Homework answers / question archive / The effects of adolescent social isolation effect on anxiety-like behaviors and The TH expression the changes in Prefrontal Cortex Abstract: Recent studies shows that adolescence is a sensitive period in the human life

The effects of adolescent social isolation effect on anxiety-like behaviors and The TH expression the changes in Prefrontal Cortex Abstract: Recent studies shows that adolescence is a sensitive period in the human life

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The effects of adolescent social isolation effect on anxiety-like behaviors and The TH expression the changes in Prefrontal Cortex Abstract: Recent studies shows that adolescence is a sensitive period in the human life. In this period, children who transitions to adulthood experience physical and social development. It also includes behavioral changes that can easily be corrupted if a person experienced stressful situations. Such situations can be social isolation, where studies shows that adolescent social isolation can lead to anxiety-like behaviors in rats. Possible reason on why there’s changes in behaviors due to isolation is the changes in the dopaminergic system, in addition, dysregulation of the 5-Ht system. It is crucial to understand the mechanisms behind anxiety-like behaviors in adolescence to better understand the diseases and the disorders that regulated by anxiety and stress. Therefore, the goal of this paper is to discuss the effects of adolescent social isolation effect on anxiety-like behaviors and monoamine release changes in different brain regions in Sprague-Dawley Rats species using multiple methods such as elevated plus maze that is used to determine certain social behaviors of the rats. Introduction Individuals mature sexually and acquire the cognitive, mental, and social skills necessary to develop freedom from their parents when they transition from childhood to adulthood (Spear, 2000). Adolescence is a period of heightened experimentation, characterized by increased thrillseeking, risk-taking, and substance use (Steinberg, 2004; Lipari and Jean-Francois, 2013). This time of complex transformation is associated with the rise of psychological conditions such as anxiety, depression, eating disorders, and schizophrenia (Kessler et al., 2005). Though the exact reason for the emergence of such diseases during puberty is unknown, one theory indicates a developmental imbalance between striatal-driven stimulus seeking (risk-taking) and prefrontal suppression of impulsivity (behavioral control). This imbalance seems to result in an increased response to both positive and negative stimuli (Casey and Jones, 2010). It has been suggested that adolescence's increased vulnerability to stress may lead to the increased prevalence of psychiatric illness during this stage of life (Grant, 1997; Grant and Dawson, 1998; Turner and Lloyd, 2004). Specific disparities in stress response affect vulnerability (vs. resilience) to various psychological conditions in adults, including depression, anxiety, and drug use disorders (SUDs). Additionally, stress's symptoms are amplified during crucial developmental stages, such as labour and neonatal development (Bagot et al., 2014; Chen and Baram, 2016). Recent research indicates that puberty is another critical developmental stage during which traumatic events can cause longlasting, even irreversible, changes in brain structure and function (Burke et al., 2017). Thus, research into the neurobiological foundations of adolescence may lead to a fundamental understanding of normative social, emotional, reproductive, and cognitive development and an understanding of the health risks and disorders associated with this life stage. Adolescence and the development of social reward. Adolescence is the transitional stage between pubescence and the acquisition of reproductive capability. Although puberty and adolescence are often used interchangeably, there are two distinct developmental stages. Puberty is known as the acquisition of adult reproductive capability and is characterized by the activation of the hypothalamic-pituitary-gonadal axis. On the other hand, adolescence is the period preceding pubertal onset, which involves a longer window through which an organism prepares for separation from its parents. During this period, specific patterns emerge that aid in the change, including play activity, changed social experiences, and intensified experimentation (Figure 1A; Steinberg, 2004; Lipari and Jean-Francois, 2013). There is a qualitative change in the importance of social reward over this time frame (Spear, 2000). In humans, adolescence is described by a shift in the quantity and nature of social encounters with peers and relatives, including a rise in peer time (Larson et al., 1996). Rather than looking on their families for social reinforcement, teenagers increasingly rely on their peers and become increasingly vulnerable to how their peers treat those (Ladd et al., 2014). Social experiences during puberty seem to impact the production and persistence of maladaptive behaviors in adults (Patterson et al., 1992; Hankin et al., 1998). Indeed, unfavorable social factors are a good indicator of adolescent depression, and adolescent depression is associated with an increased chance of developing major depressive disorder over life (MDD; Thapar et al., 2012). It is natural to think about social system reorganization during puberty exclusively in terms of developing different psychological disorders (Kessler et al., 2005). However, it is critical to remember that social organisms must undergo restructuring during puberty to evolve behavioral mechanisms required for adult survival (Gopnik et al., 2017). Additionally, it is believed that the correlation of such reorganization with pubertal emergence reduces the risk of inbreeding within a social group by increasing exposure to genetically distinct individuals at a period when reproductive habits appear (Lawson Handley and Perrin, 2007). If perturbations occur during this critical developmental period, long-term behavioral effects can continue into adulthood, affecting an organism's social and sexual competency (Schulz et al., 2009; McCormick and Green, 2013; McCormick et al., 2013). Specific Aims: It does not need to put but if there is information useful for this research keep it The specific aim of this paper is to test for anxiety-like behaviors in rats during adolescence using methods such as Hyponeophagia, elevated plus maze, and success alleys apparatus. Chronic stress in adolescence can cause changes in neurotransmission and neural metabolites that eventually provide a molecular basis for the emergence of psychopathology [9]. Chronic stress triggers changes in the dopaminergic system where researchers show that there is a decrease in the basal dopamine and the expression of D2 receptors in the nucleus acumens, ventral tegmental area, and the substantia nigra (including the amygdala and hippocampus) that can cause depression and other depressive disorders [16]. In addition, chronic stress can cause long-lasting dysregulation of the 5-Ht system [10]. Individuals with trauma exposure appear to have a decrease in 5-HT1B receptor expression in the nucleus accumbency (ACC) and amygdala [11]. This shows that chronic stress experienced during adolescence can cause dysregulations in these systems which allow the emergence of stress-induced psychopathology that regulate mood disorders and substance use disorders (SUDs) [12]. In addition, evidence shows that chronic stress leads to aberrant connectivity in corticolimbic networks. However, although chronic stress causes all of these abnormalities, it is still unknown that social isolation can also induce these abnormalities during the adolescence period. To solve this problem, the effects of adolescent social isolation effect on anxiety-like behaviors and monoamine release changes in different brain regions in Sprague-Dawley Rats species are studied using multiple methods. Data from the elevated plus maze and successive alleys apparatus will be collected after splitting the rats into two groups: socially isolated after weaning and group-housed rats that lived with their mother before reaching adolescence. Rats' brains are collected to determine the monoamine release changes in different brain regions such as the amygdala and hippocampus. The rationale behind this method is the fact that Chronic stress triggers changes in the dopaminergic system where researchers show that there is a decrease in the basal dopamine and the expression of D2 receptors in the nucleus acumens, ventral tegmental area, and the substantia nigra (including the amygdala and hippocampus) that can cause depression and other depressive disorders [16]. In addition, chronic stress can cause long-lasting dysregulation of the 5-Ht system [10]. Individuals with trauma exposure appear to have a decrease in 5-HT1B receptor expression in the nucleus accumbens (ACC) and amygdala [11]. Thus, our hypothesis is that social isolation can cause dysregulation in the 5-HT system and the dopaminergic system. This paper will visualize the changes in monoamines and the mechanisms that socially isolated rats that experience anxiety-like behaviors. Thus having a better understanding of it. Aim 1 will examine the physical effects of socially isolated rats in an elevated plus-maze. After weaning, the rats will be isolated until adolescence and will be put in an elevated plus-maze to see if they prefer the open arms or the closed ones. Where if socially isolated rats preferred the closed arms then they have anxiety-like behaviors. Aim 2 will examine the effect of social isolation on anxiety-like behaviors. After collecting the brain samples and purify them, visualization on ImageJ will be done to determine the changes in monoamines to further understand why socially isolated rats behave in certain ways. This will contribute to identifying pathways and mechanisms that lead to anxiety-like behaviors. Background and Significance: ________Doctor__Comment :mention in this section on how the TH relates to the prefrontal cortex and what this relate to chronic stress and what this relate to the dopamine Stress can be identified as the body’s response to a specific situation where the body releases hormones and signals that can be expressed as a physical, emotional, or mental response. Stress can occur when a person faces problems or unexpected changes in his or her life [1,2]. According to the American Institute of Stress, researches and statistics show that 33% of U.S citizens experience extreme stress, 77% of the population experience physical symptoms because of stress, and 73% experience psychological symptoms that are caused by stress [3,4,]. In addition, women are more likely to face stress than men with a ratio of 28% to 20% [5]. Moreover, according to the data, most of the stress comes from tensions in jobs, health crises, and relationships. There are stress-related short and long terms that impact the brain and individual behavior. The release of Cortisol, the stress hormone, is the main event that happens during stress, which turns on the fight or flight system, increases blood pressure, and controls the sleep/wake cycle. Thus, constant stress can be damaging to the body, it can lead to health problems such as heart diseases and memory problems [6]. According to Yaribeygi and collegues (2017), stress can cause functional changes to the Central Nervous System (CNS) and can structural changes in different parts of the brain (Yaribeygi et al., 2017 [7]). Moreover, according to the article, stress can affect the process of storing memories by decreasing the number of neurons, synaptic terminals, and neurogenesis processes in the Hippocampus (McEwen, 1999 [8]). Chronic stress alters adolescent development. Stress can cause behavioral changes that include cognitive and mood disorders, also decreasing the individual reaction time. Apart from that, chronic stress alters adolescent development. According to Sheth, McGlade, Yurgelun-Todd (2017), chronic stress in adolescence can cause changes in neurotransmission and neural metabolites that eventually provide a molecular basis for the emergence of psychopathology [9]. Chronic stress triggers changes in the dopaminergic system where researchers show that there is a decrease in the basal dopamine and the expression of D2 receptors in nucleus accumens, ventral tegmental are and the substantia nigra (including the amygdala and hippocampus) that can cause depression and other depressive disorders [16]. In addition, chronic stress can cause long-lasting dysregulation of the 5-Ht system [10]. Individuals with trauma exposure appear to have a decrease in 5-HT1B receptor expression in the nucleus accumbens (ACC) and amygdala [11]. This shows that chronic stress experienced during adolescence can cause dysregulations in these systems which allow the emergence of stress-induced psychopathology that regulate mood disorders and substance use disorders (SUDs) [12]. In addition, evidence shows that chronic stress leads to aberrant connectivity in corticolimbic networks. According to Sheth, McGlade, Yurgelun-Todd (2017), stress alters the connectivities of the amygdala and hippocampus with the PFC [9,13]. It is shown that adolescents with a history of violence and trauma have weak resting-state functional connectivity between the PFC regions and the amygdala and hippocampus regions [14,15]. Thus, these alterations can cause emotional regulations and depression. Overall, studies prove that chronic stress alters adolescent development that can be mediated by the aberrant connectivity in corticolimbic networks. Therefore, in this paper, I will discuss the effects of adolescent Social Isolation Stress on Anxiety and monoamine release changes in different brain regions in SpragueDawley Rats species using multiple methods. Methods: (should include in this section the subject and bee behavioral analysis and the western plot ) Sprague-Dawley Rats were used to collect data on how social isolation affects anxiety and brain changes. 12 rat males were collected were 6 of them were socially isolated and the other 6 were group-paired. Rats arrived at post-natal day 14 (P14) with Dam and weaned at P12. At P61 the rats were killed. This is corresponding with late adolescence-early adulthood. Rats were housed in plastic cages with free access to food and water when not being tested. The colony room was maintained at a temperature of 22-23 degrees Celsius on a 12-hour light-dark cycle (lights on at 7:00 AM, lights off at 7:00 PM). The Institutional Animal Care and Use Committee require that appropriate environmental enrichment be provided as part of standard animal housing unless there is scientific justification, approved by the IACUC that precludes the use of environmental enrichment materials or practices [1]. Studies show that those male rats are exposed to changes in physical stressors during adolescence [2]. Rats were housed individually or with a partner. During this time, they were tested for anxiety-like behaviors on P22 in the elevated plus-maze, on P4754, male rats were tested by hyperphagia. On P55, male rats were tested in an elevated plus-maze, and on P56, male rats were tested by hyponeophagia. Lastly at P57, male rates were tested on successive alleys apparatus. Male rats then were put down and brains were harvested on P61. The brains were dissected and multiple regions of the brain were obtained (Prefrontal cortex, Amygdala, Hippocampus, and Dorsal Raphe Nucleus). Three methods were used to measure the anxiety and stress in the rat males. Hyponeophagia, is used to determine the relationship between anxieties and feeding behaviors, this is by measuring the latency and volume for consumption in rats' home cages and a novel environment [2]. Thus, rats in this experiment were tested for hyponeophagia at P47-54 and P56. First, a very small amount of food to the experimented group, then gradually increase the amount of food. This action is since rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome potential food poisoning, rats will first ingest only little amounts of food then determine if the food is toxic or safe to ingest. After the rat has determined that the substance is safe to eat, then poison is added to a large amount of food substance. This method is highly used to measure anxiety and the latency to consume a specific amount of the new substance in a novel environment is a measure [3]. Another method is, the elevated plus-maze. This method is used as an essay to text anxiety-related behavior in rodents. In addition, it defines the brain regions and mechanisms that are responsible to produce these anxiety-related behaviors. On P22 and P55, rats were placed at the junction of the four arms of the maze, facing an open arm, and the time that rats spent in each arm is video-tracked and recorded. Rats who stay a long time in the open arm reflects anti-anxiety behaviors [4]. Finally, the last method that was used is the successive alleys test. This method is the same as the elevated plusmaze however with eliminating the central area of the plus-maze. The male rats were put in successive alleys apparatus on P57 in enclosed alley 1. Moreover, the color of the alleys changes from black (alley1), to grey (alley2), to white (alleys 3 and 4). It was observed that rats prefer dark places and areas. In addition to that, unlike, the elevated maze, this apparatus’s walls slowly decline in height. Not only that but the width of the alleys slowly decreases that exposes the rats to elevated positions. This method is used to determine a wider range of anxiety-like behaviors [5]. Once the brain is dissected and regions Prefrontal cortex, Amygdala, Hippocampus, and Dorsal Raphe Nucleus were obtained. Western blots will be performed in order to separate proteins and molecules to visualize data on ImageJ software. The first step is the synaptosomal preparation. Since synaptosomes are used to study synaptic function since they contain functional ion-channels, receptors, enzymes, and proteins. Samples will be kept in a -80 freezer until ready for use. The soluble fraction of the brain homogenate will be mixed with 0.32M HEPES sucrose, in addition to 1:1 mixed with 1.3M HEPES sucrose after second centrifugation. The sample then will be suspended in cell lysis (0.2%TritonX-100 buffer). The pellet will be discarded and the supernatant is used for protein estimation [6]. Protein estimation is then will be used to get an estimate of the total protein content in the sample. This will be performed according to manufacturer specifications. DTT and SDS will be added to the samples, they will be placed in a water bath for 7 minutes. Place gel in the box, secure them and add dH2O and SDS running buffer. Samples will be loaded and sea blue ladder will be added also and gel electrophoresis will run for 120 minutes at 125 volts. When the run stops, the wells will be cut carefully, assembled, and added to IBlot for transfer. The gel will be handled using prewet filter paper and will be placed on the stack where the gel is touching the membrane. Iblot will run for 7 minutes, 1% BSA will be added to the western tray and will be left overnight at 4C. 1 antibody will be added the next day and placed back on the rocker for 24 hours at 4C. The primary antibody will be removed and saved. 2 antibody will be added and incubated on the rocker for 1 hour. Then, 2 antibody will be discarded to develop the blot next. The developing agent will be added to each blot, saran wrap will be prepared in a dark room, and chemiluminescence will be added to the first blot and incubated for 1 minute, repeat for the second blot. The blots will be wrapped with saran wrap and tapped into the book. A film will be put into the developer and the pan will be shaken until desired darkness. The film will be washed with water a couple of times and hanged to dry [7]. Lastly, Data will be visualized using imageJ software. There are of-course some weaknesses and limitations to our methods. The experimental group that we chose is small compared to other experimental groups in another study. This can cause our results to be a non-reliable source and have a lot of errors. Thus, a bigger group can make our result significant and have less error percentage. According to Ieraci (2016), further molecular and pharmacological approaches need to be done in order to see how specific genes are regulated in isolated rats that produce stress and anxiety behaviors [10]. In addition, different variables could have made our result more significant for example using different time intervals, using drugs to determine how rats change their behaviors accordingly, or also use different species and genders. Results Put the graph from excel Figure 1: Socially Isolated and group housed rats tested for anxiety-like behaviors on elevated plus maze apparatus on P55. Rats (N=12) were either socially isolated (N=6) on P22 or group-housed (N=6). The rats were tested for anxiety-like behaviors using an elevated plusmaze apparatus and measured the time that the rats spent in either open arms or closed arms. According to Figure 1, results from this study demonstrates that socially isolated rats have a higher chance of having anxiety-like behaviors than group-housed rats after isolating them. Data shows that socially isolated rats spent less time in open arms while the group-housed rats spent longer times in the open arms. However, there is no significance to the test. Add the result I made it with the doctor (the two sample t-test was use to analysis the effect of housing and Isolated conditions on TH prefrontal cortex There is no difference between two condition levels on pair housing and the isolated on TH level, t( 10)=.75,P=.47 ( see Figure) Discussion: (Restate the goal . What did you find ? How does it relate to previous literature (why (important implication+/- future idea)limitation ?(why didn’t find what we expected ?)related to literature ) The goal of this paper is to demonstrate the effects of adolescent Social Isolation Stress on Anxiety and monoamine release changes in different brain regions in Sprague-Dawley Rats species using multiple methods. According to the data that was acquired, the isolated rate group spent significantly more time in closed arms of the elevated plus-maze than rats that were pairhoused. According to Schneider and her colleagues (2011), studies shows that rats spent a significant time in closed arms rather than in open arm [8]. However, these studies showed how rats react in different doses of midazolam and in naive rats. The rats were not isolated. Thus, I would argue that the comparison of both data is inconclusive. However, according to Butler and her colleagues (2014), who wrote about the social impact of social isolation on anxiety-like behaviors, studies show that socially isolated rats have significantly greater anxiety-like behavior than group-housed rates. The isolated rats spent more time in the closed arms, while the grouphoused rats spent more time in open arm time, exploration, and entries [9]. Although the results that we found show the exact excepted results, there are of-course some weaknesses and limitations to our methods. The experimental group that we chose is small compared to other experimental groups in another study. This can cause our results to be a nonreliable source and have a lot of errors. Thus, a bigger group can make our result significant and have less error percentage. According to Ieraci (2016), further molecular and pharmacological approaches need to be done in order to see how specific genes are regulated in isolated rats that produce stress and anxiety behaviors [10]. In addition, different variables could have made our result more significant for example using different time intervals, using drugs to determine how rats change their behaviors accordingly, or also use different species and genders. References 1- Stress and your health: MedlinePlus Medical Encyclopedia. (n.d.). Retrieved January 09, 2021, from https://medlineplus.gov/ency/article/003211.htm 2- Bhargava, H. (2020, August 14). Stress - Why It Happens and Common Causes. Retrieved January 06, 2021, from https://www.webmd.com/balance/stress-management/what-is-stress 3- Patterson, E. (2020, November 07). Stress Statistics. Retrieved January 06, 2021, from https://www.therecoveryvillage.com/mental-health/stress/related/stress-statistics/ 4- The American Institute of Stress. (2019, December 18). Retrieved January 06, 2021, from https://www.stress.org/daily-life/ 5- Gender and Stress. (n.d.). Retrieved January 06, 2021, from https://www.apa.org/news/press/releases/stress/2010/gender-stress 6- Cortisol: What It Does & How To Regulate Cortisol Levels. (2020, December 13). Retrieved January 06, 2021, from https://www.webmd.com/a-to-z-guides/what-is-cortisol 7- Yaribeygi, H., Panahi, Y., Sahraei, H., Johnston, T. P., & Sahebkar, A. (2017). The impact of stress on body function: A review. EXCLI journal, 16, 1057–1072. https://doi.org/10.17179/excli2017-480 8- McEwen BS, Weiss JM, Schwartz LS. Selective retention of corticosterone by limbic structures in rat brain. Nature. 1968;220(5170):911–912. 9- Sheth, C., McGlade, E., & Yurgelun-Todd, D. (2017). Chronic stress in adolescents and its neurobiological and psychopathological consequences: an RDoC perspective. Chronic Stress, 1, 2470547017715645. 10- Novik, AM, Forster, GL, Tejani-Butt, SM, Watt, MJ. Adolescent social defeat alters markers of adult dopaminergic function. Brain Res Bull. 2011; 86(1–2): : 123–128. 11- Murrough, JW, Czermak, C, Henry, S. The effect of early trauma exposure on serotonin type 1B receptor expression revealed by reduced selective radioligand binding. Arch Gen Psychiatry. 2011; 68(9): 892–900. 12- Markou, A, Kosten, TR, Koob, GF. Neurobiological similarities in depression and drug dependence: a self-medication hypothesis. Neuropsychopharmacology. 1998; 18(3): 135–174. 13- Barbas, H, Saha, S, Rempel-Clower, N, Ghashghaei, T. Serial pathways from primate prefrontal cortex to autonomic areas may influence emotional expression. BMC Neurosci. 2003; 4: : 25 14- Nooner, KB, Mennes, M, Brown, S. Relationship of trauma symptoms to amygdala-based functional brain changes in adolescents. J Trauma Stress. 2013; 26(6): 784–787. 15- Burghy, CA, Stodola, DE, Ruttle, PL. Developmental pathways to amygdala-prefrontal function and internalizing symptoms in adolescence. Nat Neurosci. 2012; 15(12): 1736–1741. 16- Bhatia, A., Lenchner, J. R., & Saadabadi, A. (2020). Biochemistry, Dopamine Receptors. StatPearls [Internet]. 1- (n.d.). Retrieved January 09, 2021, from https://research.uci.edu/compliance/animalcareuse/research-policies-and-guidance/environmental-enrichment.html 2-Hypophagia. (n.d.). Retrieved January 09, 2021, from https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceuticalscience/hypophagia 3-Deacon R. M. (2011). Hyponeophagia: a measure of anxiety in the mouse. Journal of visualized experiments : JoVE, (51), 2613. https://doi.org/10.3791/2613 4-Walf, A., Frye, C. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc 2, 322–328 (2007). https://doi.org/10.1038/nprot.2007.44 5- Deacon R. M. (2013). The successive alleys test of anxiety in mice and rats. Journal of visualized experiments : JoVE, (76), 2705. https://doi.org/10.3791/2705 6-Kamat, P., Kalani, A., & Tyagi, N. (2014, August 12). Method and validation of synaptosomal preparation for isolation of synaptic membrane proteins from rat brain. Retrieved January 09, 2021, from https://www.sciencedirect.com/science/article/pii/S2215016114200276 7- NEUR310/480: Western blotting Guide 8- Schneider, P., Ho, Y. J., Spanagel, R., & Pawlak, C. R. (2011). A novel elevated plus-maze procedure to avoid the one-trial tolerance problem. Frontiers in Behavioral Neuroscience, 5, 43. 9- Butler, T. R., Ariwodola, O., & Weiner, J. (2014). The impact of social isolation on HPA axis function, anxiety-like behaviors, and ethanol drinking. Frontiers in integrative neuroscience, 7, 102. The effects of adolescent social isolation effect on anxiety-like behaviors and monoamine release changes in different brain regions. Introduction Experimental Timeline Results Individuals mature sexually and acquire the cognitive, mental, and social skills necessary to develop freedom from their parents when they transition from childhood to adulthood (Spear, 2000). Though the exact reason for the emergence of such diseases during puberty is unknown, one theory indicates a developmental imbalance between striatal-driven stimulus seeking (risktaking) and prefrontal suppression of impulsivity (behavioral control). This imbalance seems to result in an increased response to both positive and negative stimuli (Casey & Jones, 2010). It has been suggested that adolescence's increased vulnerability to stress may lead to the increased prevalence of psychiatric illness during this stage of life (Turner & Lloyd, 2004). Specific disparities in stress response affect vulnerability (vs resilience) to various psychological conditions in adults, including depression, anxiety, and drug use disorders (SUDs). Male rats isolated from P21 to P43, but not from P21–P28 or P42–P63, demonstrated a greater preference for EtOH and amphetamine (AMPH), altered neuronal activity in the ventral tegmental area (VTA), increased sensitivity of VTA neurons to corticotrophin-releasing factor (CRF), and altered glutamatergic signaling in the VTA. They then demonstrated that socialization from P43 to P63 had little impact on the behavior of VTA neurons. However, conduct after resocialization was not investigated (Whitaker et al., 2013). These findings are significant because they may aid in standardizing the adolescent social isolation procedure in the field and determining sensitive windows of resistance to social stress in rodents. Once the brain is dissected, and regions Prefrontal cortex, Amygdala, Hippocampus, and Dorsal Raphe Nucleus were obtained. The first step is the synaptosomal preparation. Since synaptosomes are used to study synaptic function, they contain functional ion channels, receptors, enzymes, and proteins. Samples will be kept in a -80 freezer until ready for use. The soluble fraction of the brain homogenate will be mixed with 0.32M HEPES sucrose, in addition to 1:1 mixed with 1.3M HEPES sucrose after second centrifugation. The sample then will be suspended in cell lysis (0.2%TritonX-100 buffer).One antibody will be added the next day and placed back on the rocker for 24 hours at 4C.According to Ieraci (2016), further molecular and pharmacological approaches need to be done to see how specific genes regulate isolated rats that produce stress and anxiety behaviors (Tarazi, Tomasini, & Baldessarini, 1998). In addition, different variables could have made our result more significant, for example, using different time intervals, using drugs to determine how rats change their behaviors accordingly or also use different species and genders. Therefore, the goal of this paper is to discuss the effects of adolescent social isolation effect on anxiety-like behaviors and monoamine release changes in the Prefrontal cortex in Sprague-Dawley Rats species using multiple methods such as elevated plus maze that is used to determine certain social behaviors of the rats. Methods Sprague-Dawley Rats were used to collect data on how social isolation affects anxiety and brain changes. 12 rat males were collected were 6 of them were socially isolated and the other 6 were group-paired. Rats arrived at post-natal day (P14) with Dam and weaned at P12. At P61 the rats were killed. This is corresponding with late adolescence-early adulthood. Rats were housed in plastic cages with free access to food and water when not being tested. The colony room was maintained at a temperature of 22-23 degrees Celsius on a 12-hour light-dark cycle (lights on at 7:00 AM, lights off at 7:00 PM). The Institutional Animal Care and Use Committee require that appropriate environmental enrichment be provided as part of standard animal housing unless there is scientific justification, approved by the IACUC that precludes the use of environmental enrichment materials or practices. Studies show that those male rats are exposed to changes in physical stressors during adolescence. Results Rats (N=12) were either socially isolated (N=6) on P22 or group-housed (N=6). The rats were tested for anxiety-like behaviors using an elevated plus-maze apparatus and measured the time that the rats spent in either open arms or closed arms. Results from this study demonstrates that socially isolated rats have a higher chance of having anxiety-like behaviors than group-housed rats after isolating them. Data shows that socially isolated rats spent less time in open arms while the grouphoused rats spent longer times in the open arms. However, there is no significance to the test. Rats were housed individually or with a partner. During this time, they were tested for anxiety-like behaviours on P22 in the elevated plus-maze; on P47-54, male rats were tested by hyperphagia. On P55, male rats were tested in an elevated plus-maze, and on P56, male rats were tested by hyperphagia. Lastly, at P57, male rates were tested on successive alleys apparatus. Male rats then were put down, and brains were harvested on P61. The brains were dissected, and multiple regions of the brain were obtained (Prefrontal cortex, Amygdala, Hippocampus, and Dorsal Raphe Nucleus). Three methods were used to measure the anxiety and stress in the rat males. Hyponeophagia is used to determine the relationship between anxieties and feeding behaviours by measuring the latency and volume for consumption in rats' home cages and a novel environment (Adriani & Laviola, 2004). This method is used to determine a broader range of anxiety-like behaviours (Grossman et al., 2003). Discussion This paper aims to demonstrate the effects of adolescent Social Isolation Stress on Anxiety and monoamine release changes in different brain regions in Sprague-Dawley Rats species using multiple methods. According to the acquired data, the isolated rate group spent significantly more time in closed arms of the elevated plus-maze than rats that were pairhoused. According to Schneider and her colleagues (2011), studies shows that rats spent a significant time in closed arms rather than in open arm (arazi, Tomasini, & Baldessarini, 1998). However, these studies showed how rats react in different doses of midazolam and naive rats. The rats were not isolated. Thus, I would argue that the comparison of both data is inconclusive. However, according to Butler and her colleagues (2014), who wrote about the social impact of social isolation on anxiety-like behaviors, studies show that socially isolated rats have significantly greater anxiety-like behavior than group-housed rates. The isolated rats spent more time in the closed arms, while the grouphoused rats spent more time open arm time, exploration, and entries (Moll et al., 2014).The experimental group we chose is small compared to other experimental groups in another study. References Moll, G., Mehnert, C., Wicker, M., Bock, N., Rothenberger, A., & Ruther, E. e. (2014). Age-associated changes in the densities of presynaptic monoaminetransporters in different regions of the rat brain from early juvenile lifeto late adulthood. . Brain Res Dev Brain Res. arazi, F., Tomasini, E., & Baldessarini, R. (1998). Postnatal developmentof dopamine D4-like receptors in rat forebrain regions: comparison withD2-like receptors. . Brain Res Dev Brain Res, 110: 227–33. Tarazi, F., Tomasini, E., & Baldessarini, R. (1998). Postnatal development ofdopamine and serotonin transporters in rat caudate–putamen and nucleusaccumbens septi. Neurosci Lett , 254: 21–4. AA1 Sample ID 1 2 3 4 5 6 7 8 9 10 11 12 Location relative to ladder 12 11 10 9 7 8 6 5 4 3 2 1 AA2 Sample ID 1 2 3 4 5 6 7 8 9 10 11 12 Location relative to ladder 1 2 5 6 7 8 12 11 10 9 4 3 AA3 Sample ID 1 2 3 4 5 6 7 8 9 10 11 12 Treatment/ Condition Pair-housed Pair-housed 160% Pair-housed Pair-housed 140% Pair-housed Pair-housed 120% Isolated 100% Isolated Isolated 80% Isolated Isolated 60% Isolated 40% Control blot average The Average TH/Actin (% control +SEM) Location relative to ladder 1 2 3 4 5 6 7 8 9 10 11 12 TH optical density 1 4626.832 26172.359 2171.154 3853.497 5055.397 3900.205 9294.004 10978.317 3839.154 3511.962 1044.841 2498.447 20% Treatment/ Condition 0% Pair-housed Pair-housed Pair-housed Pair-housed Pair-housed Pair-housed Isolated Isolated Isolated Isolated Isolated Isolated Control blot average TH optical density 8305.66 Pair house 10853.539 489.92The Condtion 1731.841 3263.447 2766.79 9722.539 10475.075 3352.497 2442.426 2186.255 4651.154 Treatment/ Condition Pair-housed Pair-housed Pair-housed Pair-housed Pair-housed Pair-housed Isolated Isolated Isolated Isolated Isolated Isolated Control blot average Control Group TH optical density 5617.569 20855.915 2098.962 3638.74 4383.983 3990.326 8916.903 8145.125 4302.033 3311.497 2823.912 5763.933 USE THI Actin % Actin % control 6998.61 66% 61% 6958.075 376% 347% 6900.418 31% 29% 7985.095 48% 44% 6468.832 78% 72% 7701.953 51% 47% 7926.953 117% 108% 7736.711 142% 131% 7094.225 54% 50% 8005.66 44% 40% 6907.711 15% 14% 9374.004 27% 25% 108% Actin 12984.41 11478.39 The12338.39 Condtion 13790.53 15429.95 15161.87 18635.21 22808.33 21431.84 22700.09 21620.55 20769.43 % Actin % control 64% 179% Isolate 95% house 265% 4% 11% 13% 35% 21% 59% 18% 51% 52% 146% 46% 128% 16% 44% 11% 30% 10% 28% 22% 63% 36% Actin % Actin % control 6507.075 86% 91% 6059.539 344% 364% 9468.61 22% 23% 10177.44 36% 38% 10937.56 40% 42% 10403.15 38% 41% 12501.46 71% 75% 11534.34 71% 75% 10164.32 42% 45% 10649.27 31% 33% 10091.56 28% 30% 8911.439 65% 68% 94% Combined Data Sample ID Housing Condition 1 Pair-housed 2 Pair-housed 3 Pair-housed 4 Pair-housed 5 Pair-housed 6 Pair-housed Sample ID 7 8 9 10 11 12 Housing Condition Isolated Isolated Isolated Isolated Isolated Isolated For the graph Pair-housed average Isolate-housed average USE THIS DATA HERE! AA1 (TH/Actin % Control) AA2 (TH/Actin % Control) AA3 (TH/Actin % Control) 61% 179% 91% 347% 265% 364% 29% 11% 23% 44% 35% 38% 72% 59% 42% 47% 51% 41% AA1 (TH/Actin % Control) AA2 (TH/Actin % Control) AA3 (TH/Actin % Control) 108% 146% 75% 131% 128% 75% 50% 44% 45% 40% 30% 33% 14% 28% 30% 25% 63% 68% Need this column for the error bars 100% Pair-housed SEM 63% Isolate-housed SEM 47% 16% Grand Average (TH/actin % control) 110% 325% 21% 39% 58% 46% Grand Average (TH/actin % control) 110% 111% 46% 34% 24% 52% t-Test: Two-Sample Assuming Equal Variances Mean Variance Observations Pooled Variance Hypothesized Mean Difference df t Stat P(T