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Effects of Prolonged Social Isolation: Sex Differences in Anxiety, Depression, and Sociability Behavior in Male and Female Rats
Lake Forest College
Lake Forest, Illinois 60045
Clinically, prolonged social isolation in adolescence is regarded as a risk factor for the development of anxiety. The purpose of this research project was to determine whether the duration of social isolation, sex of the animal, or the housing condition would affect the development of depressive-like behaviors. Overall, the data from open field, conditioned place preference, appetitive conditioning, and social interaction tests revealed that prolonged social isolation in adolescence affected the sociability behavior as well as increased the anxiety behavior. Future research will focus on determining possible sex differences in the Basolateral Amygdala using the deltaFosB marker for chronic stress neuronal activation.
Key Words: social isolation, adolescence, anxiety, social behavior
In loving memory of my mom,
for the most amazing and supportive sister Jája
for the loveliest little girls and future scientists:
Mina, Matilda, and Madlenka
It is my absolute pleasure to acknowledge everyone who has supported me along my academic journey. This research would not have been possible without many important people in my life believing in me and supporting me along the journey of becoming the 1st scientist and only the 2nd woman in my family to ever graduate from college. My special thank you goes to my beloved sister, Jaja, without whom my life would have been much less colorful and joyful. Thank you for inspiring me to always do my best, to be kind and for always being my best friend and a great role model. I would also like to thank Tata, Rozarka, Deda, Babicka, Brian, Mina, Matilda, and Madlenka for always providing unconditional support and love and for inspiring me to be a better person. My special thank you also goes to my mom and although I never had the chance to share with you any of my passions, I am grateful for everything you have taught me in such a short time.
I am grateful that every country I have lived in during my life, I have met people who believed in me and positively touched my life. Without these people I would have never learned new languages, pursued science and ended up the person I am today. My special thanks go to the UWC National Committee of Czech Republic for selecting me to move to Italy, giving me a scholarship, and for becoming my role models and amazing friends.
When coming to Lake Forest College I would have never imagined, how much one person can learn in just four years. During my undergraduate time I have met, interacted with and participated in projects that taught me so many valuable lessons. First, I would like to thank Dr. Daniel Curlik who first inspired me to pursue a Neuroscience major. I would also like to thank Dr. Shubhik DebBurman who first became my Neuroscience academic advisor and who taught me how to work hard, value inter-disciplinary collaboration and education, appreciate group work and who is an inspiration of hard work and commitment to everyone at Lake Forest College. Nevertheless, it was not until the summer after my Freshmen year when I discovered my passion for Neuroscience research thanks to Dr. Jean-Marie Maddux. Thank you for your support, teaching me how to handle rats and how to talk to them, improving my critical thinking as well as your amazing mentorship during the past 3 years. I would also like to thank Dr. Virginie Bottero for helping me overcome my fear of Molecular Neuroscience and Dr. Nancy Brekke for not only making statistics exciting but also for sharing my passion for current political issues. Furthermore, my undergraduate time would not be complete without the friendship and mentorship of Beth Herbert. Thank you for always being there for all the science students, for always showing your compassion and positivity and for inspiring us all to care more about social issues. I would also like to thank professor Goldberg and professor Roberts who both changed my understanding of the world through the lenses of Art History. Thank you for believing in me and for letting me to take on a senior project combining my passion for science as well as Art History. Through the power of visual analysis and your guidance, I discovered a completely new and valuable perspective on the world of science, cultures as well as politics, which I will carry with me for the rest of my life. I also wish to thank all of my friends and study buddies who helped me get through difficult moments at LFC, always offered their unconditional support, guidance, advise and always made me laugh.
Above all, I would like to thank my Neuroscience advisor, Dr. Matt Kelley, for serving on my thesis committee, for believing in me, for being an excellent academic advisor and for encouraging me when it was most needed. Thank you for all the amazing feedback on numerous drafts, patience, support, and always reminding me that “sometimes doing less is more”.
Importantly, I have to thank everyone for supporting me during the past two years at RFUMS. Thank you to the Grace Elizabeth Groner legacy and foundation, namely Kay and Bill Marlatt, for supporting my passion for volunteering as well as research. Thank you to Mallika Padival, Brittany Avonts, Max Loh, Nicole Ferrara, Jaimie Vantrease, and Ryan Selleck for always teaching and inspiring me. I could have never asked for a friendlier and better group of people to be surrounded by during my time at RFUMS. Above all, I cannot thank Dr. Amiel Rosenkranz enough. Your guidance, mentorship, and trust put in me have given me the confidence to think and act as an informed scientist. Thank you to everyone in Rosenkranz’ lab for helping me truly fall in love with research and define my career path.
List of Abbreviations
Analysis of Variance
Conditioned Place Preference
Generalized Anxiety Disorder
Group Housed Animals
Hypothalamic Pituitary Adrenal Axis
Hypothalamic-Pituitary Gonadal axis
Major Depressive Disorder
Magnetic Resonance Imaging
Medial Prefrontal Cortex
Post Natal Day
Social Isolation Test
Socially Isolated Animals
Wistar- Kyoto Model
Effects of Social Isolation Introduction
Social Isolation as a Growing Public Health Issue
Social isolation can sneak into one’s life at the least expected moments and it does not discriminate based on who you are or where you live, whether you are an elderly woman who just lost her husband, a young man abandoned by his friends and family for his sexual orientation, or a young child being separated from his/her parents at the US-Mexico border. All of these tragic cases seem to only worsen when the people have to suffer alone. As a rising social epidemic, the effects of social isolation have slowly attracted the interest of researchers. Social isolation has been associated with poorer physical health, disrupted sleep patterns, as well as altered immune system related to the experienced chronic stress (Khullar, 2016). Social isolation thus presents a pervasive problem to be considered by the 21st-century public health policy makers and as such provides a new challenge for researchers and physicians around the world to address.
Social relationships are essential during all stages of a human life and one of the most tragic and well documented cases of isolation became the motivation for publishing one of the most famous and bestselling books ever written, The Diary of Anne Frank (The short life of Anne Frank, 2017). When Anne Frank, together with her family, went into hiding in 1942, she started writing her diary as a form of refuge from the horrors of World War II (WWII). Isolated from the rest of the world for nearly 2 years, Anne Frank wrote down all the emotions she felt as a 13-year-old girl left to her own thoughts in complete isolation (Horn, 2018). Not able to leave her hiding place, talk to her friends, or go to school, Anne, as an aspiring writer, described her feelings of despair and loneliness already after 3 months in hiding (Ozick, 1997).
‘Added to all this misery there is another, but of a more personal nature, and it pales in comparison to all of the suffering I’ve just told you about. Still, I can’t help telling you that lately I’ve begun to feel deserted. I am surrounded by too great a void. I never used to give it much thought, since my mind was filled with my friends and having a good time.’ (Frank, Mooyaart-Doubleday, & Roosevelt, 1993)
As only a 13-year-old girl, Anne Frank recognized that suffering from social isolation was one of the worst miseries she had to encounter during the dreadful time of WWII. While living in the secret annex, Anne wrote extensively, planning to publish a book about her experience of a young girl during WWII. Unfortunately, their hiding place was discovered, and Anne passed away in 1946 in Bergen-Belsen, Germany (Berenbaum, 2019). After the war, Anne’s father decided to fulfill her wish and published her diary. Since then, her diary, accounts of a teenage girl isolated from the rest of the world, was translated into more than 70 languages and became one of the most sold books in the world (Horn, 2018). Even today, Anne’s diary leaves readers all around the world mesmerized by the tragic life story of a girl who spent her teenage years in social isolation, hiding from the rest of the world (Frank, Mooyaart-Doubleday, & Roosevelt, 1993).
The tragic story of Anne Frank is just one of the many heartbreaking stories from WWII; nevertheless, even today in 2019, social isolation is one of the leading problems that our modern society faces (Khullar, 2016). This growing issue was not left unnoticed by prominent politicians and in 2017, the British Prime Minister Theresa May stated,
“For far too many people, loneliness is the sad reality of modern life” (Yeginsu, 2018, January 17).
Following her quote, Theresa May appointed a minister of loneliness, Tracey Crouch, to confront the challenge of social isolation (Yeginsu, 2018, January 17).Recent data show the pervasiveness of this issue in Western societies where one-person households account for more than 40% of all households (data from Scandinavia)and more than half of people older than 45 report feeling lonely (USA data; Klinenberg, 2016; John, 2018). Politicians across the world slowly have started to recognize the rising prevalence of social isolation and loneliness among the aging population (John, 2018).
For the purpose of this thesis, it is important to define the differences between living alone, being socially isolated, and feeling lonely. According to Klinenberg (2016), these terms are often misused by the press, scholars, and health providers, and although the terms might seem related, it is important to distinguish between them (Hawkley & Kocherginsky, 2018). Loneliness refers to perceived isolation as well as the subjective feelings of distress in the absence of social contact or belongingness. As a concept, loneliness is more of a measure of the quality of social contact rather than its quantity (Beller & Wagner, 2018). Loneliness in human studies is often identified in individuals who live alone, have limited social networks, or lack a partnership (Holt-Lunstad & Smith, 2015) and it is most frequently measured using the UCLA Loneliness Scale (Elphinstone, 2017). In contrast, social isolation refers to the lack of social contact and, compared to subjective loneliness, offers an objective measure that can be manipulated in research using animal models (discussed in the following section in greater detail; Beutel, Klein, Brahler, Reiner, Junger, Michal, Wiltink, Wild, Munzel, Lackner, & Tibubos, 2017; Che, Wei, Reuben, & Bee Hoon, 2017).
Although the subjective feeling of loneliness among adults has been linked to social status, culture, age, and gender, factors determining the occurrence of social isolation have been also been identified. One of the most important factors affecting the prevalence of social isolation is age (Yeginsu, 2018, January 17). As Katie Hafner mentioned in her article, more than a third of Americans older than 65 years live alone (Hafner, 2016). This phenomenon among the aging generation has been associated with increased risks for heart diseases, occurrence of strokes, altered sleep patterns, dementia, and prevalence of anxiety and depression (Steptoe, Shankar, Demakakos, & Wardle, 2013). Furthermore, social isolation as a chronic stressor has been shown to disrupt the functioning of hypothalamic pituitary axis (HPA) across all age groups (further discussed in next sections; Gunnar, 2007).
Although social isolation has been associated with many physical manifestations, it is not just the elderly generation who struggle with the high pervasiveness of social isolation (Cacioppo, 2015). Adolescents are not immune to the negative effects of social isolation either. According to the British Office for National Statistics, people aged 16-24 years-old often feel more isolated than those aged 65-74 (John, 2018). Thus, despite our age, motivation to belong is a fundamental human behavior (Beutel et al., 2017). Although the effects of social isolation on physical and mental health have been well established for the elderly generation (Vozikaki, Papadaki, Linardakis, & Philalithis, 2018), the effects of early social isolation on the adolescents still motivate many new research questions (Witvliet & Brendgen, 2010). Indeed, many longitudinal studies have shown that socially isolated children and teenagers were at a higher risk of developing cardiovascular disorders, showed elevated cholesterol levels, were often overweight, and had elevated blood pressure (discussed in the following sections in greater detail; Caspi, Harrington, Moffitt, et al., 2006).
In her diary, Anne Frank described her struggles with social isolation during WWII, but as previously mentioned, even today many teenagers across the world face social isolation in their everyday lives. Given the pervasive negative impacts that social isolation has on a growing number of people across borders, religions, and ages, it presents a new problem to be considered by the 21st-century public health policy makers and as such provides a new challenge for researchers and physicians around the world to address (Cacioppo, 2015).
Review of Adolescent Psychopathology and Social Isolation
As previously mentioned, most studies focus on the effects of social isolation on mental and physical health of late adulthood (Beutel et al., 2017). With older age, the vulnerability for social isolation increases and is often associated with poorer health outcomes as well as increased morbidity and mortality risks (Hawkley & Kocherginsky, 2018; Holt-Lundstad & Smith, 2015). It is expected that by 2030 over 70 million Americans will be over 65-years old and thus, it is important to understand how to prevent the frequently negative effects of social isolation. The effects of social isolation have been widely analyzed among the elderly population, yet there is still much unknown about the effects of social isolation among the adolescent population.
Regardless of our age, motivation to belong is a fundamental human behavior, which when dissatisfied, can compromise one’s psychological and physical well-being (Beutel et al., 2017). According to the belongingness hypothesis, evolutionary selection is responsible for the internal mechanisms that orient humans toward the preference for social interactions and propel us for close relationships (Laursen & Halrtl, 2013). In fact, adolescence, as a transition period between childhood and adulthood, can be characterized by many changes in social interaction, hormonal changes, increased risk-taking behavior, and novelty seeking, as well as the maturation of cognitive abilities (Caballero, Granberg, & Tseng, 2016; Laursen & Hartl, 2013).
As a period characterized by the development of social behaviors, adolescence is a crucial developmental step for obtaining social relationships and for social learning (Shetty & Sadanda, 2017). According to Rudolph et al. (2008), problematic social relationships can escalate the risk for depressive symptoms during adolescence. Following the interpersonal theory of depression, the absence of social interaction can play a role in the development of further anxiety or depression symptoms (Witvliet & Brendgen, 2010). Therefore, researchers have established that peer relationships during adolescence can have powerful impact on the development of depression in adolescence, as well as in childhood (Caballero et al., 2016). Moreover, adolescence is a period characterized by academic stress and performance, isolation from familial environment, and peer pressure; the combination of all these negative factors can have devastating effects on one’s mental health (Rao & Chen, 2009).
According to longitudinal human studies, the prevalence of depression and anxiety increases sharply during adolescence, more specifically after the age of 15 (Witvliet & Brendgen, 2010), and as Qualter et al.’s 8-year longitudinal study showed, isolation in childhood is a predictor of depressive symptoms in later adulthood (Qualter, Brown, Munn, & Rotenberg, 2009). As such, it has been established that adolescent depression affects more than 5% of the teenage population; this percentage increases with age and acts as a strong predictor of depression in adulthood (Shetty & Sadanda, 2017). This increase of depression and anxiety prevalence in adolescence is also linked with an increased risk of developing alcohol or drug addictions (Spear, 2013).
As a developmental stage, adolescence is a critical period for the cognitive development of many brain areas (Caballero et al., 2016). For instance, the brain region directly associated with the development of social behaviors is the pre-frontal cortex (PFC), which has not yet matured during adolescence. Similarly, other regions such as the hippocampus and amygdala undergo major changes in volume from late childhood to adulthood (Caballero et al., 2016). These particular regions are often affected by chronic stress, which puts the maturation of these regions at risk. For example, the amygdala—the brain region associated with emotion processing and in orchestrating responses to stress (Gunnar, 2007)—sends glutamatergic inputs to the PFC, which controls the consolidation of learning and memory, as well as the regulation of emotions needed for the development of social relationships (Caballero et al., 2016). Further, chronic stress such as prolonged social isolation can lead to the dysregulation of the proper maturation of the PFC-Amygdala axis (Caballero et al., 2016). The effects of social isolation on various brain regions will be further discussed in the following sections.
The current study focused on the effects of prolonged social isolation on depression, anxiety, and sociability behavior in both male and female adolescent rat animal models. Chronic stressors can be manipulated in adolescent animal models using environmental and social stressors, as well as with pharmacological manipulations. In rats, chronic stress can be achieved by social defeat, cage tilt, water deprivation, or social isolation (B. Tannenbaum, G. Tannenbaum, Sudom, & Anisman, 2002). Thus, as a well-known stressor, the effects of social isolation were examined during rats’ adolescence period. As Figure 1 shows, adolescence as a developmental stage can be identified across the majority of mammalian species, but the precise definition of adolescence changes based on the organism of study (Spear, 2013). In humans, adolescence has been identified as the period between 10-20 years of age, whereas in primates, adolescence is between 2-4 years of age (Cacioppo, 2015). In rodents, as displayed in Figure 1, the life period corresponding to adolescence in humans has been defined as starting on postnatal day 30 and ending on postnatal day 50 (Caballero et al., 2016) but some studies refer to the period of adolescence as 28-42 days (Spear, 2013). Importantly, these age boundaries differ between male and female rats and many different factors can play a role on the maturation of each individual (Caballero, et al., 2016). The reasons for using rodents in this study will be further discussed in the next sections. Given the rise of anxiety and depression diagnoses particularly during adolescence, it is important to first understand the symptomology of these two mental disorders in both humans and rodents.
Review of Symptomology of Anxiety and Depression
According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-V), major depressive disorder (MDD) is characterized by a nearly every day depressed mood, loss of interest, loss of energy, inability to concentrate, and in some cases recurrent thoughts of death. Generalized Anxiety Disorder (GAD) in the DSM-V is defined as a condition where patient shows excessive periods of worry, restlessness, fatigue, irritability, muscle tension, sleep disturbances, and difficulty concentrating (Diagnostic and statistical manual of mental disorders: DSM-5, 2013). Both depression and anxiety are the most commonly diagnosed mental disorders across all world regions as well as cultures (Degenhardt, Ferrari, Calabria, Hall, Norman, McGrath, Flaxman, Engell, Freedman, Whiteford & Vos, 2013). Research has established that nearly 5% of adolescents are affected by depression (Shetty & Sadananda, 2016). Meanwhile, GAD is the most common psychiatric disorder, occurring in an estimated 21% of adults (Patriquin & Matthew, 2017).
Chronic stress, such as social isolation in adolescence, can significantly affect the homeostatic biological system by increasing stress responses in our body. Stress has been unquestionably linked to the onset of depression (Young, 1998) and increased cumulative stress has been shown to lead to many adverse health consequences such as GAD or MDD (Patriquin & Matthew, 2017). Furthermore, numerous fMRI human studies on depression and anxiety have shown reductions in grey matter of hippocampus, amygdala, prefrontal cortex as well as white matter reductions in the frontal cortex (Pinel & Barnes, 2018).
The symptoms of depression increase sharply with age in adolescence (Witvliet, Brendgen, van Lier, Koot, & Vitaro, 2010). Among the most gripping symptoms of depression are the prolonged periods of negative mood, anhedonia, increased reactivity to stress, deficits in memory, difficulties with attention, and problems with emotional processing (Hayes, Yasinski, Barnes & Bockting, 2015). As one of the most widely diagnosed disorders, depression not only affects individuals but also their families and communities, thus posing a burden on health care systems worldwide (Degenhardt et al., 2013).
As Figure 2 shows, anxiety and depression are two distinct disorders, but their symptoms can often overlap and consequently complicate an accurate diagnosis. According to the tripartite model, anxiety and depression can be distinguished based on anhedonia and hyperarousal, nonetheless, mild forms of anxiety and depression are difficult to distinguish (Kemp & Felmingham, 2008; Degenhardt et al., 2013). Anxiety is a chronic fear that persists even in the absence of a threat and is a common psychological correlate with chronic stress (Mahan & Ressler, 2012). Although both anxiety and depression are frequently diagnosed as separate entities, they can co-occur in nearly half of the adolescent patients (Krueger et al., 2003). In fact, generalized anxiety disorder occurs in high rates of comorbidity with MDD, which ranges from 40% to 98% in treatment studies (Patriquin & Matthew, 2017).
Given the specification of symptomology of anxiety and depression in humans, rodent animal models act as excellent research subjects in this field (Gorman, 1996). Over the decades, scientists have developed a wide variety of behavioral paradigms and the next section will discuss the prominent animal studies in the field of social isolation research.
Animal Studies on Social Isolation
Motivation to belong is a fundamental human behavior but can also be observed in many other mammalian species (Beutel et al., 2017). Since rodents show an elaborate social behavior characterized by play, high-frequency communication, and social learning, they are the most frequently used animal model for the study of chronic social isolation (Vanderschuren & Trezza, 2013). As previously mentioned, social isolation refers to the lack of social contact and, in contrast to loneliness, social isolation offers an objective manipulation in research (Beutel et al., 2017). Although the behavior of primates shows the closest resemblance to the human behavior, with respect to the principle of “Three Rs” (animal replacement for non-animals, reduction of the number of animals, and refinement of the procedures to minimize pain and stress), use of rodents represents a most cost and time effective animal model for this study (Warnick, & Sufka, 2008). Furthermore, rats’ social behavior, as well as their more elaborate brain structure (compared to mice), helps to ensure that rats are a frequently used animal model in the anxiety and depression behavioral paradigms (Beutel et al., 2017). Thus, since rats, like humans, are social animals, we expected a comparable negative effect of social isolation during adolescence on behavior as well as brain development. Furthermore, research using structural MRI in a rodent model as well as human samples by Nikolova, Miscquitta, Rocco, Prevot, Knodt, Ellegood, Voneskos, Lerch, Hariri, Sibille & Banasr (2018) examined stress effects on brain structures conserved across species. Their research provided clinical evidence for structural covariance of amygdala as well as basal ganglia after periods of mild stress (Nikolova et al., 2018). Moreover, research has showed conservation of key sequential events in brain maturation, such as neurogenesis, synaptogenesis, and gliogenesis in both human and rodent adolescents (Semple, Blomgren, Grimlin, Ferriero, & Noble-Hausslein, 2013). Given the conservation of development as well as function of rodents’ brain structures to human’s, rats are a prevailing animal model in research.
Although this thesis research focused solely on social isolation during adolescence, it is important to note that research in rodents also has shown that the quality of maternal care during the first days of a rodent’s life can have robust effects on the development of stress-response behaviors by affecting early the gene expression as part of the HPA-axis (Biggio et al., 2004). Pre-weaning social isolation depleted the number of neurons in the PFC and post-weaning introduction to social interaction did not recover the neurons in the PFC (McCormick, Mongillo, & Simone, 2013; Pascual & Zamora-Leon, 2007). The behavioral effects of pre-weaning social isolation on depression and anxiety can be assessed using various different behavioral tests. The following studies highlight the main effects of pre-weaning social isolation on rats’ behavior.
A study on neonatal maternal deprivation showed that the separation (PND6-21) not only resulted in a reduction in the dendritic material in the PFC but also lead to an anxiety-like behavior in the elevated plus-maze, which is a standard assay for rodent anxiety behavior (Biggio et al., 2004). Pre-weaning social isolation also has been shown to increase rearing, hyperactivity and exploration inside a novel environment in male rats (Tanas, Ostaszewski, & Iwan, 2015). Particularly the increased hyperactivity has been previously associated with anxiety symptoms in rats (Shetty & Sadanda, 2017). In another behavioral test, pre-weaning socially isolated rats compared to group housed rats showed increased social interaction but no effect on a novel object exploration, which has previously been associated with anxiety behavior in rats (Tanas, Ostaszewski, & Iwan, 2015). Furthermore, when weaning was done too early, it resulted in an increased anxiety-like and aggression behavior during adulthood (Kikusui & Mori, 2009). These studies demonstrate the importance of maternal social interaction for the brain development of the rodent pups and the development of their social behavior.
Although the maternal separation was shown to be an effective stressor, social isolation in the post-weaning period has shown similar effects (Biggio et al., 2004). Early environmental and social experiences can have profound effects on behavior and brain development. Thus, social isolation, as a form of adverse experience and chronic stress in humans, can also be modeled in rats by a post-weaning social isolation procedure not just the maternal deprivation during PND6-21 (Tanas, Ostaszewski, & Iwan, 2015).
One of the most widely used animal models of depression and anxiety are Wistar-Kyoto (WKY) rats. Although this animal model was not used as part of the current thesis research, it offers a well-established model of depression in rats. In a study by Shetty and Sadananda (2017), WKY rats were socially isolated during an early adolescence period and were tested for novel environment-induced hyperactivity and for anxiety-related behaviors. This pivotal study also found that depression-like symptoms in socially isolated rats persisted into the predisposed WKY rats’ adulthood (Shetty & Sadanda, 2017). Another study using the WKY animal model found that socially isolated male rats exhibited anhedonia and agoraphobia, as well as reduced social contact calls (i.e., 50- kHz ultrasonic vocalizations), which shows that depression might be linked to sociability behavior alterations in rats (Seffer, Rippberger, Scharting, & Wohr, 2015).
As the post-weaning period in rats is characterized by a peak in social play, this social depletion can show robust effects on the development of anxiety and depression (Einon and Morgan, 1977). For instance, Robbins, Jones, and Wilkinson (1996) found that when rats were isolated at PND20, they became hyperactive in an open field test, exhibited abnormal responses to novelty, cognitive impairments, and altered responses to stressors as adults. Furthermore, group housed adolescent rats compared to socially isolated adolescent rats showed decreased preference for the engagement with an unfamiliar rat (Templer, Wise, J. Dayaw, & K. Dayaw, 2018). Djodjevic and colleagues used an open field test and showed that socially isolated animals avoided the exploration of the open field center zone, which is an indicator of depression-like behavior (Djordjevic, Djordjevic, Adzic, & Radojcic, 2012, Belovicova, Bogi, Csatlosova, & Dubovicky, 2018).
Prolonged social isolation in adolescence study also showed that socially isolated male rats had longer latency to enter the open arm of the elevated plus maze (increased depression like behavior) as well as decreased social interaction time with novel rats (Green, Barnes, & McCormick, 2012). Furthermore, social isolation in adolescence was shown to increase the instances of freezing (fear measure) in female rats, whereas socially isolated adult rats showed no change in their freezing behavior compared to group housed female rats (McCormick, Mongillo, & Simone, 2013). Given all the negative effects social isolation has on rats’ behavior, Hellemans, Benge, and Olmstead (2004) studied how the introduction of cage enrichment can alleviate some of the negative aspects of social isolation during adolescence. Although researchers are now focusing on the possible reversal of the negative effects of social isolation during the critical period of adolescence, reversal of these symptoms often appears unlikely (Schrijver, Bahr, Weiss, & Würbel, 2002).
In summary, research findings show a wide range of social isolation effects. Both pre- and post-weaning social isolation can have irreversible effects on one’s sociability behavior and induce depression-like behaviors. However, social isolation duration has been associated with contradicting results on the sociability behavior (Templer et al., 2018, Robbins et al., 1996) and there are still many questions remaining to be answered.
Effects of Chronic Stress on the Brain and Physiology
As briefly mentioned in the previous sections, social isolation during adolescence depleted the correct development of the PFC and has been associated with the dysregulation of the proper maturation of the PFC-Amygdala axis (Caballero et al., 2016). Despite these clues, the exact mechanisms that link social isolation and depression remain poorly understood (Shankar, McMunn, & Steptoe, 2011). Yet, in the study of depression, there are three major hypotheses explaining the observed pathology, which include the HPA axis dysregulation hypothesis, the monoamine hypothesis, and the neurotrophic factor BDNF hypothesis (Keller, Gomez, Williams, Lembke, Lazzeroni, Murphy, & Schatzberg, 2017). The HPA axis dysregulation hypothesis suggests that, during depression, the HPA axis becomes overactivated which causes an increased release of cortisol. In contrast, the monoamine hypothesis argues that depression is caused by decreased serotonin function in the brain (Owens & Nemeroff, 1994). Furthermore, according to this theory, monoamine oxidase inhibitors, selective norepinephrine-reuptake inhibitors, and selective serotonin-reuptake inhibitors are all agonists of serotonin and thus associated with serotonergic underactivity in depression (Pinel & Barnes, 2018). Similarly, the neurotrophic factor BDNF hypothesis argues that the differentiation of serotonin releasing neurons in decreasing BDNF expression will instigate a negative impact on the survival of serotonin neurons (Martinowich and Lu, 2008). Research has shown that, in depression, the hippocampus and the medial prefrontal cortex (mPFC) can downregulate the function of BDNF but upregulate the nucleus accumbens and amygdala (Caballero et al., 2016). Although the three hypotheses seemingly describe different pathophysiology, together they provide an integrative approach to the depression symptoms.
The molecular and signaling pathways connecting chronic stress and depression have been widely researched, suggesting that depression has been associated with dysregulation of the hypothalamic pituitary-adrenal axis (HPA) (Keller, Gomez, & Schatzberg, 2017). As Figure 3 shows, stress can initiate a hormonal cascade stimulating corticotropin-releasing hormone (CRH) as well as utilizing the gonadal steroids role modulated by the HPA (Young, 1998).
In greater detail, chronic stress such as prolonged social isolation during adolescence activates the response of the hypothalamus paraventricular region thus releasing the corticotropin releasing hormone (CRH) and the arginine vasopressin (Gunnar, 2007).
Through vascular systems, these hormones affect the anterior part of the pituitary gland and stimulate the release of adrenocorticotropic hormone (ACTH), which is then released into the blood stream to the cortex of the adrenal glands. Here the ACTH triggers the release of cortisol in humans (equivalent to corticosterone in rats). It is also important to note, that the central nucleus of the amygdala also participates in the release of the corticotropin releasing hormone (CRH) thus directly participating in the HPA axis.
The HPA-axis becomes hyperactivated in depression as the final common pathway in the stress response. This hyperactivation may either be related to genetic factors or to aversive stimuli in one’s life, such as social isolation (Swaab, Bao, & Lucassen, 2005). This hormone then acts as a powerful endogenous feedback compound in the signaling pathway for inflammation (Lacey, Kumari, & Bartley, 2014; Rivest & Rivier, 1995) and exhibits multiple roles in the physiology under chronic stress conditions (Gunnar, 2007).
These changes are achieved by a signaling pathway where corticosterone binds to the receptors on the outside of the cells and are carried into the nucleus by glucocorticoids receptive elements (GRE). Here they interact with other gene regulatory signals, thus affecting the gene transcription (Shirk, 2007). This mechanism then shows suppressive effects on the immune system function, as well as other chronic stress responses (Gunnar, 2007).
Figure 4 shows the well-researched effects the increased level of cortisol has on brain development as well as physiological changes. For the purpose of this thesis, it is not crucial to understand the regulatory molecular pathways of the effects of cortisol, but it is important to link the chronic stress and social isolation to the physiological changes during adolescence. On the physiological level, chronic stress (social isolation during different development stages) showed that increased levels of cortisol lead to an increased blood pressure in adolescents (Maslova & Bulygina, 2010), which was also documented in the elderly population (Caspi, et al., 2006). Furthermore, increased levels of cortisol have been linked to obesity, changed appetite in both males and females (Epel, Lapidus, McEwen, & Brownell, 2001).
Figure 4 also shows that the increased release of cortisol has also been associated with many changes in different brain regions. Previous research showed that early life stress affecting the HPA axis contributed to the increased volume of amygdala and decreased hippocampal volume in teenagers (Pagliaccio, Luby, Bogdan, Agrawal, Gaffrey, Belden, Botteron, Harms, & Barch, 2014). Furthermore, chronic stress during adolescence has also been associated with hypoactivity in in-vivo firing of the function of the neurons in the medical nucleus of the amygdala (Adams & Rosenkranz, 2016). In the PFC, increased levels of cortisol release have been associated with the shortening of the dendritic branching (Biggio et al., 2004).
Given the direct effects chronic stress has via the increased release of cortisol on the physiology and brain development, social isolation in adolescence shows similar effects (Swaab, Bao, & Lukassen, 2005). Nowland et al. proposed that loneliness and social isolation can have an impact on health by increasing the perception of social threat. They conducted an important research focusing on undergraduate participants which showed that loneliness group had higher levels of perceived stress and increased social threat sensitivity (2018). This study thus related the HPA stress reactivity to perceived stress and social threat sensitivity (Nowland, Robinson, Bradley, Summers, & Qualter, 2018).
It has also been previously shown that early life stress of neonatal isolation results in enhanced corticosterone levels and stress responsivity in both male and female adolescent rats (Kosten, Miserendino, Bombace, Lee, & Kim, 2005). Similarly, to the data from pre-
weaning isolation, a brief social isolation (PND30-35) in both male and female Sprague- Dawley rats revealed that female rats showed a significant decrease in synaptic plasticity proteins in the PFC as well as increased climbing behavior as a sign of depression in the forced swim test (Hong, Flashner, Chiu, ver Hoeve, Luz, & Bhatnagar, 2011). Previous studies have also suggested that post-weaning social isolation of male rats led to sensitization of serotonergic system and an increase anxiety-like behavior in adulthood (Lukkes, Engelman, Zelin, Hale, & Lowry, 2012).
Using Prairie voles as a model organism, it has been shown that social interaction plays an important role on the HPA axis. By separating the male prairie voles from their female partners, data showed that corticotropin releasing factor in these animals was dysregulated by social isolation (Bosch, Nair, Ahern, Neumann, & Young, 2009). Furthermore, female prairie voles isolated for four weeks showed increased heart rate at rest and reduced heart rate variability, which during a psychosocial resident-intruder challenge translated into an exaggerated cardiac response that took three times as long (compared to pair-housed voles) to return to pre-stress baseline (Grippo, Lamb, Carter, & Porges, 2007a).
Given the common link between the pathology of chronic stress, social isolation, and depression, it is obvious that early social isolation can have profound effects on the physiology and brain development in rodents (Gunnar, 2007). We have previously established the main findings of the effects of social isolation during adolescence on depression, anxiety and sociability nevertheless, an important variable instigating prominent differences in the effects of social isolation are sex differences.
Review of Sex Differences in Anxiety, Depression and Sociability Behavior
Research has shown that sex and gender are important determinants of one’s well-being and mental health (Heidari, Babor, Castro, Tort, & Curno, 2016). Nevertheless, often in research the terms sex and gender are often interchanged. This thesis solely focuses on sex differences and attributes, which are associated with physiological features such as chromosomes, hormonal function, and reproductive/sexual anatomy. In contrast, gender signifies the socially constructed roles and behaviors and how that influences the distribution of power and resources in society (Heidari, Babor, Castro, Tort, & Curno, 2016), but gender will not be discussed in this research. Although sex can be a critical determinant of health, in the past, research on sex differences has been far from optimal (Nieuwenhoven & Klingen, 2010). In order to combat the sex imbalance in research, the NIH in 1993 decided to require grant applicants to describe how researchers plan to include both male and female cells or animals in pre-clinical studies (Correa de Araujo, 2006). In order to obtain optimal health care for both men and women, the differences and similarities in their health need to be properly understood and addressed (Nieuwenhoven & Klingen, 2010).
Anxiety disorders are one of the most commonly diagnosed mental disorders as nearly 1 in 5 adults in the US live with a form of anxiety disorder. According to NIH data, American women show consistently higher prevalence of anxiety disorders as well as depression (McLean, Asnaani, Litz, & Hoffman, 2011, Young, 1998). However, also reported symptoms vary between men and women. According to Rao and Chen (2009), female patients reported more frequent symptoms such as changes in appetite, sleep problems, feelings of failure, guilt, and poor self-esteem. On the contrary, male adolescents were more likely to report symptoms of anhedonia, social withdrawal, and work impairment (Rao & Chen, 2009). Moreover, anxiety diagnosis among women was more likely to be linked to other anxiety disorders such as bulimia nervosa or major depressive disorder (MDD) thus suggesting that anxiety disorders can be more disabling among women compared to men (McLean, Asnaani, Litz, & Hoffman, 2011).
Human research has shown that social isolation is more prevalent in men compared to women (Warner, Adams, & Anderson, 2018), but that there is a higher prevalence of anxiety disorders in women than men. Similar disparities between sexes were found in animals studies showing that female rats were more effected by various forms of chronic stress induced by social isolation, defeat or restraint during adolescence (Bourke & Neigh, 2011). Interestingly, however, most studies focusing on anxiety have solely focused on male rats (Lukkes, Engelman, Zelin, Hale, & Lowry, 2012), although, as detailed below, some relevant research has examined sex differences. Thus, the objective of the current thesis project was to also consider possible sex differences between the effects of social isolation during adolescence.
Young (1988) hypothesized that the sex difference pronounced in the prevalence of stress-related disorders has been linked to the interaction between the hypothalamic-pituitary gonadal HPG axis and the HPA axis. As previous research showed, when female rats were socially isolated for 3 weeks, immunohistochemistry showed an increased expression of c-Fos in the BLA in socially isolated rats injected with FG-7142. This suggests an increased sensitization of the basolateral amygdala to stress-related stimuli in female socially isolated rats (Lukkes, Engelman, Zelin, Hale, &Lowry, 2012). Interestingly, when both male and female mice pups underwent a maternal separation before weaning period, only male pups showed increased myelination in the amygdala, decreased brain neurotrophic factor protein levels in the hippocampus and PFC (Kiksui & Mori, 2009). This interestingly suggested that male rats were more affected by maternal separation compared to female pups.
As established above, the prevalence and symptoms of anxiety and depression vary between sexes in both humans as well as rodent animal models. Therefore, the inclusion of female rats in this study was crucial to determine the possible differences between male and female rats in sociability behavior and depression-like behavior in adolescence.
Social Isolation in Adolescence Hypothesis
Our present research was designed to study the effects of social isolation in adolescence on depression and anxiety. First, we hypothesized that the effects of social isolation will become more pronounced with the increased duration of social isolation period. Furthermore, the earlier in the rat’s adolescent life the isolation started, the more depression-like symptoms could be observed using various different behavioral paradigms. Next, given the higher prevalence of anxiety and depression among women, we hypothesized that chronic stress (social isolation) would impact female rats significantly more compared to male rats. Based on our hypotheses we predicted that prolonged-social isolation (SI) female rats would show lowered social interaction in social interaction test (SIt) as well as conditioned place preference paradigm (CPP) compared to GH males, GH females as well as prolonged SI males and 1-week SI females. Furthermore, we hypothesized that prolonged SI female and SI male rats would show decreased exploration, decreased interest for reward seeking as a response to a stressful stimulus, but increased anhedonia and increased interaction with novel objects rather than novel rats compared to GH male and female rats.
Materials and Methods
Thirty-two male and female Charles River (K72 colony, SAS:SD strain) rats were used for this research project. Rats tested during summer 2017 arrived at the Rosalind Franklin University of Medicine and Science vivarium at 21-23 days postnatal (PND). Initially all the rats were group housed until PND45, when half of the male and half of the female rats were randomly assigned to become socially isolated in individual cages. During summer 2018, rats arrived on PND21 and immediately upon the arrival, half of all the rats were socially isolated (SI) and the rest were housed in groups of two per cage (GH). Rats had no enrichment present in their cages. Every rat was provided water and food (Rodent Diet 2020x pelleted feed, Harlan Teklad) ad libitum. All cages were placed in the same room with 12/12h reverse light-dark cycle. SI rats remained in the same environment as the group housed rats, with a similar level of exposure to olfactory, visual, and auditory stimuli to prevent any additional variation. The socially isolated rats remained in visual contact with the neighboring cages but could not interact with them.
Temperature was consistently maintained at 64-69F with humidity ranging from 30-70%. Rats were habituated to the new facility for a week prior to testing and were all handled daily at least 3 days before the first test (open field). All rats were handled by more than just one person during their lifetime. Furthermore, the occurrence of porphyrin stains as a marker of stress response were noted throughout the entire duration of the experiments.
All tests were performed in accordance with the Institutional Animal Care and Use Committee of Rosalind Franklin University of Medicine and Science and followed the Guide of the Care and Use of Laboratory Animals published by the US National Institutes of Health. Experiments in both 1-week and 4-week social isolation conditions began on rats’ PND52. This was to ensure that the age did not influence their social behavior performance. Figure 5 displays a visual overview of the experimental timeline. The remainder of this section provides a brief verbal overview of the timeline and the sections to follow will provide the essential details of each task previewed in this section.
Overall, the experiment employed three independent variables (IV) including: duration of social isolation with 2 levels (IV: 1-week SI vs. 4-week SI), housing condition with 2 levels (group housed vs. socially isolated), and sex with 2 levels (male vs. female). A variety of dependent variables assessed anxiety-like behavior using various different behavioral tests, which are detailed below. For each behavioral test, rats were transported from their home cages in the animal colony room using the transport cages to the testing rooms.
Figure 5 displays a visual overview of the experimental timeline. On PND52, 1-week SI males and females, 4-week SI males and females, and all of the GH rats began with a 5-minute open field (OF) test immediately followed by a 5-minute social interaction test (SIt) during which each rat was randomly placed with a novel group housed rat of the same sex. On the next day, all experimental rats were exposed to the Conditioned Place Preference (CPP) chamber and they were allowed to freely explore the novel environment (pre-test, PND53). For the next 4 days the CPP chamber included a novel rat and a novel object on each side of the chamber (consistent placement randomly assigned for each rat) letting the experimental rat explore the chamber and associate each side with either an object or a social contact (PND53-57). On the last day of CPP (PND58, Test Day), rats were placed again into the same chamber but without the presence of any novel object nor the novel rat; everything else inside the chamber remained unchanged. After the end of CPP (PND59), rats were introduced to the appetitive conditioning chamber on a Fixed Ratio 2 schedule (FR2), in which two active nose pokes were required for obtaining one pellet of chocolate and were conditioned each day for an hour until reaching at least 50 pellets in two consecutive sessions (PND60-65). After all rats reached at least 50 pellets (FR2), OFF1/ON/OFF2 conditioning with anxiogenic bright light continued for 3 consecutive days (PND66-69). All animals were sacrificed using the perfusion protocol on PND74-75.
Inducing Chronic Stress by Social Isolation
As Figure 6 shows, 1-week socially isolated rats arrived on PND21; nevertheless, until their PND45 all rats were group housed. As indicated in Figure 6, on PND45 all rats were randomly assigned to either GH or SI condition. 4-week socially isolated rats arrived on PND21 and were immediately isolated upon their arrival into the GH or SI condition. There were an equal number of rats in each condition (male vs. female, GH vs. SI, and 1-week vs.4-week). In order to keep the number of animals consistent, it is important to note that four 1-week condition animals (2 GH male and 2 SI male) were tested during summer 2018 whereas the other 16 1-week condition animals were tested during summer of 2017. All 4-week SI animals were tested during the summer of 2018.
Open Field (OF)
Rats were individually placed into an open field (San Diego Instruments, 100x100x38 cm) in a room with red light with the presence of a computer screen eliciting dim white light (20 lux). Video was captured from the moment of the placement of a test rat into the open field and for the next 5 minutes with an IR sensitive camera (Fire I, Unibrain). As depicted in Figure 7, for the analysis, the open field was divided into 25 boxes (20x20 cm) with a center zone defined by 1 box, a central area defined as the middle 9 boxes (50x50 cm), and the periphery zone of the open field (outside ring of 16 boxes, 25x25 cm). The center of the rat’s body was tracked by the software. After the administration of the test, the data was checked for anomalies to ensure a correct software measurement.
Furthermore, the OF chambers were wiped with ethanol solution before each animal was placed in for the testing session. All OF tests occurred on PND52. Exploration in the open field was quantified as the amount of time spent in each zone, number of line crossings, total distance traveled, and the number of entries to the central zone and center area (AnyMaze software). As previously mentioned, the thesis experiment spanned over 2 years and during this time a new OF chamber was purchased as female rats tended to escape the lower walls of the OF chamber. The newly purchased OF chamber was divided by the same software into the same number of boxes (total 25). Sixteen 1-week condition rats (summer 2017) were analyzed using the lower wall OF whereas the new OF with higher walls was used for all of the four 1-week SI animals as well as all the eighteen 4-week condition animals during summer 2018.
Social Interaction Test (SIt)
In both the 1-week and 4-week conditions, a social interaction test (SIt) was conducted on PND52 immediately after the open field 5-minute test ended (for a schematic, see Figure 8). All novel rats were group housed and their backs and tails were labeled with a black marker to distinguish them from the test animals. The duration of the test was 5-minutes and was conducted in the OF chamber (San Diego Instruments, 100x100x38 cm). A novel rat (always same sex as the test rat, with a body weight within 50g of the test rat) was placed in the opposite side of the chamber to where the experimental rat was at the time of placing the novel rat. Immediately after the placement of the novel rat, an IR sensitive camera (Fire I, Unibrain) recorded the entire duration of the test.
All the videos were analyzed blindly to the condition by me as well as by a second independent rater. The criteria for social contact were a direct face to face approach by the experimental rat towards the novel rat or chasing of the novel rat by the experimental rat. Only social interactions initiated by the experimental rat were counted. All the videos were blindly analyzed twice to account for possible errors in the analysis thus providing an interrater reliability. These data were then compared using the % agreement. When there were discrepancies in the videos analyzed, the videos were rescored in order to come to a consensus. The social interactions yielded t quantifiable data for the following 5 dependent measures: the latency to first contact, the duration of first contact, the total number of contacts, the time spent in social contact, and the percentage time spent interacting.
Furthermore, it is important to note that 28 1-week condition rats (summer 2017) were tested in a lower wall OF chamber whereas the rest of the rats were tested in the new, higher wall OF chamber. An issue arose with the lower wall OF chamber during the video analysis as some of the female experimental and novel rats climbed out of the chamber, which is a potential confound in the experiment. The time spent climbing the walls of the open field chamber was eliminated from the data analysis.
Conditioned Place Preference (CPP)
The Conditioned Place Preference (CPP) chamber was 60x30 cm and divided into 3 distinct parts by transparent plastic walls (see Figure 9). The novel object and novel rat zones (25x30 cm) were separated by a middle neutral zone (10x30cm). The CPP test lasted for 10 minutes. All rats were initially placed into the middle zone and then were able to freely move through the chamber. The novel object zone, as well as the novel rat zone, were distinguished by different visual and tactile patterns. These tactile differences were designed to help the rats build an association between the environment and the presence of either a novel object or a novel rat (which was always group housed). All rats were randomly assigned to groups either with the constant placement of a novel rat on the left side and a novel object on the right side or to the counterbalanced placement (novel rat on right and novel object on left).
During each of the four conditioning days, the novel rats were placed into the appropriate side of the chamber and were enclosed inside a small metal wired cage so that they could not freely move around the chamber but were still able to interact with the experimental rat through the holes in the metal wire. After the novel rat was placed in its cage, the experimental rat was placed inside the neutral zone. Immediately, the camera started recording and analyzing the movement of the rat’s body center. To ensure that the rats did not climb out of the chamber, a transparent plastic lid was placed on the chamber during the 10-minute session.
On PND58, a posttest was run during which neither the novel rat nor the novel object were present inside the chamber and the experimental rat was allowed to freely explore the chamber. This posttest data (time spent in each zone of the CPP chamber (s) and number of entries to each zone) was compared to the pretest data to account for any possible preference a rat could have for a side inside the CPP chamber regardless of the presence of the novel object or a novel rat. All rats (summer 2017 and summer 2018) were tested using the same CPP chamber.
Appetitive Conditioning with OFF1, ON, OFF2 Aversive Light Stimuli
One day before the appetitive conditioning began (PND72), three chocolate pellets per rat were placed inside their home cages to extinguish any neophobia confounds in rats. All rats were initially trained for at least one week each day for 60 minutes. After they reached at least 50 pellets in an hour in two consecutive sessions with FR2, they were started with the OFF1, ON, OFF2 paradigm. Each rat was randomly assigned to either having an active nose poking area on the right or on the left (counterbalanced) and this side was kept constant for each rat during every training session as well as during the OFF1, ON, OFF2 (see Figure 10). None of the rats were food deprived prior to the testing session and rats were always run simultaneously in groups of 4 in a counterbalanced order.
Most of the rats learned to nose poke correctly during the first 5 days of the 60-minute training. One day after two consecutive training sessions in which more than 50 pellets were obtained, all rats were placed into a chamber with a software program protocol preset for the OFF1, ON, OFF2 session. Each session was 3 minutes in duration. Exactly 3 minutes after the start of OFF1 phase, a bright anxiogenic light (approximately 200 lux) turned on and stayed activated for the next 3 minutes, after which the bright light turned off again for the remaining 3 minutes. The number of nose pokes during each session was counted and compared across the three (OFF1, ON, OFF2 ) 3-minute intervals.
Tables 1 summarizes all of the dependent measures for all four of the tests described above: open field (OF) (total distance, number of entries to center area/center zone, time in the center area/center zone), conditioned place preference (CPP) (total distance, time in the novel object zone/middle zone/novel rat zone, number of entries to the novel object zone/middle zone/novel rat zone) as well as the number of active nose pokes were gathered by the AnyMaze software. In addition, as previously mentioned the recorded videos of the social interaction test were analyzed manually by 2 independent scorers blind to the testing conditions. Furthermore, in order to keep the number of animals consistent in each group two 1-week GH males and two 1-week SI males were added to the 2017 collected data in 2018. There was no significant difference in the performance between the 2017 and 2018 1-week condition therefore, it was acceptable to include the 4 extra animals in the overall analysis (p>0.05).
Table 2 reports the number of animals in each group (n) as well and highlights the groups of animals that were added to the 2017 data. It also shows that all data were analyzed for significant differences between-subjects (male vs. female, GH vs. SI, and 1-week vs. 4-week condition) using a series of multifactor analysis of variance (3-Way ANOVA). A p-value of less than 0.05 was considered as a statistically significant result. In order to compare means, independent-samples t-tests were performed, assuming that all rows sampled from populations with similar standard deviations (SD). The Sidak-Bonferroni method was used to control the alpha level for the family of tests (i.e., multiple comparisons). When applicable, all post hoc comparisons were made using the Tukey-Kramer’s post hoc test, which compared every mean with other mean allowing for the possibility of unequal sample.
All of the results are discussed in the chronological order of the tests corresponding to the PND of the rats (see Figure 5). All of the data were from purely behavioral experiments and, with the exception of social interaction, the data were scored using the AnyMaze software and analyzed in Prism 7 and Prism 8. The social interaction test was analyzed by independent scorers who were blind to the testing conditions.
The combination of all the tests/measurements (see Table 1 and Table 2) was intended to examine diverse aspects of anxiety and depression resulting from a prolonged social isolation during adolescence. Generally, we hypothesized that the long (4 week) social isolation condition would result in more pronounced anxiety-like behaviors and that female adolescent rats would show more pronounced anxiety symptoms.
Overall, the Open Field (OF) test should indicate how anxiety plays a role in an exploratory behavior in a novel environment. A high anxiety rat would show increased hyperactivity, thigmotaxic behavior and decreased center area exploration. The social interaction test (SIt) should show how social behavior would either be depressed or amplified after a period of prolonged social isolation, with a high anxiety rat showing decreased interest in social interaction. After observing the direct social interaction behavior, the conditioned place preference (CPP) test should indicate a preference for either social contact or for the exploration of a novel inanimate object. Often anxious or depressed rats would spend more time interacting with novel object, decreased preference for social interaction and increased hyperactivity. Lastly, the appetitive conditioning with the OFF1, ON, OFF2 stages should determine the balance between appetitive and anxiogenic stimuli. This measure will then indicate possible decrease in reward seeking behavior in the presence of an acute stressor.
As previously mentioned in the Materials and Methods section, 2017 1-week condition cohort of rats was tested inside a smaller open field chamber with lower walls. The four rats added in 2018 (2 SI male and 2 GH male rats) showed no significant difference in the number of meters traveled and therefore, there was no need of excluding the 2017 cohort from the overall data analysis (p > 0.05). Thus, the analyses below represent data from both cohorts of rats.
Figure 11.1 displays the total distance traveled in an open field chamber. Generally, female rats are known to be more active and therefore we can notice an observable trend of female rats showing more activity inside the OF chamber (Lipatova, Campolattaro, Dixon, & Durak, 2018). Indeed, according to the data analysis, female rats showed a significantly increased activity inside the OF chamber, with a main effect of sex, F(1, 24) = 6.96, p = 0.01). Furthermore, given that the rats were assigned randomly to each condition, we did not expect any significant difference between any of the conditions, and in fact, the 3-way ANOVA (sex vs. social isolation duration vs housing condition) demonstrated that there were no differences in the exploratory behavior as measured by the number of meters traveled inside the open field, F(1, 24) = 0.01, p = 0.92. Furthermore, neither the 1-week nor the prolonged 4-week social isolation had an effect on the activity of the rats, F(1, 24) = 0.65, p= 0.43.
After determining that there was no significant difference in the overall distance traveled inside the open field chamber between the GH control groups, we needed to establish whether social isolation affected the number of entries or the time spent in the center area (Figure 11.2). Recall that this area consisted of 9 boxes and formed the entire central part of the open field including the center zone, as displayed in Figure 7.
Figure 11.2 shows the total number of entries to the center area. The ANOVA found no significant effect of the duration of social isolation, F(1, 24) = 0.05, p = 0.83, nor sex, F(1, 24) = 2.34, p = 0.14. However, there was a significant main effect of the housing condition (GH vs. SI) on the number of entries to the center area, F(1, 24) = 4.78, p = 0.04. In particular, the effect was driven by the 4-week prolonged social isolation condition. Both male and female 4-week SI rats showed decreased number of entries to the center area compared to GH males and females, F(1, 12) = 13.55, p = 0.003. There was no significant difference between the 4-week SI male (M = 2.75) and 4-week SI female rats (M = 3.25), p > 0.99.
Interestingly, the analysis of the total number of entries to the center zone (1 box, see Figure 11.3), time spent in the center area was not significant across any group. None of the main effects for the time spent in the center area yielded statistical significance [housing condition: F(1, 24) = 0.02, p = 0.89; social isolation duration: F(1, 24) = 3.49, p = 0.07; sex: F(1, 24) = 0.70, p = 0.41].
Two additional ANOVAs were run—one on the time spent in the periphery zone and one examining the number of line crossings between zones. Neither ANOVA yielded any significant results (all p’s > 0.05). Given the null results, we decided to not report these data in graphical form in this thesis.
Social Interaction Test
Tested immediately after the open field (OF) test, rats were allowed to freely move through the open field chamber (refer to Figures 5 and 8). Raters (DePaul University summer student and I) blindly analyzed the recorded videos and before the data was compiled, the rater showed more than 70% concordance rate (external reliability) in the tabulations of the behaviors scored. Using Cohen’s Kappa, a measure of internal reliability with a value of 1.00 reflecting complete agreement between scorers and 0.00 reflecting complete disagreement, in our analysis we yielded a respectable value of k=0.74. As expected, the total interaction time among GH males and females in both 1-week and 4-week did not significantly differ thus showing that the changed open field chamber from 2017 to 2018 had no effects on the social interaction behavior among the rats (p > 0.05). Thus, the results below reflect the data from both cohorts of rats.
Figure 12.1 displays the total number of social interactions for each condition. Overall, an ANOVA revealed that no significant main effects of social isolation duration or housing condition on the number of social interactions. The main effect of sex revealed that female rats interacted more than male rats, F(1, 24) = 17.81, p = 0.003. Furthermore, the interaction between social isolation duration and housing condition was statistically significant, F(1, 24) = 14.26, p = 0.009.
Interestingly, 1-week GH male rats (M = 15.25) showed significantly fewer social interactions compared to 1-week SI female rats (M = 29.25), p = 0.03. In the 1-week condition, there was no significant effect of sex nor the housing condition. However, in the prolonged social isolation condition, both sex, F (1, 12) = 13.93, p = 0.003, and housing condition, F(1, 12) = 7.39, p = 0.02, affected the number of social interactions. As Figure 12.1 shows, 4-week SI female (M = 28.25) preferred significantly less social interaction compared to 4-week GH female (M = 34.75), p =.004.
Figure 12.2 shows the total time spent interacting, as measured by independent raters from video replay. Both social isolation duration, F(1, 24) = 26.74, p < 0.001, as well as the housing condition, F(1, 24) = 10.02, p = 0.004) had significant main effects on the total time spent socially interacting. Furthermore, an interaction between social isolation duration and housing condition showed significant effects on the interaction time,
F(1, 24) = 15.23, p < 0.001. Interestingly, the difference in the total time spent interacting was significantly different between 1-week SI (M = 4.055) and 1-week GH males (M = 2.663), p =.007. Furthermore, 4-week SI female rats (M= 4.080) interacted significantly more compared to 1-week GH male rats (M = 2.663), p=.005. Although, 4-week SI female rats showed lowered numbers of social interactions compared to GH female rats (Figure 12.1), there was no significant difference in the overall time interacting (Figure 12.2).
Raters also scored the latency to first contact (Figure 12.3) as well as the initial contact duration (Figure 12.4). Overall, the housing condition (GH vs SI) had a main effect on the latency to first contact, F(1, 24) = 7.11, p = 0.02, whereas sex, F(1, 24) = 1.63, p = 0.21, and social isolation duration, F(1, 24) = 0.06, p = 0.80, had no main effects. As shown in Figure 12.3, 4-week SI female rats (M = 15.75) took more time before the first social interaction compared to 4-week SI male rats (M = 4.40), p = 0.49. Furthermore, there was a significantly lower latency to first contact in 4-week group housed female (M = 3.75) compared to 4-week socially isolated female rats (M = 15.75), p=0.03.
Figure 12.4 shows that there was no significant main effect of any of the independent variables: sex, F(1, 24) = 3.96, p = 0.06; social isolation duration, F(1, 24) = 2.65, p = 0.12); and housing condition, F(1, 24) = 0.38, p = 0.54, on the duration of the first social interaction.
Conditioned Place Preference (CPP)
Similar to the open field analyses, first we needed to determine that there was no significant difference in the total distance traveled between the GH animals (Figure 13.1) as the control group. There was no significant difference between the GH males or females,
p > 0.05. An ANOVA showed a significant main effect of sex, F(1, 24) = 18.76, p = 0.001, and social isolation duration, F(1, 24) = 18.13, p = .001, on the total distance traveled inside the CPP chamber thus suggesting an increased hyperactivity and exploration. The interaction between sex and social isolation duration was also significant, F(1, 24) = 5.67, p = 0.03). In the 1-week condition, socially isolated female rats (M = 53.23) were significantly more active compared to 4-week SI male (M = 30.84), p = 0.003, which further supported the trend of higher activity among female rats. In addition, 4-week SI female rats (M = 53.23) showed decreased overall movement through the CPP chamber compared to the 1-week SI female rats (M = 36.21), who showed more exploratory behavior inside the 3 compartments of the chamber, p = 0.006. Furthermore, there was a significant difference between 1-week SI female (M = 53.23) compared to 4-week GH female (M = 32.75), p = 0.008 (see Figure 13.1).
Figure 13.2 shows the overall time spent in the middle neutral zone of the CPP chamber, which divided the novel rat area from the novel object area (see Figure 9 for a schematic). There were no significant main effects of any of the independent variables on the time spent inside the neutral middle zone (all p’s > 0.05). Nevertheless, a three-way interaction (sex vs. social isolation duration vs. housing condition) was found to be statistically significant, F(1, 24) = 4.91, p = 0.04.
Next, we examined whether social isolation and sex had any impacts on the preference between social contact and exploration of an inanimate novel object. Neither the sex, F(1, 24) = 0.01 p = 0.91, nor the social isolation duration, F(1, 24) = 1.64 p = 0.21, main effects were found significant. Yet the housing condition had a main effect on the time spent in the novel object zone, F(1, 24) = 16.27, p < 0.001. Furthermore, a significant interaction was found between social isolation duration and housing condition, F(1, 24) = 16.27,
p < 0.001). 1-week GH male rats (M = 267.50) spent less time interacting in the novel object zone compared to 1-week SI male rats (M = 172.10), p = 0.04. The same trend was observed in the 1-week female condition, with GH females (M = 303.10) significantly higher than SI females (M = 174.90), p= .003.
When looking at Figure 13.3, there might appear to be a trend that 4-week socially isolated male and female rats spent more time in the novel object zone compared to 1-week socially isolated male and female rats. However, this trend did not reach statistical significance, p > 0.05.
The ANOVA showed that all of the independent variables had significant effects on the time spent in the novel rat zone: sex, F(1, 24) = 11.02, p = 0.003, social isolation duration, F(1, 24) = 47.30, p = .013, and housing condition, F(1, 24) = 13.02, p = 0.001. Only the interaction between social isolation duration and housing condition was found significant, F(1, 24) = 6.03, p = .02. 1-week GH male rats (M = 239.40) spent less time interacting with the novel rat compared to 1-week SI male rats (M = 343.30), p = 0.02. Furthermore, there was a significant difference between 1-week GH female and 1-week SI female rats, p = 0.04. Interestingly, in the 4-week prolonged social isolation condition, no significant differences between groups were found, p > 0.05. Furthermore, there was no significant difference between socially isolated females in the 1-week condition compared to the prolonged social isolation, p > 0.05. In Figure 13.3, there appeared to be an interesting trend showing that the prolonged socially isolated males and females tended to spend less time in the novel rat zone compared to the 1-week socially isolated males and females. Unfortunately, this trend was not statistically significant (p > 0.05).
In regard to the other measure (Figure 13.4)—the number of entries to each zone—the ANOVA determined that there was a significant main effect of social isolation duration, F(1, 24) = 78.08 p < 0.001, as well as housing condition, F(1, 24) =4.58, p = 0.04, on the number of entries to the novel object zone. Furthermore, there was a significant difference between 1-week SI (M = 27) male and 4-week SI male (M = 6.75), p = 0.18, as well as between 1-week SI female rats (M = 31.75) and 4-week SI female rats (M = 6.75), p=0.002.
Both sex, F(1, 24) = 4.82, p = 0.04, and social isolation duration, F(1, 24) = 166.60,
p = .001, had significant main effects on the number of entries into the novel rat zone. Furthermore, a significant interaction between sex and social isolation duration was found, F(1, 24) = 7.41, p = 0.01.
Rats were initially conditioned to nose poke for chocolate sucrose pellets until they all reached at least 50 pellets in one session with FR2. Therefore, all rats started from the same baseline and showed no significant differences between their baseline, p > 0.05. All rats displayed acquisition of nose poking for chocolate pellets and therefore all were included in the analysis. Random assignment to housing condition groups confirmed that there was no significant difference between groups on active nose pokes or inactive nose pokes during training, p > 0.05.
As seen in Figure 14.1, the anxiogenic bright light phase did not significantly reduce the active nose poking compared to the light off Phase 1 or light off Phase 3. Furthermore, as Figure 14.1 indicated there was no effect of stress (social isolation) on nose poking during the initial light-off phase, p > 0.05. There was no significant different between initial nose poking in Phase 1 (light off) and final Phase 3 (light off). Nonetheless, a decreasing trend in the number of active nose pokes over the period of 9 minutes was observed which might have related to an increase in overall satiety since the rats were not food deprived prior to the testing. Overall, short social isolation in adolescence had no effect on the effectiveness of anxiogenic stimulus in reducing appetitive behavior.
Although there were no significant differences observed in appetitive behavior after just 1 week of social isolation, as shown in Figure 14.2, prolonged social deprivation resulted in a significant decrease of chocolate pellet consumption during Phase 2 (light on). Our data showed that when bright light was turned on after 3 minutes of free nose poking, socially isolated female and socially isolated male rats nose poked significantly less when compared to GH males and females, p = 0.041. Furthermore, when anxiogenic bright light was turned off, both socially isolated males and females returned to their baseline pellet consumption. There was no significant difference between the Phase 1 and Phase 3, p > 0.05.
Given the robust decline in nose poking when anxiogenic bright light was turned on, we can assume the effectiveness of bright light in reducing appetitive behavior and natural reward seeking in chronically stressed animals. Furthermore, there were no significant differences between male and female adolescent rats during Phase 2 with anxiogenic bright light, p > 0.05. Moreover, there was no effect of housing condition, social isolation duration or sex on the Phase 1 (light off), p > 0.05. The effect of bright light was significantly greater in socially isolated rats compared to the group housed in the prolonged social isolation condition. Although it was expected that the bright light would cause a more long-lasting effects on the appetitive behavior, as we can see, there was a significant tendency to return to the nose poking base line for both socially isolated female rats as well as for socially isolated male rats.
Overall, anxiogenic bright light had no effect on the appetitive behavior of 1-week socially isolated rats but its presence significantly decreased natural reward seeking in both male and female 4-week socially isolated rats. Interestingly there was no significant difference between male and female prolonged socially isolated rats.
Previous research has shown associations between social isolation and depression, poor social skills, anxiety, premature death, and worsened immune system (Khullar, 2016). The present thesis research was designed to study the effects of social isolation in adolescence on depression, anxiety, and sociability behavior. Using four distinct behavioral tests (open field, social interaction test, conditioned place preference, and appetitive conditioning with anxiogenic stimulus), we found that prolonged social isolation affects hyperactivity, exploration behavior, social interaction, and anhedonia, as well as reward seeking in the presence of anxiogenic stimulus. Overall, our research showed that chronic social isolation had no effects on novel object exploration, but significantly decreased social interaction in both social interaction test and conditioned place preference. Furthermore, social isolation lead to decreased exploration and increased thigmotaxic behavior. Lastly, we reported that anxiogenic stimulus only had an effect on the reward seeking behavior of chronically isolated male and female rats but no effect on 1-week SI rats. Interestingly, our research found no sex differences between the chronically isolated animals (4-week SI condition) as well as between 1-week SI animals. As part of future studies, effects of resocialization as well as effects of social isolation on ΔFosB expression in the BLA could be assessed and will be discussed in the next sections.
Effects of Social Isolation on Sociability
Motivation to belong is a fundamental human behavior but can also be observed in rodents (Beutel et al., 2017). Our experiment manipulated social isolation in both late (PND44) and early adolescence (PND21), thus interfering with the developmental period characterized both by play and by social learning (Witvliet & Brendgen, 2010). Using two different behavioral tests (SIt and CPP), we intended to determine what the effects of chronic social isolation are on rats’ motivation to socially interact. The main findings on the effects of social isolation on sociability behavior are summarized in Figure 15.
First, using the social isolation test (SIt) on PND52, all socially isolated male and female rats were allowed to freely interact with a novel rat (same sex) in an open field chamber. Previous research showed that anxiolytic-like behavior is inferred by an increase in social interaction but no effects on the hyperactivity in the chamber, whereas anxiogenic behavior is characterized by a decrease in social interaction (Campos, Fogaca, Aguiar, & Guimares, 2013).
Overall, our research showed that female rats interacted more compared to male rats regardless of their housing condition or the social isolation duration. Our research also found that prolonged socially isolated animals interacted less compared to group housed rats. This finding supported our hypothesis claiming that prolonged social isolation has effects of sociability behavior (number of interactions) nevertheless, there were no sex differences found between the chronically isolated animals (4-week SI condition). Consistent with our findings, Shetty and Sadananda (2017) found that social isolation in adolescence rapidly decreases preference for social interaction in SIt. Similarly, McCormick, Mongillo, and Simone (2013) found that socially isolated female Long Evans rats tested on PND60 decreased their interaction time with novel rats. Interestingly, our results showed no significant difference in interaction time between female rats but found that short social isolation increased preference for social interaction (longer time interacting) among male rats. This finding corresponds to findings by Templer, Wise, and Dayaw (2018) who showed that short adolescence social isolation leads to an increased preference for the engagement with an unfamiliar rat. This unusual behavior might suggest that a brief social isolation increases the motivation of rats to socially play and interact.
Furthermore, it is important to note that in our study, the increased number of social interactions did not correspond to an increased time interacting (min). Although, there was a significant difference between the number of interactions between 4-week socially isolated female rats compared to GH rats, there was no significant difference in the overall interaction time. When closely analyzing the interaction time plot, we can notice that overall the 4-week GH and SI animals interacted a majority of the test time (5 minutes). Nevertheless, as the number of interactions plot displays, SI female rats showed lower number of social interactions, which might mean that overall each interaction lasted longer compared to the GH female rats.
According to Tanas, Ostasewski, and Iwan (2015), when rats were socially isolated already during pre-weaning period (PND2-21), socially isolated rat pups showed increased social interaction. Therefore, these discrepancies in findings show that the effects of social isolation are dependent on the timing as well as post-natal day of isolation initiation. Overall, social interaction test results indicated that prolonged social isolation in early adolescence impairs social behavior in rats’ in early adulthood.
Secondly, preference for social interaction was also evaluated using the conditioned place preference test (CPP). Data collected between PND53 and PND58 rats were evaluated for their preference for either the novel object side or the novel rat side of the CPP chamber. As expected, there was no significant difference between the time spent in the middle neutral zone as a function of sex, housing condition, or the duration of social isolation. However, there was significant difference in the time spend in the novel object zone where both GH male and females spend more time in this zone compared to 1-week SI male and female rats. This finding seems to suggest that short social isolation might decrease the curiosity and exploration in a novel object zone. Accordingly, one might assume that an opposite result would be observed when interacting in a novel rat zone. However, only 1-week SI isolated male rats showed an increased time spent in the novel rat zone. In a study by Douglas, Varlinskaya and Spear (2003), researchers found that responses to novel objects were elevated among male adolescents but less in male adults and the preference for objects among female adolescents and adults was not significant.
Although adolescence is characterized by the motivation of seeking novelty (Douglas, Varlinskaya, & Spear, 2003) there was no significant difference between time spent in the novel object zone versus novel rat zone in the chronic social isolation condition. This finding was supported by Tanas et al. (2015) who also found no significant effect on novel object exploration in the CPP. This might suggest that prolonged social isolation does not affect the interaction with inanimate objects but as was previously mentioned has an effect on social interaction with same-sex novel rats.
Effects of Social Isolation on Hyperactivity, Anxiety, and Novelty Seeking
Although sociability behavior can be a useful indicator of anxiety and depression, in our hypothesis we tested other factors indicating the effects of social isolation in adolescence. As Figure 16 shows, using open field, we found that there were no significant differences in the total distance traveled as a function of sex, housing condition, or the duration of social isolation. However, there was a main effect of sex where female rats showed increased hyperactivity inside the open field chamber. Our findings were in contrast to Robbinson, Jones and Wilkinson (1996) and J. Djordjevic, A. Djordjevic, Adzic, & Radojcic (2012) who found increased hyperactivity in socially isolated both male and female rats. The reason for the discrepancy in our results compared to other research findings is not immediately obvious and will require further exploration.
Total distance traveled was also evaluated in the conditioned place preference (CPP) test. The results from CPP were more consistent with the field of research as they showed increased hyperactivity in the 1-week socially isolated female rats compared to 1-week SI male rats. We also found that 4 week- SI female rats showed decreased activity inside the CPP chamber compared to 1-week SI females. This finding is interesting as it might suggest a possible relationship between chronic social isolation and decreased activity. Consistent with our CPP results, Douglas, Varlinskaya, and Spear (2003) reported suppression in activity in an open field test.
Using the open field (OF) paradigm, we also assessed the exploration behavior inside the chamber. Assessing the thigmotaxic behavior, which reflects the finding that rats rarely venture away from the walls of the OF chamber (Pinel & Barnes, 2018), we measured the number of entries to center area and center zone. Our results showed that chronically socially isolated male and female rats made significantly fewer entrances to the center area compared to GH male and female rats indicating fearfulness and anxiety. Our results were congruent with the findings of Djordjevic et al, (2012), who also showed that socially isolated rats avoided the exploration of the open field center zone.
Nevertheless, when analyzing the time spend in the center area, we found no significant differences, suggesting that although GH rats entered the center zone more frequently compared to SI rats, they did not spend significantly more time in the center area or center zone.
Effects of Social Isolation on Reward Seeking in the Presence of a Stressor
According to the tripartite model, anhedonia is one of the symptoms of depression and can be both observed in humans as well as rats (Kemp & Felmingham, 2008). Anhedonia, characterized as a general inability to experience pleasure, was assessed by appetitive conditioning in the presence of anxiogenic bright light. In our experiment, we adapted the procedure of Jaisinghani and Rosenkranz (2015) with a few important modifications. In our experiment, rats were placed in the operant chamber for 9 minutes (instead of 15 minutes), which allowed us to divide the session into 3 distinct phases each lasting 3 minutes. Furthermore, in our experimental design, rats were conditioned to nose poke, whereas Jaisinghani and Rosenkranz (2015) employed lever pressing. These changes in the procedure were made in an effort to increase the effects of the presence of anxiogenic bright light in the 2nd phase of this behavioral paradigm. Our research found that, only after 4 weeks of social isolation, SI male and SI female rats showed a decrease in the number of nose pokes suggesting a decrease in the appetitive behavior. When the bright light turned off at the beginning of the 3rd phase (light off), both SI male and SI female rats returned to their baseline nose poking. These results were consistent with Jaisinghani and Rosenkranz (2015) who reported that socially defeated rats decreased lever pressing during the bright light session but recovered to lever pressing baseline when light was turned back off.
Previous studies demonstrated that chronic social isolation suppresses unconditional approach of appetitive stimuli, in our case chocolate sucrose pellets, which often is identified as anhedonia-like behavior in rats. Since sucrose is highly palatable in rats, any decrease in consumption of this natural reward reflect anhedonia in rats. Similarly, to our findings, Seffer et al. (2015) found that socially isolated male WKY rats exhibited anhedonia in appetitive conditioning after post-weaning social isolation as well as after post-adolescent social isolation (after PND50).
Effects of Social Isolation Summary
Overall, our data supported our hypothesis that chronic social isolation in early adolescence (4-week SI, start on PND21) leads to a decrease in the number of social interactions as well as an increased latency to social contact in 4-week SI female rats. This finding suggests that sociability behavior might be more impacted among female adolescents compared to males. Furthermore, only short social isolation resulted in a decreased interest in the novel object exploration in both 1-week SI male and female adolescents, and only 1-week male adolescents showed elevated preference for the interaction with a novel rat. As predicted, socially isolated females showed elevated hyperactivity in the CPP chamber, but no increased activity was observed in an open field. Furthermore, anhedonia was observed after prolonged social isolation in both male and female adolescents. After the anxiogenic stimulus was removed, the appetitive behavior returned to its baseline.
Important limitation to consider concerns the timeline of the experiment. As discussed earlier, one group of rats was isolated for the entire duration of adolescence (start PND21) whereas the 1-week of SI rats were isolated at the end of their adolescence (PND45). All behavioral tests were thus tested in early adulthood and thus we cannot make any conclusions about the effects of social isolation on the depression, anxiety, and sociability during adolescence. Moreover, since female rats were used in this experiment and behavioral tests were run during their adulthood, it would be necessary to obtain female vaginal samples to assess their estrous cycle. From previous research, we know that hormones have effects on the HPA axis (Young, 1998, Gunnar, 2007) (see Introduction, chapter: Effects of Chronic Stress on the Brain and Physiology) and thus play role in possible sex differences in responses to chronic stressors during adolescence (Young, 1988). Using the estrous cycle data, we would be able to assess the effects of changes in hormonal levels on the effects of social isolation in adolescence.
Moreover, the timeline of the experiment was designed so that both groups of SI rats (1 week and 4 week) and GH rats were always tested on exactly the same PND. This was to prevent any possible differences caused by developmental changes during adolescence. Both social interaction test (SIt) and CPP tested the effects of social isolation on sociability. When SIt was run on PND52, the socially isolated rats were naïve to any social interaction prior to this test. Nevertheless, when CPP was run following SIt, all the socially isolated rats were no longer naïve to social interaction, which might have affected our results. In order to overcome this issue, different groups of rats should be used for each behavioral test so that all rats were naïve to novel rats or another possibility would be to run only one of the sociability tests in the future experiments.
Another interesting aspect of this social isolation research was the possible visual contact between all the rats housed in the same room in the vivarium. Since all the rats were able to see other rats through the transparent walls of their cages, to what extent were they socially isolated? The premise of this study was to socially isolate adolescent rats to prevent the development of social play, yet the presence of rats in surrounding cages might have affected social development during adolescence as well as social play but possibly to a lesser extent. Moreover, since all rats were housed in the same room, we cannot exclude the possibility of socially transmitted vocalization about threats and rewards among the rats housed in the behavioral room (Masuda, Narikiyo, Someya, & Aou, 2013). Another possible confound might have been housing female and male rats together in the same room, which might have stimulated sexual desire among the rats. Furthermore, all the rats were handled daily by a group of summer students and it is a question to what extend did everyday human contact affect the perils of social isolation.
Furthermore, it is important to note that this animal study solely focused on determining the effects of social isolation, but not loneliness. As previously discussed, loneliness refers to perceived isolation and the subjective feelings of distress in the absence of social contact (Beller & Wagner, 2018). Given that human studies often employ a loneliness questionnaire whereas animal studies focus on measurable behavioral effects of social interaction, the two terms get often misused in research. This distinction between the two terms makes then it difficult to generalize findings from animal studies to the growing reports of loneliness in many of Western societies (Rona & Dury, 2014).
Furthermore, it is also important to note that there are often individual differences among depression and anxiety patients and our study focused only on a subsection of the DSM-V diagnostic spectra of major depressive disorder and anxiety. Although rats have different social behaviors compared to humans, as we have shown when exposed to a chronic stressor such as social isolation, rats demonstrate comparable symptoms as human with respect to anhedonia, hyperactivity, and alterations in social behaviors.
Another important limitation is the generalizability of our research in the understanding of anxiety and depression among human adolescent patients. Animal models for anxiety-related disorders are based on an assumption that anxiety in rats is comparable to anxiety symptoms in humans (Ohl, 2005). Although both MDD and GAD can be characterized by a wide range of pathologies and symptoms (Patriquin & Matthew, 2017), our study provides an important puzzle piece in improving our understanding of mental disorders stemming from an increasing global epidemic of social isolation in adolescence.
Future Behavioral Studies
As previously established, behavioral tests such as open field, conditioned place preference, social interaction, appetitive conditioning with bright light are a good indicator of anxiety and depression-like behavior in rodents. Decreased reward seeking (anhedonia), changes in motor activity, and social avoidance are characteristic of increased anxiety behavior in male and female rats which corresponds to our findings. Nevertheless, in our study, we did not find significant sex differences as we expected and thus it would be promising to increase the number of animals used in this study to further confirm or repudiate the absence of sex differences after chronic social isolation in adolescence.
In our study, we used male and female Charles River (K72 colony, SAS:SD strain) adolescent rats. Using a different rat strain could underline possible effects of chronic social isolation effects in adolescence. As previous research showed, rat strain Wistar-Kyoto (WKY) exhibit decreased activity in the open field test (assessing exploration and anxiety behavior) as well as decreased consumption of a sugar-based reward in response to chronic stressors (enhanced anhedonia) (Edwards & King, 2009). Furthermore, research by Tanas, Ostasewski, and Iwan (2015) and by Hong et al. (2012) showed that prolonged social isolation in WKY adolescents resulted in an altered exploration behavior and anhedonia in appetitive conditioning in male rats. Using this rat model might help to enhance possible sex differences associated with chronic social isolation.
Since anxiety and depression show a wide variety of symptoms, employing different behavioral paradigms and measures could help further establish the effects of social isolation on the behavior of rats. One of the possible measures previously assessed by different research groups with focus on social isolation was an increased prevalence of aggressive behavior among male rats (Burke, McCormick, Pellis, & Lukkes, 2017). In our social interaction test (SIt), any type of interaction was counted. It would be interesting to assess interactions based on its type: play-fighting, chasing, defensive behavior, or aggression attacks. This more detailed analysis could uncover important behavioral differences resulting from prolonged social isolation as well as possible behavioral differences between male and female adolescent rats.
Another interesting measure concerns the effects of social isolation on vocalization in socially isolated male rats. As was also researched among socially isolated children (in extreme cases of feral children), social isolation in childhood and adolescence irreversibly impacted their language skills development (Favazza, 1977). We know that simple communication (ultrasonic vocalization) is an important component of rat’s social behavior and that researchers have established that high-frequency (50kHz) occur only in appetitive situation or in a social approach behavior (Seffer, Roppberger, Schwarting, & Wohr, 2015). Therefore, measuring these high-frequency ultrasonic vocalizations during the CPP and SIt could act as an indicator of altered social behavior after prolonged social isolation in adolescence. Furthermore, understanding the effects of social isolation on the rats’ communication could reveal important differences between male and female social behavior after social deprivation.
As previous research showed, resocialization after prolonged social isolation in adolescence can have reversal effects on some of the negative aspects of chronic stress. For instance, Tanas, Ostasewski, and Iwan (2015) showed that play-related behavior acts as a protective factor from anxiety and depression in young adulthood. Another study by Tulogdi, Toth, Barsvari, Biro, Mikics, and Haller (2014) showed that resocialization in adulthood after a period of prolonged social isolation in adolescence rescued social deficits such as abnormal defensive behavior however, elevated aggression behavior was resilient to the treatment of resocialization. These specific behavioral assessments were evaluated only in male rats and thus the question about the resocialization of female rats remains open. Therefore, it would be noteworthy to determine the effects of resocialization in adulthood on behaviors assessed in this study: hyperactivity, sociability, exploration, and anhedonia.
Another form of rescue during social isolation is environmental enrichment, when rats are singly housed but their cages include an inanimate object, a toy. Previous research has shown that adolescent enrichment reversed the negative effects of prolonged social isolation on spatial learning, locomotor activity, and anhedonia in male rats (Hellemans, Benge, & Olmstead, 2004). The findings were also supported by Tanas, Ostaszewski, and Iwan (2015) who found that environmental enrichment ameliorates socially isolated male rats’ exploratory behavior as well as social play behavior. More research needs to be conducted to understand the effects of environmental enrichment on female rats’ behavior. Using our set of paradigms, we could reveal possible sex differences in hyperactivity, exploration, anxiety, sociability or anhedonia after the introduction of environmental enrichment.
Another important field of future research related to prolonged social isolation in adolescence and its negative impacts focusses on substance abuse. Human studies, such as research by Copeland, Fisher, Moody, and Feinberg (2018), showed that social isolation and social disengagement in 9th grade students were associated with increased risky behavior, cigarette, and marijuana use in both boys and girls. Furthermore, rat model research by Venniro, Zhang, Caprioli, Hoots, Golden, Heins, Morales, Epstein, and Shaham (2017) highlighted the importance of incorporating social factors in the study of drug addiction. Their research introduced an operant paradigm in which rats were given a choice between drug administration or social interaction with a same sex familiar rat. This study revealed that social reward prevented drug self-administration in both male and female rats.
Therefore, using a model of prolonged post-weaning social isolation during adolescence (start PND21) could reveal possible negative effects of social isolation on the preference between social reward and drug self-administration. If social isolation in adolescence impacted the positive role of social reward in addiction, could environmental enrichment or resocialization reverse its negative effects? Given the rising prevalence of loneliness and social isolation reported among adolescents across the world, research using rat models could be pivotal in understanding the negative impacts social deprivation has on the rising number of MDD and GAD diagnoses. Furthermore, research on social isolation in animal models can help us understand possible benefits of social interaction in addiction treatment during adolescence.
Future Pharmacological Studies
Although behavioral paradigms are useful in determining differences in behavior between male and female rats employing a non-behavioral experiment could enhance our findings. While our research did not reveal any major sex differences in behavior, previous research has shown possible sex differences in rodents associated with prolonged social isolation in adolescence. This section will discuss two possible future studies first study focusing on immunohistochemistry with ΔFosB as an indicator of neuronal activation and second study using CRF 1 antagonist for depression.
After all of the behavioral tests in our research were performed, all of the animals were sacrificed, and their brain tissue preserved so that molecular staining could be performed. Using ΔFosB, a transcription factor that indicates neuronal activation in response to chronic stress such as prolonged social isolation, we could determine the effects of prolonged social isolation on the basolateral amygdala. The BLA has been previously shown to act as a site of long-term fear memory storage (Gale, Anagnostaras, Godsil, Mitchell, & Fanselow, 2004), together with NPY expression plays a role in long-term resilience in male rats (Silveira Villarroel, Bompolaki, Mackay, Tapia, Michaelson, Leitermann, Marr, Urban, & Colmers, 2018) and it also contributes to the regulation of the HPA axis (Pagliaccio et al., 2014). As explained in the introduction chapter of this thesis, the HPA axis has been previously shown to be disrupted by chronic social isolation or social disturbances (Gunnar, 2007).
Targeting the amygdala, we hypothesize that ΔFosB, as an indicator of neuronal activation in response to chronic stress, would be upregulated particularly in BLA. Previously research showed a greater expression of ΔFosB in the BLA after social defeat in mice (Bourne, Mohan, Stone, Pham, Schultz, Meyerhoff, & Lumley, 2013). In addiction studies, ΔFosB has been shown to increase in nucleus accumbens shell and cingulate cortex (El Rawas, Klement, Salti, Fritz, Dechant, Saria, & Zernig, 2012). Previously, the effects of chronic stress in both male and female adolescent rats on the expression of ΔFosB in the amygdala as well as nucleus accumbens have not been researched. Therefore, using this molecular staining technique, we could offer new insights on the possible effects of chronic social isolation.
Based on previous research another possible future study could incorporate Corticotropin releasing factor 1 (CRF1) receptor antagonist as a rescue for depression symptoms in rodents. Expression of CRF1 receptor protein has previously been associated with an individual’s risk of developing MDD and contributes to the regulation of long-term responses to social and environmental stressors (Waters, Rivalan, Bangasser, Deussing, Ising, Wood, Holsboer, & Summers, 2015). Research by Bosch, Nair, Ahern, Neumann, and Young (2009) focused on determining the potential involvement of CRF system as an indicator of depression and anxiety in socially deprived prairie voles. Their research found that after just 4-day long social isolation, CRF mRNA elevated particularly in the bed nucleus of stria terminalis thus increasing the circulating corticosteroid in blood samples as well as increased adrenal gland. As discussed in the Introduction, disturbances in social relationships have been previously associated with the dysregulation of HPA axis by upregulation of the corticosteroids. Thus, examining the role of CRF1 in rats after chronic social isolation could help us understand possible sex differences observed in the HPA regulation (Bosch, Nair, Ahern, Neumann, & Young, 2009).
Previous research also focused on possible benefits associated with CRF1 receptor antagonists on the prevention of the behavioral effects of chronic adolescent stress. Using social defeat as a chronic stress model in male rats, Bourke, Glasper, and Neigh (2014) discovered that CRF1 receptor antagonist, GSK876008 partially rescued the negative behavioral effects of social defeat as rats’ anhedonia and anxiety were attenuated (Kehne, Hoffman, & Baron, 2005). Therefore, using the same CRF1 antagonist may be a viable treatment of anxiety and depressive behavior resulting from chronic social isolation in adolescence.
As a rising social epidemic, social isolation has been associated with the increased risk of obesity, dementia, insomnia, and cardiovascular disorders, as well as anxiety and depression. Adolescence is particularly vulnerable to the negative effects of social deprivation. As such, adolescent depression affects more than 5% of the teenage population and this number seems to exacerbate with age resulting in more than 21% of adults diagnosed with either MDD or GAD during their lifetime.
The present study focused on determining the effects of social isolation in male and female adolescent rats on anxiety, depression, and sociability in young adulthood. Overall, our study found that chronically isolated rats displayed decreased interest in social interaction, increased anxiety, decreased exploratory behavior, and decreased interest in rewards (anhedonia). No major sex differences were found to be associated with the effects of chronic social isolation.
Consequences of prison solitary confinement during adolescence, separation of young teenagers and children across borders, as well as the increase in popularity of technology lacking face-to-face contact can have serious effects on one’s life. Therefore, acknowledging the dire effects chronic social isolation has on one’s development, it is important to advance the field of scientific research on chronic social isolation. Only in response to new research discoveries can novel biomedical treatments be developed to reverse the destructive effects social isolation can have on one’s life. Furthermore, advancing our understanding of the perils of social isolation can help to change social policies and laws purposefully putting teenagers in social isolation increasing their risks of developing anxiety and depression in adulthood.
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