis that OT manipulation of postnatal prairie voles changes the production of OT in the hypothalamus. Results indicated that both males and females exhibited an increase in OT expression in the PVN of the hypothalamus when treated with both OT and OTA (Yamamoto et al., 2004). Although OT is produced in both the PVN and the SON, the PVN is responsible for hormone release from both the anterior and posterior pituitary gland (Sawchenko and Swanson, 1983). These two studies illustrate that a single treatment of OT at birth can have both behavioral impacts and changes to the production of OT in prairie voles, and early exposure to OT has the potential to augment or alter the functions of this peptide into adulthood (Bales and Carter, 2003; Bales et al., 2004; Yamamoto et al., 2004).

NEUROCHEMISTRY OF SOCIAL RECOGNITION

OT also has been shown to have varied effects on memory, both enhancing and diminishing memories (Nelson and Kriegsfeld, 2017). Benelli et al. (1995) performed an experiment to study the effects of OT on social recognition memory. Adult male rats were given central injections of OT after an encounter with a juvenile and then they were tested with the same juvenile a second time two hours later. Results indicated that OT treatment significantly reduced investigation time during the second encounter. This illustrates that OT has a specific effect on short term memory, with OT exposure enhancing social memory. Using female OT knockout mice (OTKO), Choleris et al. (2003) determined that they exhibited impaired social recognition as well as diminished habituation to a repeatedly presented conspecific. Additionally, social recognition in adult OTKO mice shows recovery with OT treatment. This suggests that the

5 reason there is impairment of a mouse’s social memory is due to the absence of OT activity in the brain as opposed to disruptions in development (Gabor et al., 2012). Ferguson, Young, and

Insel (2001) performed a similar experiment with OTKO mice, in which the authors tested the timing of OT injections to the medial amygdala to determine whether OT is required for the acquisition and processing of social cues or for information recall. The researchers concluded that OT treatment in a male OTKO mouse must be given before the initial exposure to a female in order for social recognition to be restored for the second exposure. If OT treatment was given after the first meeting, investigation time of the female would not decline, suggesting that the male did not recognize her. Thus, OT must be present during the initial exposure between the two individuals for social memories to form (Ferguson et al., 2001). In another study, Ferguson et al. (2002) concluded that male OTKO mice are still able to process olfactory cues when cotton balls were used as the stimulus instead of a conspecific, yet they could not recognize individuals they had previously encountered. Therefore, OT is essential for short term social recognition in mice.

NEUROANATOMY OF SOCIAL RECOGNITION

The recognition of other individuals requires the learning of a specific chemosensory profile, which requires changes in neural processing (Brennen and Kendrick, 2006). There are two olfactory structures that are responsible for this— the vomeronasal system which is comprised of the accessory olfactory blub (AOB) and the main olfactory system which includes the main olfactory bulb (MOB). Mitral cells of the MOB send fibers to different nuclei which form structures connected to higher cortical brain centers that facilitate the sense of smell. AOB mitral cells transmit to brain areas making up the limbic system, such as the amygdala, hippocampus, hypothalamus and the bed nucleus of the stria terminalis (BNST). These regions

6 are associated with homeostasis, reproduction, aggression, and parental and sexual behavior

(Dulac and Wagner, 2006; Nelson and Kriegsfeld, 2017). However, the MOB is also connected to areas of the limbic system, shown in Figure 1. (Dulac and Wagner, 2006; Sanchez-Andrade and Kendrick, 2008). Therefore, the areas making up the limbic system are critical for the integration of sensory information that is key in the initiation of essential behaviors (Dulac and

Wagner, 2006; Nelson and Kriegsfeld, 2017).

Figure 1: Schematic diagram of rodent main olfactory system. The main olfactory bulb receives input from the main olfactory epithelium and projects the scent information to the anterior olfactory nucleus (AON), tenia tecta (TT), olfactory tubercle (OT), the piriform cortex olfactory cortex (PIR), entorhinal cortex (ENT) and the amygdala. Some second order connections are made to the amygdala, hypothalamus, bed nucleus of the stria terminalis (BNST) and the hippocampus (noted by the blue arrows) which illustrate how cortical and limbic brain areas receive olfactory information. Adapted from Sanchez-Andrade and Kendrick 2008. Dluzen, Muraoka, Engelmann, and Landgraf (2000) report that OT receptors in the olfactory bulb of male rats must be functioning in order for an appropriate social recognition response to be made. By simulating birth and/or mating on female rats and then testing their response to a juvenile stimulus animal, Larrazolo-lópez et al. (2008) determined that vaginocervical stimulation prolongs an olfactory-caused social recognition memory in female rats and increases OT release into the olfactory bulb. Dluzen, Muraoka, Engelmann, Ebner, and

Landgraf (1998) also showed that OT administration to the olfactory bulb maintains social

7 recognition responses in male rats. Olfactory bulb removal is another way in which the roles of the olfactory system in behavior can be studied. Carter et al. (1992) cite unpublished data from

Witt and Insel in which they discovered OT receptors located in the olfactory bulbs of prairie voles. Williams Insel, Harbaugh, and Carter (1994) performed olfactory bulbectomies on female prairie voles which eliminated partner preferences, decreased social contact, and reduced the majority of females who showed behavioral estrus. Bulb removal had no effect on maternal behavior. However, this response varies among other rodents and differs from male prairie voles who underwent bulbectomies and show reduced paternal care, suggesting that the olfactory system is still essential for parental behavior (Williams et al., 1994).

The hippocampus, forming part of the limbic system, is one of the oldest parts of the brain (Cherubini and Miles, 2015). The hippocampus is critical for the recall of memories

(Nakashiba et al., 2012). Studies with famous patient H.M., who suffered from damage to his hippocampus, proved that damage to this region is enough to produce short term memory impairment (Squire and Wixted, 2011). The hippocampus as a whole plays a role in social memory formation, with each region contributing to learning and memory (Hitti and Siegelbaum,

2014; McHugh, Blum, Tsien, Tonegawa, and Wilson, 1996). It is divided into different regions that are termed the CA1, the CA2, the CA3, and the dentate gyrus (DG). OT and/or OT receptors are expressed differently in each region. Overall, there is increasing evidence demonstrating the role of the hippocampus in social recognition memory from rodent studies which indicates that the CA1, CA2, and CA3 regions have high levels of OT receptors (Lin and Hsu, 2018; Maniezzi,

Talpo, Spaiardim Toselli, and Biella, 2019). Specifically, the CA1 is responsible for certain types of memory formation, the CA3 contributes to episodic memory, and the CA2 serves as a link between the two, with the whole region appearing necessary for social memory formation

(Garrido Zinn et al., 2016; Sekino, Obata, Tanifuji, Mizuno, and Murayama, 1997; Tsien,

Huerta, and Tonegawa, 1996; Rolls, 2013). Further, there is evidence that the CA2 serves as a key function in social recognition memory (Lopatina et al., 2018). For instance, Stevenson and

Caldwell (2014) hypothesized that lesioning the CA2 region in mice would impact their social recognition memory. Results indicated that CA2-lesioned animals did have impaired social recognition, further supporting the contention that the CA2 region of the hippocampus is vital for social recognition. Lastly, the DG is responsible for creating pattern separation as lesions to the

DG significantly impair a rat’s ability to spatially separate objects (Rolls, 2013; Gilbert, Kesner, and Lee, 2001). Figure 2 is a mouse hippocampus which highlights the regions mentioned above.

Figure 2: Mouse hippocampus with left side nissil stained and right side depicting the regions that make up the hippocampus. Image credit: Allen Institute, Allen Mouse Brain Atlas (2004) SEROTONIN

Serotonin is a key neurotransmitter that plays a role in functions such as cognition, appetite and digestion, memory, aggression, mood and social behavior, sexual desire and function, and the sleep-wake cycle (Lucki, 1998; Campbell, 2018). In humans, the regulation of serotonin has been found to influence a spectrum of psychiatric disorders such as anxiety,

9 depression, and schizophrenia (Lucki, 1998). Additionally, having fewer serotonin reuptake transporters has been associated with aggressive and violent behaviors for those who suffer from impulse related disorders such as alcoholics and addicts (Lucki, 1998; Coe and Mckenna, 2016).

Furthermore, low quantities of serotonin transporter correlates to impulsive and antisocial behaviors, depression, suicidal inclinations, and homicidal actions (Coe and Mckenna, 2016).

INTERACTIONS BETWEEN OXYTOCIN AND SEROTONIN

Serotonin plays a role in regulating OT levels and expression. It acts on OT by way of

5HT receptors that are located in the PVN and SON (Gabor et al., 2012; Eaton et al., 2011).

Further studies have also shown that OT secretion can be affected by the central infusion of 5HT or 5HT agonists (Jørgensen et al., 2003). Eaton et al. (2011) tested the hypothesis that OT can influence organization on the serotonin system in the brain during early postnatal development in prairie voles. On postnatal day one, male prairie voles were randomly assigned to a treatment of

3.0 µg of OT, 0.3 µg of OT, 0.3 µg of OT antagonist, or a saline control. On postnatal day 21, the voles were sacrificed, and the densities of 5-HT-ir axons were analyzed in various brain regions involved in aggression and social behavior. These included the medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN), cortical amygdala (CoA), anterior hypothalamus (AH), and ventromedial hypothalamus (VMH). Their results supported the hypothesis that OT can have an organizational effect on the serotonergic system during early development, specifically with the treatment of 3.0 µg of OT illustrating a site-specific increase of 5-HT in the AH, CoA, and the VMH. Figure 3 shows 5-HT staining in the brain tissue of a vole from their study.

Figure 3: 5HT staining of vole brain at (A) low and (B) high magnification from Eaton et al., 2011

Knowing that OT enhances serotonin concentration in rodents, Lefevre et al. (2017) designed an experiment to test if OT administered directly into the brains of macaques would also alter serotonin concentrations. Two radiotracers— [11C]DASB which binds to the serotonin

18 transporter SERT and [ F]MPPF the 5-HT1AR marker—were used to track changes induced by

OT at both the serotonin concentration and receptor levels, as both SERT and 5-HT1AR are widespread across the brain. The authors predicted that OT would both induce an increase of

MPPF in the limbic regions because these are associated with emotions and decrease DASB, which would be consistent with an increase in 5HT concentration. Results showed OT directly injected into the lateral ventricle decreased the binding of [11C]DASB to the SERT and increased

18 binding of [ F]MPPF to the 5-HT1AR. These effects were observed in regions of the brain important for social functions, such as the hippocampus, which suggest that OT is modulating the serotonergic system in nonhuman primates.

Because serotonin and OT are two neuromodulators involved in sociality among humans,

Mottolese, Redoute, Costes, Le Bars, and Sirigu (2014) intranasally administered either OT or a placebo to human males and performed a PET scan to map their cerebral 5-HT system to determine whether OT and serotonin interact in the regulation of emotion-based behaviors.

Results showed that OT does in fact modulate the serotoninergic system, with the binding

11 localized to the right amygdala/hippocampus/parahippocampus complex. This suggested that the amygdala and hippocampus create a common ground where interactions between OT and serotonin could control the response to stress and anxiety.

SYNTHETIC OXYTOCIN AND OXYTOCIN ANTAGONISTS

OT acts through the serotoninergic system and Eaton et al. (2011) demonstrated that any disruption to the OT system during important stages of postnatal development can result in enduring changes to serotonin expression in regions of adult brains involved in aggression and social behavior. Because OT has a wide variety of functions in both males and females, and because many aspects of medicine and child rearing change OT concentrations, it has the potential to influence the reproductive, cardiovascular, and immune systems (Carter, 2003). OT can cross the placenta and may even cross the infant’s blood brain barrier during birth (Wahl,

2004; Malek, Blann, and Mattison, 1996). There is also the suggestion that autism is linked to a disruption of OT or OT receptors during early development (Carter, 2007). In humans, synthetic

OT (i.e., Pitocin) is used to induce childbirth and OT antagonists may be used to inhibit premature labor and contractions (reviewed in Carter, 2003). To study the use of OT in a hospital setting, Selin, Almström, Wallin, and Berg (2010) looked at the number of women treated with

Pitocin for augmentation of labor at a Swedish hospital from 2000-2001. The authors found that

55% of women received OT to hasten their labor (2010). However, the use of Pitocin to induce labor has also been shown to cause complications with the central nervous system development

(Carter, 2003; Carter, 2007). Overall, more studies need to be done to study the long-term effects of synthetic OT and OT antagonists during the birthing process.

In rodent experiments, early postnatal exposure to OT has shown to have behavioral and physiological consequences in the long term (Carter, 2003). Bales et al. (2004) cautions against

12 the use of OT antagonists in clinical settings as their research has shown both male and female prairie voles treated with OTA exhibit high attack behaviors towards pups. Further, a single treatment of either OT or OTA on the first day of life affected partner preference expression in prairie voles (Bales and Carter, 2003). Results from Yanamoto et al.’s (2004) experiment testing neonatal OT manipulation also indicates that administration of OT or OTA showed a region- specific effect on OT production. Long-term changes to behavior or physiology indicate that OT has an organizational effect on the central nervous system.

Monogamy is most often seen with behaviors of selective affiliation with a single partner, high levels of parental behavior, and aggressive actions towards nonfamily members for the defense of a territory, nest, mate, or young (Young and Wang 2004; Insel, Preston, and Winslow,

1995; Williams et al. 1994; Ophir, Gessel, Zheng, and Phelps, 2012). However, this is relatively rare in mammals, as only 3-5% exhibit the aforementioned characteristics (Kleiman, 1977;

Young and Wang, 2004). Prairie voles are socially monogamous; they live and reproduce with a single partner (Getz and Carter, 1996). They also exhibit behaviors such as pair bond formation, nest sharing, biparental care of offspring, and alloparental behavior. Similar to humans, voles show great variation in social organization, which makes them valuable models to study affiliation (Nelson and Kriegsfeld, 2017; Young and Wang 2004).

CURRENT STUDY AND HYPOTHESIS

As reviewed above, OT and serotonin work in concert to affect social behavior in primates and rodents, which relies on intact social recognition ability. The hippocampus plays a vital role in social recognition and OT and 5HT act within this region to influence memory and social behaviors. Previous research discovered that OT manipulation to male prairie voles on the first day of life altered serotonin expression in adult brain regions involved in social behaviors,

13 especially aggression. The current project will build on these findings by evaluating whether early exposure to OT or its antagonist alters serotonin within the hippocampus of prairie voles.

We expect that there will be differences in the amount of serotonin based on the treatment of either high OT, low OT, OTA, or the saline control in regions of the hippocampus.

CHAPTER 2: METHODS

Specimens

Male prairie voles stemming from Urbana, IL, USA were housed and bred in a temperature and humidity-controlled environment at the University of Illinois at Chicago (UIC).

The treatments and tissue collection of the prairie voles was also performed there, with the approval by the UIC Animal Care and Use Committee in was in accordance with the National

Institutes of Health Guidelines for Care and Use of Animals (Eaton et al., 2011).

Treatment

Seventeen male pups were assigned to one of four treatment conditions within 24 hours of their birth (P1). These included a single intraperitoneal (IP) injection of one of the following: high OT (3.0 µg OT), low OT (.03 µg OT) .3 µg of OTA, or saline used as the control. Of the seventeen voles, three were injected with high OT, four were injected with low OT, four were injected with OTA, and six were injected with saline. Treatments were chosen based on previous research and were part of prior studies (Eaton et al., 2011).

Tissue Collection

On postnatal day 21 the male voles were sacrificed after being deeply anesthetized and then decapitated. Brains were removed and fixed in 4% paraformaldehyde using the spinning immersion described in Yamamoto et al. (2004). After fixation, the brains were stored in 30% sucrose until they were sectioned on a freezing slide microtome at 20 µm and then placed in cryoprotectant and stored at -20º C until immunohistochemical staining was performed (Eaton et

15 al., 2011). The use of cryoprotectant allows for long term preservation and storage of brain tissue

(Watson, Wiegand, Clough, and Hoffman, 1986).

Immunohistochemistry

Using the avidin-biotin peroxidase method, free-floating tissue sections from each experimental male vole (n= 4 per treatment) were immunohistochemically stained for 5HT using a rat anti-serotonin monoclonal antibody (MAB352, Millipore), as described in Eaton et al.

(2011).

Regions of Interest

The focus of this present study was the hippocampus (CA1, CA2, CA3, and DG regions), as it is involved in learning and memory as well as social memory, which is key for social recognition in rodent species (Lin and Hsu, 2018; Hitti and Siegelbaum, 2014; McHugh et al.,

1996). Either the right or the left hippocampus was used, depending on the condition of the brain tissue. The regions were demarcated based on the Allen Brain Atlas of a mouse brain (see

Figure 4).

Figure 4: Mouse hippocampus demarcated with the regions of interest. CA1 in aqua, CA2 in hot pink, CA3 in red, and the DG in brown. Image credit: Allen Institute, Allen Mouse Brain Atlas (2004).

Data Collection

Serotonin axon length density was obtained using StereoInvestigator software (MBF

Bioscience, Williston, VT, USA, version 9) on an Olympus BX-51 photomicroscope equipped with a Ludl X, Y motorized stage, Heidenhain z-axis encoder, and digital camera that projects images onto a 24-inch LCD flat panel monitor. Three sections spaced 10 sections apart were quantified for each individual. The CA1, CA2, CA3, and DG regions were traced at 4x magnification and the Spaceballs program was used to quantify 5HT-ir axon length at 100x magnification with a grid size of 90 x 90 μm. Using the hemisphere setting with a radius of 8

μm, section thickness was measured every 5th sampling site with the probe height at 8 μm and the guard zone at 2 μm. The total estimated axon length was divided by planimetric volume to obtain axon length densities.

All slides were coded so that treatment group was blinded during data collection. All data were collected by one observer (EJ).

Statistical Analyses

5HT-ir axon length density was compared among treatment groups using a one-way analysis of variance (ANOVA) with repeated measures (IBM SPSS, version nine). The within- subject variable was area (CA1, CA2, CA3, or DG) and the between-subject variable was treatment.

CHAPTER 3: RESULTS

All data are listed in Table 1. Figure 5 provides examples of 5HT immunohistochemical staining of the hippocampus of a prairie vole treated with high OT at low and high magnification. The mean number of sampling sites per area/individual was 81.6 with a standard deviation of 19.5. The mean coefficient of error was .093 with a standard deviation of .021.

A B C

Figure 5 A-C: Scan of 5HT Immunohistochemical staining of prairie vole brain, hippocampus at 4x magnification, and 100x magnification. All images were of voles treated with HOT. A one-way repeated-measures ANVOA was used to test for treatment differences in 5HT axon length densities in different regions of the hippocampus and results showed that there was not a significant main effect of treatment on the quantity of serotonin axons in the hippocampus regions studied (F9,39 = .816, p=.604) seen in Figure 6. However, there is evidence that there is a significance in the main effect of area (F3,39 = 5.847, p=.002), which correlates to findings from previous studies in which lesions to different regions of the hippocampus lead to impairments in memory or object separation (Stevenson and Caldwell, 2014; Rolls, 2013; Gilbert, Kesner, and

Lee, 2001). Figure 7 illustrates the mean 5HT-ir axon lengths for each hippocampus region broken down by treatment.

5HT

Figure 6: One-way repeated-measures ANOVA table

Figure 7: Mean 5HT-ir axon length densities for each hippocampus region broken down by treatment. HOT (blue): high oxytocin, LOT (red): low oxytocin, OTA (green): oxytocin antagonist, Saline (orange): saline control. Error bars: 95% CI

Table 1: Average 5HT-ir axon length densities for each hippocampus area by treatment

Area Treatment Mean 5HT-ir axon length density

CA1 HOT .0508 +/- .02617

LOT .0588 +/- .01632

OTA .0398 +/- .01093

Saline .0348 +/- .00784

CA2 HOT .0506 +/- .02670

LOT .0507 +/- .01794

OTA .0451 +/- .02028

SALINE .0473 +/- .01868

CA3 HOT .0560 +/- .03697

LOT .0617 +/- .04397

OTA .0542 +/- .02840

SALINE .0679 +/- .03416

DG HOT .0625 +/- .05164

LOT .0820 +/- .05781

OTA .0675 +/- .02077

SALINE .0584 +/- .02698

CHAPTER 4: DISCUSSION

Eaton et al. (2011) found that voles treated with high OT (3.0 µg) had significantly higher

5HT-ir axon length density in the AH, CoA, and VMH when compared to those treated with saline. This suggests that early OT manipulation has a lasting impact on the organization of serotonin in brain regions involved in aggressive behaviors. The goal of the present study was to determine if OT manipulation also impacted 5HT expression within the adult hippocampus, potentially affecting social recognition. Using the same sample used by Eaton et al. (2011), we quantified 5HT in hippocampal regions CA1, CA2, CA3, and DG of seventeen male prairie voles treated with high OT, low OT, OT antagonist, or saline on the first day of life. Our results indicated that OT manipulation does not have an effect on serotonin expression in the adult hippocampus.

The hippocampus has been shown to be responsible in social recognition memory, and previous studies have indicated that within it and surrounding brain areas, there is a relationship between OT and serotonin (Lin and Hsu, 2018; Mottolese et al., 2014; Lefevre et al. 2017;

Jørgensen et al. 2003; Eaton et al. 2011). Mottolese et al.’s (2014) study supports the idea that

OT modulates the serotoninergic system as intranasal administration of OT in humans shows binding of OT within the right amygdala/hippocampus/parahippocampus complex. Their results also suggest that the hippocampus and amygdala are regions in which OT and 5HT interactions may manage the response to anxiety and stress. Because OT enhances serotonin concentration in rodents and because OT and OT receptors are expressed differently in each region of the hippocampus, it was hypothesized that an early exposure to OT or its antagonist would alter

21 serotonin within different regions of the hippocampus (Lin and Hsu, 2018; Lefevre et al. 2017).

Further, Lefevre et al. (2017) illustrated that OT administration to nonhuman primates prompts the release of serotonin in areas which make up the limbic system, specifically, the hippocampus.

Arginine vasopressin (AVP), like OT, is a nonapeptide, consists of nine amino acids which are identical in seven out of the nine residues and is a hormone released from the posterior pituitary gland (Nishimori et al., 1996; de Wied, Wimersma Greidanus, Bohus, Urban, and

Gispen, 1976; Nelson and Kriegsfeld 2017; Acher, 1985). Both AVP and OT share an ancestral peptide, arginine vasotocin (AVT) (Acher, 1985; Nelson and Kriegsfeld 2017). The evolution of

AVT from its ancestral state to the nonapeptides known today is due to a duplication of the gene with subsequent mutations. At low concentrations, AVP acts to conserve the amount of water in the body, however high concentrations have been found to be involved in memory processes (de

Weid et al., 1976). Moreover, just like OT, AVP can affect social recognition in mammalian species (Dluzen et al., 1998; Young and Wang, 2004; Choleris et al., 2003; Ferguson et al., 2001:

Benelli et al., 1995). Additionally, Dluzen et al. (1998) reported that AVP administration to the olfactory bulb has been shown to preserve social recognition responses in male rats. DeVito et al.

(2009) also determined that AVP knockout mice show an impairment in memory and social recognition. Stevenson and Caldwell’s (2014) study on lesioning the CA2 region of the hippocampus supports the hypothesis that the CA2 is key for normal displays of social recognition. Smith, Williams Avram, Cymerblit-Sabba, Song, and Wong (2016) specifically wanted to analyze the relationship of AVP signaling in the CA2 and how it affects social memory. By exciting the AVP pathway from the PVN to the CA2 during the acquisition of a new social memory, authors were able to demonstrate the enhancement of social memory, lasting as long as seven days. This suggests that the new social memory can then form a long-term

22 memory and indicates that the CA2 region of the hippocampus can increase the prominence of social signals, such as those important in social recognition memory (Smith et al., 2016). While there are OT receptors in the hippocampus, and early OT manipulation affects 5HT in brain regions involved in aggression, our results show that OT manipulation does not affect 5HT within the hippocampus. It is possible that AVP plays the dominant role within hippocampal regions for social recognition memory.

As previously mentioned, synthetic OT and synthetic OTA are commonly used in hospitals to hasten or inhibit childbirth (reviewed in Carter, 2003). However, not much is known about the long-term repercussions of OT or OTA exposure to humans. Therefore, rodent models are useful to study the effects of such treatment. Moreover, because prairie voles are socially monogamous and live and reproduce with a single partner, they are vital candidates to study the effects of OT treatments to humans (Getz and Carter, 1996).

Future directions

It is known that OT induces uterine contractions and regulates milk ejection in mothers, which plays an important role in mother-infant bonding (Nishimori et al., 1996; Nelson and

Kriegsfeld 2017). One future direction would be to expand the current study to females to determine if there is a sex-specific effect. There is some evidence that suggests this as a possibility: For example, dams who are deficient in OT fail to nurse their offspring, yet exhibit normal maternal behaviors such as successful delivery, nest building, cleaning young, and retrieval (Nishimori et al., 1996). Recognition of offspring is necessary for an appropriate response to be made (Gabor et al., 2012). Otherwise, female rodents may exhibit maternal aggression with behaviors such as tail rattling, lunge-bite attack behavior, pushing, chasing, or

23 contact with the intruder (Rich, deCárdenas, Lee, and Caldwell, 2014; Nelson and Kriegsfeld,

2017).

The present study focused on male prairie voles and how OT or OTA affected the amount of serotonin in their hippocampi. Eaton et al. (2011) determined that any disruption to the OT system during important stages of postnatal development can result in lasting changes to serotonin expression in some regions of adult brains involved in aggression and social behavior.

However, when examining the hippocampus of the same sample, we can conclude that there are not changes to 5HT within the adult hippocampus with treatment of OT or OTA.

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