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Repeated adolescent activity-based anorexia influences central estrogen signaling and adulthood anxiety-like behaviors in rats
Accepted Manuscript Repeated adolescent activity-based anorexia influences central estrogen signaling and adulthood depressive-like behaviors in rats
Tien-Jui Lee, Caroline S. Johnson, Kimberly P. Kinzig PII: DOI: Reference:
S0031-9384(16)30745-4 doi: 10.1016/j.physbeh.2016.12.039 PHB 11618
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
28 August 2016 28 November 2016 17 December 2016
Please cite this article as: Tien-Jui Lee, Caroline S. Johnson, Kimberly P. Kinzig , Repeated adolescent activity-based anorexia influences central estrogen signaling and adulthood depressive-like behaviors in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phb(2017), doi: 10.1016/ j.physbeh.2016.12.039
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ACCEPTED MANUSCRIPT Title Repeated adolescent activity-based anorexia influences central estrogen signaling and adulthood depressive-like behaviors in rats
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Author names and affiliations Tien-Jui Lee, Caroline S. Johnson1, and Kimberly P. Kinzig* Department of Psychological Sciences, Purdue University, 703 Third Street, West Lafayette, IN 47907, United States
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E-mail addresses: Tien-Jui Lee: [email protected] Caroline S. Johnson: [email protected] Kimberly P. Kinzig: [email protected]
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Present address Caroline S. Johnson Hedco Neuroscience Building, HNB 120 University of Southern California University Park, MC 2520 Los Angeles, CA 90089
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*Corresponding author: Kimberly P. Kinzig
ACCEPTED MANUSCRIPT 1. Introduction
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Anorexia nervosa (AN) is a highly treatment resistant eating disorder. It is marked by refusal to maintain one’s body weight at or above a minimally normal weight for age and height, intense fear of gaining weight or becoming fat despite being underweight, and disturbance in the way in which one’s body weight is experienced. Further, individuals experiencing AN commonly display hyperactivity, and postmenarcheal females often develop amenorrhea. Rates of recovery from AN are low; statistics demonstrate that only 50% will recover, while 20-30% remain chronically ill and > 10% will eventually die from the disorder [1, 2], thereby assigning AN the highest mortality rate among psychiatric disorders [3, 4].
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More than 90 percent of individuals with AN are female [5], and AN is typically diagnosed during adolescence. Onset of AN follows a bimodal distribution with peak occurrences at ages 14.5 and 18 years old [6]. This time coincides with the median age for the development of other psychiatric conditions; more individuals develop mood and anxiety disorders during adolescence than any other age [7, 8]. Many of these conditions are comorbid with AN [9-12] and persist beyond weight restoration and elimination of AN symptomology [12-18], often worsening after recovery [18-20]. It is likely that the adolescent onset of eating disorders plays a role in their enduring effects as regions of the CNS that mediate stress responsivity and mood regulation, including the hypothalamus, basal ganglia, amygdala, hippocampus, and medial prefrontal cortex, undergo significant development during this time. Many of these developmental changes depend on the pubertal surge of sex steroids, which leads to both activation of sex steroid receptors and changes in the structural organization of the brain [21].
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Because AN most frequently occurs in adolescent females, ovarian hormones have been suggested to play a role in its etiology [22]. Estrogens are anorexigenic [23], and ovariectomized (OVX) rats have increased food intake and body weight, compared to controls [23-25]. Given the suppressant effect of estradiol on food intake, it has been hypothesized that eating disorders result from a lack of typical neurological desensitization to estradiol during adolescence [26]. Indeed, women with Turner’s syndrome, who are sexually immature and have low levels of sex steroids, are more likely than women in the general population to develop AN immediately following estradiol treatment. This bout of disordered eating is transient and ceases rapidly upon treatment cessation [27]. Likewise, disordered eating scores obtained from the Minnesota Eating Disorder Behavior Survey (MEBS), were associated with higher levels of salivary estradiol [28]. However, individuals currently ill with AN have lower levels of 17-β estradiol [2932], estrone, and progesterone [29], the combination of which results in amenorrhea. Estrogen receptors (ER) and corticotropin-releasing hormone (CRH) regulate activity of the HPA axis [36], and the HPA axis is altered in AN and in response to activity-based anorexia (ABA) [34, 35]. Among the subtypes of estrogen receptors, both ERα and ERβ are involved in mediating anxiety. In rodent brains, ERβ is thought to be involved in anxiety-like behavior [36] and depression [37-39], whereas ERα is considered anxiogenic but mainly plays a role in reproductive function [40-42]. Although these two estrogen receptors are both widely expressed in several brain regions and often overlap with each other, only ERβ is expressed in PVN and the dorsal raphe nucleus in the midbrain [43-45]. ERβ is also expressed in other emotion-related and stress-sensitive brain regions, such as hippocampus, amygdala, and prefrontal cortex [46], suggesting the role of ERβ in mediating anxiety- and stress-related behaviors. Accordingly, ERβ knockout female mice show increased anxiety compared to wild-type littermates [47, 48]. Treatment with ERβ-selective agonists in OVX wild-type rats or mice is sufficient to reduce anxiety like behavior, but not in ERβ knockout mice [49-51]. Further, chronic unpredictable mild
ACCEPTED MANUSCRIPT stress significantly reduced ERβ expression in the prefrontal cortex, hippocampus and amygdala in male mice [52]. These studies imply an important anxiolytic role of ERβ.
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Because adolescent females are at the greatest risk for the development of AN, more research is required to develop a foundation for determining mechanisms underlying the relationship between AN and anxiety in order to foster development of novel treatment approaches and decrease AN relapse. The activity based anorexia (ABA) model has been used to model AN for decades [53], as it allows for evaluation and elucidation of neurobiological and behavioral factors involved in the onset, maintenance, and recovery from AN-like behaviors. ABA is considered an isomorphic model of AN due to its ability to induce both voluntary suppression of food intake and increased physical activity [54, 55]. In the ABA paradigm, rats are allowed timerestricted access to food paired with unlimited access to a running wheel. Without the running wheel, time-restricted access to food results in adaptation to the feeding schedule, however the addition of the running wheel results in hyperactivity and voluntary suppression of food intake even when it is available [56].
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Our previous research assessed whether exposure to ABA during adolescence (defined as postnatal day 21-60 [57]) affected adult anxiety-like behavior and activity of the HPA axis in females. We found that ABA in adolescence had enduring effects on anxiety-like behavior and stress responsivity; ABA in adolescent females consistently induces a highly anxious phenotype in adult female rats, which is relevant not only to AN, but to affective disorders that more frequently affect females [35]. In our current studies, we further characterized the short- and long-term consequences of ABA in adolescent females. We first evaluated whether two exposures to ABA in adolescence were required to produce long-term increased anxiety-like behavior, and then evaluated the long-term consequences of ABA in adolescence on depressive-like behavior. Subsequently, because AN and ABA often disrupt ovarian hormones [56, 58, 59], we determined whether ovarian hormones modulate the development of ABA, and the long-term behavioral consequences of ovariectomy prior to ABA exposure. Finally, we hypothesized that ERβ expression levels would be altered by ABA, as it has recently been shown to be involved in mediating anxiety-like behavior, and compounds that agonize this receptor are currently under active investigation for the treatment of anxiety [49-51].
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2. Materials and Methods
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2.1 Animals and housing Female Long Evan rats were purchased from Envigo (Indianapolis, IN) and arrived on postnatal day 25 (P25). Rats were housed individually in plexiglass cages and maintained at a constant temperature (20.5 ± 1°C) on a 12:12 h light/dark cycle, with the dark cycle beginning at 4:00 PM. Individual stainless steel feeders were attached to plexiglass cages (26.7 x 48.3 x 20.3 cm) lined with Aspen bedding (Envigo, Indianapolis, IN). For groups of animals allowed access to a running wheel, cages of the same dimension/material as described above were fitted with attached running wheels (Lafayette Instruments, Lafayette, IN). Running wheel activity was computer monitored and recorded (Activity Wheel Monitor, Lafayette Instruments, Lafayette, IN) hourly for the duration of experiments. Water and rodent chow (Harlan Teklad 2018 18% protein diet, Envigo, Indianapolis, IN) was available to all rats ad libitum, except where noted. Food intake and body weights were recorded for all rats daily, throughout the experiments. Bedding was sifted each day to collect food spillage. All procedures were approved by the Purdue Animal Care and Use Committee.
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2.2a Mid- or late adolescent activity-based anorexia and anxiety-like behavior Adolescent female rats were assigned into two large subgroups (n=32 each subgroup) for either mid- or late adolescent ABA study. Within each study, rats were further divided into four groups (n=8 per group) on P30. Group 1 was allowed access to a running wheel and ad libitum access to chow for the duration of the experiment (wheel/ad lib). In the second group, each rat had a running wheel attached to its cage and access to food limited to one hour before dark cycle begins each day (ABA) for four days starting from either P38 (mid-adolescence) or P48 (late adolescence). Food was returned to rats at the beginning of dark cycle when the four-day ABA period finished and animals had ad libitum access, and running wheels were locked. The third group did not have access to a running wheel (sedentary) and was allowed ad libitum access to food (sed/ad lib). The final group did not have access to a running wheel and had restricted access to food on the days the ABA group was restricted (1 h/day, sed/restrict). After P52, all rats were placed individually in plexiglass cages and had ad libitum access to food and water for the duration of the experiment.
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2.2b Elevated plus maze Rats were tested for anxiety-like behavior on an elevated plus maze (EPM) on P90. The EPM consisted of two open arms (50 cm x 10 cm) and two enclosed arms (50 cm x 10 cm). On testing day each rat was removed from the home cage within 2 h prior to the onset of the dark cycle and placed individually in the center of the EPM for 5 minutes, during which time its behavior was video-recorded for later scoring by an observer blinded to the conditions of the experiment.
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2.3 Ovarian hormones and activity-based anorexia In this experiment, 23 days old rats were weight-matched and divided into two groups. Half of each group was bilaterally ovariectomized (OVX) on P25 and the other half was sham operated based on previously described procedures [60]. In brief, animals were anesthetized with 5% isoflurane induction and maintained on 2%. The ovaries were lifted and removed following clamp of uterine blood vessels. Sham animals had their ovaries exposed but not removed.
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Following recovery of weight lost due to surgery, rats within each surgical group were weightmatched and divided into 4 groups, as described above (section 2.2a). The same protocol was followed: Two bouts of ABA occurred for the ABA animals 4 days beginning at P37 and P49. All rats had ad libitum access to food following the second bout of ABA, and no access to running wheels. Rats were tested for anxiety-like behavior as described above (section 2.2b).
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2.4a Activity-based anorexia: adolescent females and ERβ. For this experiment, wheel/ad lib and ABA rats were used to determine whether ABA affected ERβ expression levels in the amygdala. These animals underwent the same two-bout ABA paradigm as described in Figure 1E. These animals were sacrificed and ERβ expression level in the amygdala was measured by quantitative real-time PCR (qPCR) one week after the second bout of ABA. 2.4b Quantitative real-time PCR. For gene expression assays, amygdala samples were collected by tissue punch followed by homogenized and extracted for RNA by TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. RNA was quantified by spectrophotometer at 260 nm absorbance. First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA) was used to synthesize cDNA from 2 μg total RNA. Real-time quantitative PCR (qPCR) was performed with Maxima SYBR Green/Fluorescein qPCR Master Mix (ThermoScientific,
ACCEPTED MANUSCRIPT Waltham, MA) on iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). The primer sequences are: esr2, forward, AAA GCC AAG AGA AAC GGT GGG CAT, reverse, GCC AAT CAT GTG CAC CAG TTC CT; gapdh, forward, ACAGCAACAGGGTGG TGGAC, reverse, TTTGAGGGTGCAGCGAACTT. Results were detected and analyzed with MyiQ software (Bio-Rad Laboratories, Hercules, CA). esr2 expression level was normalized to GAPDH by ΔΔCt method.
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2.5 Statistical Analysis For the effects of one bout of ABA during adolescence, total time spent in the open arm of the EPM was analyzed by one-way ANOVA. Data for experiment 2.3 were analyzed by two -way repeated measures ANOVA. For the effects of ABA on body weight, repeated measures ANOVA was performed with treatment (group assignment) as a between subjects factor and time as a within subjects factor. Significant interactions and main effects were further analyzed by post-hoc Tukey’s multiple comparisons tests. Similar analyses were used to evaluate food intake and running wheel activity. For all comparisons, α-level was set at p < 0.05. Expression of ERβ was evaluated by t-test. The level of significance was set at p < 0.05, and stat are represented as mean and standard error of the mean (SEM).
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3. Results
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3.1 Mid-or late adolescent activity-based anorexia and anxiety-like behavior In order to determine whether two bouts of ABA were necessary during adolescence to produce long-term effects on anxiety-like behavior [35], adolescent female rats experienced a single bout of ABA during either mid- (P38-42) or late adolescence (P48-52). Anxiety-like behavior was evaluated on the EPM at P90. As depicted in Figure 2, one bout of ABA in mid-adolescence (Figure 2A) or late adolescence (Figure 2B) did not result in differences in the amount of time spent in the open arms of the EPM.
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3.2 Ovarian hormones and activity-based anorexia Adolescent female rats were sham operated or ovariectomized at P25. Two bouts of ABA were implemented, as described above and shown in Figure 1D. As depicted in Figures 3A and 3B, ABA and sed/restrict rats lost weight during periods of restriction. During the days of restriction there was a significant interaction (F(49,280)= 6.997, p < 0.001), as well as a effect of time (F(7,280)= 11.05, p < 0.001) and treatment groups (F(7,40)= 12.61, p <0.001). Post-hoc analyses revealed that for sham rats (Figure 3A), change in body weight for the ABA rats was significantly lower than wheel/ad lib rats during ABA bouts (p < 0.05). Sham ABA change in body weight was also significantly lower than sham sed/ad lib rats on (p < 0.01). There were no differences in change in body weight between sham ABA and sham sed/restrict groups. Comparison of sham ABA to OVX ABA revealed similar changes in body weight over the course of the experiment, although on P52, sham ABA change in body weight was significantly different from OVX change in body weight (p < 0. 05). Food intake was reduced and running wheel activity was increased for ABA and Sed/restrict rats during both bouts of restriction. As depicted in Figure 4, there were no differences in intake during the two bouts of ABA between sham ABA and OVX ABA groups. There were, however, differences in running wheel activity based on surgical treatment. For running wheel activity during restricted access to food, there was a significant interaction (F(7, 70)= 2.262, p<0.05, as well as a main effects of time (F(7,70)=2.673, p < 0.05 and treatment group (F(1,10) = 6.532, p <0.05). Post-hoc analyses revealed that sham ABA rats ran significantly more than OVX ABA
ACCEPTED MANUSCRIPT rats for the first two days of the first bout of ABA (p < 0.05 for both days) and all four days of the second bout of ABA (p < 0.05 for all days), as shown in Figure 5C. While sham ABA rats ran more than OVX ABA, OVX ABA ran significantly greater distance than OVX wheel/ad lib rats (Figure 5B) for all four days of the first bout of ABA (p < 0.01 for all four days) and for day 3 and 4 of the second bout of ABA (p < 0.01).
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On P90, as in previous experiments, all rats were tested for anxiety-like behavior on the EPM. As shown in Figure 6, ABA during adolescence in sham-operated females spent significantly less time in the open arm of the EPM than controls (p < 0.05). Ovariectomy prior to ABA treatment in adolescence did not affect the long-term anxiety-like behavior observed in intact rats; OVX ABA rats spent significantly less time in the open arm of the EPM than did OVX controls (p < 0.05).
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3.3 Activity-based anorexia: adolescent females and ERβ For this experiment, intact wheel/ad lib and ABA rats were treated as described in Figure 1E. When rats were 60 days old, they were sacrificed and mRNA expression levels of ERβ in the amygdala were analyzed by qPCR. As depicted in Figure 7, two bouts of ABA during adolescence resulted in long-term differences in ERβ expression in the amygdala; levels were significantly lower than those in wheel/ad lib controls (p < 0.05).
4. Discussion
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AN and anxiety disorders are highly comorbid [11, 12, 61, 62], and more commonly present in adolescent females than in males or females at any other developmental stage [8]. Our previous research has demonstrated long-term effects of experiencing ABA in adolescence. When adolescent, but not adult, females experienced ABA, anxiety-like behavior and activity of the HPA axis was increased long after ABA exposure ended [35]. Here, we further characterize the long-term effects of exposure to ABA in adolescent females and demonstrate effects beyond anxiety and HPA activity.
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More than 20% of AN patients remain chronically ill or repeatedly undergo a recovery and illness cycle [1]. Our previous research using ABA to investigate short- and long-term consequences of AN in adolescence addressed the effects of ABA when females experienced it in mid- and late adolescence, times when human females are most likely to develop AN.
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Anorexia nervosa develops after repeated attempts of food restriction many females in mid- to late adolescence. We therefore exposed animals to two brief bouts of ABA to model this occurrence. One goal of the present research was to determine whether mid- or late adolescent ABA was sufficient to produce long-term behavioral effects. Identification of a specific developmental period of vulnerability would allow for future investigation of changes taking place in the period that may foster development of adverse consequences of adolescent AN. However, our data demonstrate that only one bout of ABA in either mid-or late-adolescence is insufficient for producing increased anxiety in adulthood, relative to controls. These time periods were chosen because the highest AN occurrence age is 14.5 and 18 [6]. This result shows that more than a single bout of ABA during adolescence is required for development of long-term consequences. During adolescence, the CNS undergoes important developmental growth and modification, including changes in regions involved in the mediation of stress reactivity and mood regulation. It is possible that the long-term effects on anxiety- and depressive-like behaviors are due to cumulative effects of multiple exposures to ABA, or other
ACCEPTED MANUSCRIPT stressful physiological or psychological conditions, and that animals are resilient to short-term effects.
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In order to investigate the role of ovarian hormones in ABA and their role in the long-term behavioral consequences, rats were ovariectomized prior to puberty and underwent two bouts of ABA in adolescence. Because the onset of AN is most commonly during adolescence and there is an increased prevalence of anxiety- and depressive disorders in females after puberty, whereas rates are similar to those in males prior to puberty, we hypothesized that removal of ovarian hormones would attenuate the long-term increased anxiety-like behavior. Based on the literature, it was hypothesized that OVX ABA and OVX wheel/ad lib rats would run less than their intact counterparts, and OVX ABA and OVX sed/restrict rats would lose less body weight during restriction than their intact counterparts. While there were no differences in change in body weight between OVX and intact groups (OVX ABA: sham ABA, OVX sed/restrict: sham sed/restrict, etc.), OVX rats ran significantly less than intact rats did, thus the severity of ABA differed between surgical treatment groups. Regardless, the lack of ovarian hormones resulted in increased anxiety-like behavior in adulthood, OVX ABA rats spent significantly less time in the open arm of the EPM than controls, suggesting increased anxiety in responses to adolescent ABA despite a lack of ovarian hormones. One possible explanation for these data is that decreased ovarian hormones due to ABA [56] or OVX plays a role in the long-term behavioral consequences. This is only a partial explanation, however. If a lack of or diminished ovarian hormones during adolescence was responsible for increased anxiety-like behavior in adulthood, results for all of the OVX groups would have been similar, regardless of treatment during adolescence. Future research will include hormone replacement during ABA to address how ovarian hormones may play a role in the long-term consequences of adolescent ABA.
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Several studies have shown elevated estrogen decreased both ERα and ERβ mRNA or protein levels in several brain regions, including hypothalamus and lateral, medial and basolateral amygdala [63-65]. On the other hand, OVX resulted in opposite outcomes. Due to the fact that estradiol has similar binding affinity to ERα and ERβ [66], and the antagonized effects on anxiety of these two receptors, it is hard to dissociate the anxiogenic effects from estradiol and estrogen receptor signalings. However, interestingly, a decrease in physical activity after OVX has been found in female animals [67], which is similar to what is found in present study. Considering the effects of OVX on increased ERβ expression and reduced physical activity, one may predict that the ABA development should be attenuated because of lacking estrogen. However, elevated food intake and body weight gain were also found in OVX females [68]. In this condition, the 1 h food access may become more restricted and a greater stressor for OVX rats than sham rats. From this point of view, OVX rats may have a greater chance to develop ABA. This is opposite to what is discussed above and can be one of the possibilities that we do not see significant differences between ABA and anxiety development between OVX and sham rats. Furthermore, due to the fact that 90% of AN occurs in females [5], estrogen signaling has been proposed to play a role in AN development [22]. Here, we provide evidence that the ERβ expression level is significantly reduced in amygdala one week after two bouts of ABA compared to wheel/ad lib group. ERβ is known to be involved in anxiety-like behavior [36] and depression [37-39, 49]. To our knowledge, there are currently no data suggesting that exercise or food restriction alone influences ERβ expression in the amygdala. Unfortunately, our experimental groups only included ABA and wheel/ad lib animals, so the possibility that food restriction during adolescence may have an effect on ERβ expression in the amygdala remains and requires attention in future research. Although previous studies showed that exercise
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increased estrogen level in the hippocampus [69] and food restriction reduced CRH in the ventral hippocampus [70], the relationship between these changes and future anxiety or depressive behaviors have not been examined. Our finding reveals a possibility that during adolescence, an important period of brain development, repeated exposure to ABA causes deficits in ERβ signaling and leads to enduring elevated anxiety and depression. This biochemical change also explains why those psychological disorders persist after the elimination of AN symptomology. However, the change in ERβ expression may only reveal part of the mechanism and more research is required to clarify the role of ERβ expression change in amygdala after ABA exposure.
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Collectively, we demonstrate that two bouts of ABA during adolescence are required to produce enduring behavioral changes in female rats, and expand on our previous research on the relationship between adolescent ABA and adult behavioral and neural responses, and identify a potential role for ERβ signaling in mediating long-term behavioral consequences of adolescent ABA. Because adolescence is a period of developmental vulnerability and marked by changes in the activity of the HPG axis, the ABA paradigm provides a tool to further identify neurobiological and hormonal factors mediating the relationships between AN-like behaviors and their short- and long-term effects on the CNS and behavior.
5. Acknowledgements
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Perspecitves. In closing, I (KPK) wish to acknowledge the contributions of Randall Sakai for the positive impact he made on many scientists, including mine. He was an incredibly supportive mentor who went out of his way to ensure his trainees, whether they were members of his lab or not, were seen and heard as they found their way in graduate school and on to labs where they would receive the best post-doctoral training, and eventually develop their independent careers.
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6. References
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The authors wish to acknowledge the contributions of Meredith Cobb, Sara L. Hargrave, and Mary Ann Honors to the data contained within this manuscript. Each contributed greatly to data collection and analyses for these experiments.
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Figure 1. Scheme of activity-based anorexia (ABA) designs. (A) Female Long Evans rats were assigned to one of the four groups and experienced one bout ABA for four days on P38. Elevated plus maze (EPM) was performed on P90 to evaluate their anxiety-like behaviors in adulthood. Rats were sacrificed after EPM. (B) Female rats in late adolescent ABA study only experienced one bout of four-day ABA on P48. (C) Rats experienced two bouts of ABA during adolescence, starting on P38 and P48. Porsolt forced swim test (FST) were performed to test depressive like behaviors on P82 to P84 followed by sacrifice. (D) Ovariectomy or sham operation (OP) were performed on P25, followed by two bouts of four-day ABA on P37 and P49 in order to test the role of ovarian hormones on adolescent ABA development and adulthood anxiety-like behaviors. (E) Rats were sacrificed one week after two bouts of ABA in order to determine whether ABA affected ERβ expression levels in the amygdala.
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Figure 2. Adulthood anxiety-like behaviors after one-time exposure of adolescent ABA. The percentage of time spent in open arms on the elevated plus maze from rats experienced a single, four-day bout of ABA paradigm during either (A) mid- or (B) late adolescence. Data are shown as mean ± SEM.
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Figure 3. Body weight change in OVX and sham rats during two-bout ABA. Both (A) sham and (B) OVX adolescent female rats experienced ABA during P37-41 and P49-53. Data are shown as mean ± SEM.
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Figure 4. Food intake in OVX and sham rats during two-bout ABA. Both (A) sham and (B) OVX adolescent female rats experienced ABA during P37-41 and P49-53. Data are shown as mean ± SEM.
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Figure 5. Wheel running distance in OVX and sham rats during two-bout ABA. Adolescent female rats experienced ABA during P37-41 and P49-53. Daily running distance was monitored in both (A) sham and (B) OVX rats. (C) Comparison between sham vs. OVX ABA groups. Data are shown as mean ± SEM. *p < 0.05
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Figure 6. Adulthood anxiety like behaviors in OVX and sham rats after two-bout ABA during adolescence. Adolescent female rats experienced ABA during P37-41 and P49-53. The percentage of time spent in the open arms on elevated plus maze of (A) Sham and (B) OVX rats at P90. Data are shown as mean ± SEM. *p < 0.05
Figure 7. ERβ expression level in amygdala. The expression level was measured by qPCR one week after two bouts of ABA. Data are shown as mean ± SEM. *p < 0.05
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Highlights ● Experiencing activity-based anorexia (ABA) twice in adolescent females increases anxiety- like behavior in adulthood.
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● Repeated bouts of the ABA paradigm during adolescence are required for long-term
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behavioral effects.
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● ABA-induced changes in CNS ERβ signaling may play a role in the long-term consequences of adolescent exposure to ABA.
Tien-Jui Lee, Caroline S. Johnson, Kimberly P. Kinzig PII: DOI: Reference:
S0031-9384(16)30745-4 doi: 10.1016/j.physbeh.2016.12.039 PHB 11618
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
28 August 2016 28 November 2016 17 December 2016
Please cite this article as: Tien-Jui Lee, Caroline S. Johnson, Kimberly P. Kinzig , Repeated adolescent activity-based anorexia influences central estrogen signaling and adulthood depressive-like behaviors in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phb(2017), doi: 10.1016/ j.physbeh.2016.12.039
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ACCEPTED MANUSCRIPT Title Repeated adolescent activity-based anorexia influences central estrogen signaling and adulthood depressive-like behaviors in rats
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Author names and affiliations Tien-Jui Lee, Caroline S. Johnson1, and Kimberly P. Kinzig* Department of Psychological Sciences, Purdue University, 703 Third Street, West Lafayette, IN 47907, United States
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E-mail addresses: Tien-Jui Lee: [email protected] Caroline S. Johnson: [email protected] Kimberly P. Kinzig: [email protected]
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Present address Caroline S. Johnson Hedco Neuroscience Building, HNB 120 University of Southern California University Park, MC 2520 Los Angeles, CA 90089
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*Corresponding author: Kimberly P. Kinzig
ACCEPTED MANUSCRIPT 1. Introduction
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Anorexia nervosa (AN) is a highly treatment resistant eating disorder. It is marked by refusal to maintain one’s body weight at or above a minimally normal weight for age and height, intense fear of gaining weight or becoming fat despite being underweight, and disturbance in the way in which one’s body weight is experienced. Further, individuals experiencing AN commonly display hyperactivity, and postmenarcheal females often develop amenorrhea. Rates of recovery from AN are low; statistics demonstrate that only 50% will recover, while 20-30% remain chronically ill and > 10% will eventually die from the disorder [1, 2], thereby assigning AN the highest mortality rate among psychiatric disorders [3, 4].
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More than 90 percent of individuals with AN are female [5], and AN is typically diagnosed during adolescence. Onset of AN follows a bimodal distribution with peak occurrences at ages 14.5 and 18 years old [6]. This time coincides with the median age for the development of other psychiatric conditions; more individuals develop mood and anxiety disorders during adolescence than any other age [7, 8]. Many of these conditions are comorbid with AN [9-12] and persist beyond weight restoration and elimination of AN symptomology [12-18], often worsening after recovery [18-20]. It is likely that the adolescent onset of eating disorders plays a role in their enduring effects as regions of the CNS that mediate stress responsivity and mood regulation, including the hypothalamus, basal ganglia, amygdala, hippocampus, and medial prefrontal cortex, undergo significant development during this time. Many of these developmental changes depend on the pubertal surge of sex steroids, which leads to both activation of sex steroid receptors and changes in the structural organization of the brain [21].
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Because AN most frequently occurs in adolescent females, ovarian hormones have been suggested to play a role in its etiology [22]. Estrogens are anorexigenic [23], and ovariectomized (OVX) rats have increased food intake and body weight, compared to controls [23-25]. Given the suppressant effect of estradiol on food intake, it has been hypothesized that eating disorders result from a lack of typical neurological desensitization to estradiol during adolescence [26]. Indeed, women with Turner’s syndrome, who are sexually immature and have low levels of sex steroids, are more likely than women in the general population to develop AN immediately following estradiol treatment. This bout of disordered eating is transient and ceases rapidly upon treatment cessation [27]. Likewise, disordered eating scores obtained from the Minnesota Eating Disorder Behavior Survey (MEBS), were associated with higher levels of salivary estradiol [28]. However, individuals currently ill with AN have lower levels of 17-β estradiol [2932], estrone, and progesterone [29], the combination of which results in amenorrhea. Estrogen receptors (ER) and corticotropin-releasing hormone (CRH) regulate activity of the HPA axis [36], and the HPA axis is altered in AN and in response to activity-based anorexia (ABA) [34, 35]. Among the subtypes of estrogen receptors, both ERα and ERβ are involved in mediating anxiety. In rodent brains, ERβ is thought to be involved in anxiety-like behavior [36] and depression [37-39], whereas ERα is considered anxiogenic but mainly plays a role in reproductive function [40-42]. Although these two estrogen receptors are both widely expressed in several brain regions and often overlap with each other, only ERβ is expressed in PVN and the dorsal raphe nucleus in the midbrain [43-45]. ERβ is also expressed in other emotion-related and stress-sensitive brain regions, such as hippocampus, amygdala, and prefrontal cortex [46], suggesting the role of ERβ in mediating anxiety- and stress-related behaviors. Accordingly, ERβ knockout female mice show increased anxiety compared to wild-type littermates [47, 48]. Treatment with ERβ-selective agonists in OVX wild-type rats or mice is sufficient to reduce anxiety like behavior, but not in ERβ knockout mice [49-51]. Further, chronic unpredictable mild
ACCEPTED MANUSCRIPT stress significantly reduced ERβ expression in the prefrontal cortex, hippocampus and amygdala in male mice [52]. These studies imply an important anxiolytic role of ERβ.
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Because adolescent females are at the greatest risk for the development of AN, more research is required to develop a foundation for determining mechanisms underlying the relationship between AN and anxiety in order to foster development of novel treatment approaches and decrease AN relapse. The activity based anorexia (ABA) model has been used to model AN for decades [53], as it allows for evaluation and elucidation of neurobiological and behavioral factors involved in the onset, maintenance, and recovery from AN-like behaviors. ABA is considered an isomorphic model of AN due to its ability to induce both voluntary suppression of food intake and increased physical activity [54, 55]. In the ABA paradigm, rats are allowed timerestricted access to food paired with unlimited access to a running wheel. Without the running wheel, time-restricted access to food results in adaptation to the feeding schedule, however the addition of the running wheel results in hyperactivity and voluntary suppression of food intake even when it is available [56].
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Our previous research assessed whether exposure to ABA during adolescence (defined as postnatal day 21-60 [57]) affected adult anxiety-like behavior and activity of the HPA axis in females. We found that ABA in adolescence had enduring effects on anxiety-like behavior and stress responsivity; ABA in adolescent females consistently induces a highly anxious phenotype in adult female rats, which is relevant not only to AN, but to affective disorders that more frequently affect females [35]. In our current studies, we further characterized the short- and long-term consequences of ABA in adolescent females. We first evaluated whether two exposures to ABA in adolescence were required to produce long-term increased anxiety-like behavior, and then evaluated the long-term consequences of ABA in adolescence on depressive-like behavior. Subsequently, because AN and ABA often disrupt ovarian hormones [56, 58, 59], we determined whether ovarian hormones modulate the development of ABA, and the long-term behavioral consequences of ovariectomy prior to ABA exposure. Finally, we hypothesized that ERβ expression levels would be altered by ABA, as it has recently been shown to be involved in mediating anxiety-like behavior, and compounds that agonize this receptor are currently under active investigation for the treatment of anxiety [49-51].
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2. Materials and Methods
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2.1 Animals and housing Female Long Evan rats were purchased from Envigo (Indianapolis, IN) and arrived on postnatal day 25 (P25). Rats were housed individually in plexiglass cages and maintained at a constant temperature (20.5 ± 1°C) on a 12:12 h light/dark cycle, with the dark cycle beginning at 4:00 PM. Individual stainless steel feeders were attached to plexiglass cages (26.7 x 48.3 x 20.3 cm) lined with Aspen bedding (Envigo, Indianapolis, IN). For groups of animals allowed access to a running wheel, cages of the same dimension/material as described above were fitted with attached running wheels (Lafayette Instruments, Lafayette, IN). Running wheel activity was computer monitored and recorded (Activity Wheel Monitor, Lafayette Instruments, Lafayette, IN) hourly for the duration of experiments. Water and rodent chow (Harlan Teklad 2018 18% protein diet, Envigo, Indianapolis, IN) was available to all rats ad libitum, except where noted. Food intake and body weights were recorded for all rats daily, throughout the experiments. Bedding was sifted each day to collect food spillage. All procedures were approved by the Purdue Animal Care and Use Committee.
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2.2a Mid- or late adolescent activity-based anorexia and anxiety-like behavior Adolescent female rats were assigned into two large subgroups (n=32 each subgroup) for either mid- or late adolescent ABA study. Within each study, rats were further divided into four groups (n=8 per group) on P30. Group 1 was allowed access to a running wheel and ad libitum access to chow for the duration of the experiment (wheel/ad lib). In the second group, each rat had a running wheel attached to its cage and access to food limited to one hour before dark cycle begins each day (ABA) for four days starting from either P38 (mid-adolescence) or P48 (late adolescence). Food was returned to rats at the beginning of dark cycle when the four-day ABA period finished and animals had ad libitum access, and running wheels were locked. The third group did not have access to a running wheel (sedentary) and was allowed ad libitum access to food (sed/ad lib). The final group did not have access to a running wheel and had restricted access to food on the days the ABA group was restricted (1 h/day, sed/restrict). After P52, all rats were placed individually in plexiglass cages and had ad libitum access to food and water for the duration of the experiment.
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2.2b Elevated plus maze Rats were tested for anxiety-like behavior on an elevated plus maze (EPM) on P90. The EPM consisted of two open arms (50 cm x 10 cm) and two enclosed arms (50 cm x 10 cm). On testing day each rat was removed from the home cage within 2 h prior to the onset of the dark cycle and placed individually in the center of the EPM for 5 minutes, during which time its behavior was video-recorded for later scoring by an observer blinded to the conditions of the experiment.
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2.3 Ovarian hormones and activity-based anorexia In this experiment, 23 days old rats were weight-matched and divided into two groups. Half of each group was bilaterally ovariectomized (OVX) on P25 and the other half was sham operated based on previously described procedures [60]. In brief, animals were anesthetized with 5% isoflurane induction and maintained on 2%. The ovaries were lifted and removed following clamp of uterine blood vessels. Sham animals had their ovaries exposed but not removed.
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Following recovery of weight lost due to surgery, rats within each surgical group were weightmatched and divided into 4 groups, as described above (section 2.2a). The same protocol was followed: Two bouts of ABA occurred for the ABA animals 4 days beginning at P37 and P49. All rats had ad libitum access to food following the second bout of ABA, and no access to running wheels. Rats were tested for anxiety-like behavior as described above (section 2.2b).
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2.4a Activity-based anorexia: adolescent females and ERβ. For this experiment, wheel/ad lib and ABA rats were used to determine whether ABA affected ERβ expression levels in the amygdala. These animals underwent the same two-bout ABA paradigm as described in Figure 1E. These animals were sacrificed and ERβ expression level in the amygdala was measured by quantitative real-time PCR (qPCR) one week after the second bout of ABA. 2.4b Quantitative real-time PCR. For gene expression assays, amygdala samples were collected by tissue punch followed by homogenized and extracted for RNA by TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. RNA was quantified by spectrophotometer at 260 nm absorbance. First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA) was used to synthesize cDNA from 2 μg total RNA. Real-time quantitative PCR (qPCR) was performed with Maxima SYBR Green/Fluorescein qPCR Master Mix (ThermoScientific,
ACCEPTED MANUSCRIPT Waltham, MA) on iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). The primer sequences are: esr2, forward, AAA GCC AAG AGA AAC GGT GGG CAT, reverse, GCC AAT CAT GTG CAC CAG TTC CT; gapdh, forward, ACAGCAACAGGGTGG TGGAC, reverse, TTTGAGGGTGCAGCGAACTT. Results were detected and analyzed with MyiQ software (Bio-Rad Laboratories, Hercules, CA). esr2 expression level was normalized to GAPDH by ΔΔCt method.
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2.5 Statistical Analysis For the effects of one bout of ABA during adolescence, total time spent in the open arm of the EPM was analyzed by one-way ANOVA. Data for experiment 2.3 were analyzed by two -way repeated measures ANOVA. For the effects of ABA on body weight, repeated measures ANOVA was performed with treatment (group assignment) as a between subjects factor and time as a within subjects factor. Significant interactions and main effects were further analyzed by post-hoc Tukey’s multiple comparisons tests. Similar analyses were used to evaluate food intake and running wheel activity. For all comparisons, α-level was set at p < 0.05. Expression of ERβ was evaluated by t-test. The level of significance was set at p < 0.05, and stat are represented as mean and standard error of the mean (SEM).
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3. Results
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3.1 Mid-or late adolescent activity-based anorexia and anxiety-like behavior In order to determine whether two bouts of ABA were necessary during adolescence to produce long-term effects on anxiety-like behavior [35], adolescent female rats experienced a single bout of ABA during either mid- (P38-42) or late adolescence (P48-52). Anxiety-like behavior was evaluated on the EPM at P90. As depicted in Figure 2, one bout of ABA in mid-adolescence (Figure 2A) or late adolescence (Figure 2B) did not result in differences in the amount of time spent in the open arms of the EPM.
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3.2 Ovarian hormones and activity-based anorexia Adolescent female rats were sham operated or ovariectomized at P25. Two bouts of ABA were implemented, as described above and shown in Figure 1D. As depicted in Figures 3A and 3B, ABA and sed/restrict rats lost weight during periods of restriction. During the days of restriction there was a significant interaction (F(49,280)= 6.997, p < 0.001), as well as a effect of time (F(7,280)= 11.05, p < 0.001) and treatment groups (F(7,40)= 12.61, p <0.001). Post-hoc analyses revealed that for sham rats (Figure 3A), change in body weight for the ABA rats was significantly lower than wheel/ad lib rats during ABA bouts (p < 0.05). Sham ABA change in body weight was also significantly lower than sham sed/ad lib rats on (p < 0.01). There were no differences in change in body weight between sham ABA and sham sed/restrict groups. Comparison of sham ABA to OVX ABA revealed similar changes in body weight over the course of the experiment, although on P52, sham ABA change in body weight was significantly different from OVX change in body weight (p < 0. 05). Food intake was reduced and running wheel activity was increased for ABA and Sed/restrict rats during both bouts of restriction. As depicted in Figure 4, there were no differences in intake during the two bouts of ABA between sham ABA and OVX ABA groups. There were, however, differences in running wheel activity based on surgical treatment. For running wheel activity during restricted access to food, there was a significant interaction (F(7, 70)= 2.262, p<0.05, as well as a main effects of time (F(7,70)=2.673, p < 0.05 and treatment group (F(1,10) = 6.532, p <0.05). Post-hoc analyses revealed that sham ABA rats ran significantly more than OVX ABA
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On P90, as in previous experiments, all rats were tested for anxiety-like behavior on the EPM. As shown in Figure 6, ABA during adolescence in sham-operated females spent significantly less time in the open arm of the EPM than controls (p < 0.05). Ovariectomy prior to ABA treatment in adolescence did not affect the long-term anxiety-like behavior observed in intact rats; OVX ABA rats spent significantly less time in the open arm of the EPM than did OVX controls (p < 0.05).
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3.3 Activity-based anorexia: adolescent females and ERβ For this experiment, intact wheel/ad lib and ABA rats were treated as described in Figure 1E. When rats were 60 days old, they were sacrificed and mRNA expression levels of ERβ in the amygdala were analyzed by qPCR. As depicted in Figure 7, two bouts of ABA during adolescence resulted in long-term differences in ERβ expression in the amygdala; levels were significantly lower than those in wheel/ad lib controls (p < 0.05).
4. Discussion
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AN and anxiety disorders are highly comorbid [11, 12, 61, 62], and more commonly present in adolescent females than in males or females at any other developmental stage [8]. Our previous research has demonstrated long-term effects of experiencing ABA in adolescence. When adolescent, but not adult, females experienced ABA, anxiety-like behavior and activity of the HPA axis was increased long after ABA exposure ended [35]. Here, we further characterize the long-term effects of exposure to ABA in adolescent females and demonstrate effects beyond anxiety and HPA activity.
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More than 20% of AN patients remain chronically ill or repeatedly undergo a recovery and illness cycle [1]. Our previous research using ABA to investigate short- and long-term consequences of AN in adolescence addressed the effects of ABA when females experienced it in mid- and late adolescence, times when human females are most likely to develop AN.
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Anorexia nervosa develops after repeated attempts of food restriction many females in mid- to late adolescence. We therefore exposed animals to two brief bouts of ABA to model this occurrence. One goal of the present research was to determine whether mid- or late adolescent ABA was sufficient to produce long-term behavioral effects. Identification of a specific developmental period of vulnerability would allow for future investigation of changes taking place in the period that may foster development of adverse consequences of adolescent AN. However, our data demonstrate that only one bout of ABA in either mid-or late-adolescence is insufficient for producing increased anxiety in adulthood, relative to controls. These time periods were chosen because the highest AN occurrence age is 14.5 and 18 [6]. This result shows that more than a single bout of ABA during adolescence is required for development of long-term consequences. During adolescence, the CNS undergoes important developmental growth and modification, including changes in regions involved in the mediation of stress reactivity and mood regulation. It is possible that the long-term effects on anxiety- and depressive-like behaviors are due to cumulative effects of multiple exposures to ABA, or other
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In order to investigate the role of ovarian hormones in ABA and their role in the long-term behavioral consequences, rats were ovariectomized prior to puberty and underwent two bouts of ABA in adolescence. Because the onset of AN is most commonly during adolescence and there is an increased prevalence of anxiety- and depressive disorders in females after puberty, whereas rates are similar to those in males prior to puberty, we hypothesized that removal of ovarian hormones would attenuate the long-term increased anxiety-like behavior. Based on the literature, it was hypothesized that OVX ABA and OVX wheel/ad lib rats would run less than their intact counterparts, and OVX ABA and OVX sed/restrict rats would lose less body weight during restriction than their intact counterparts. While there were no differences in change in body weight between OVX and intact groups (OVX ABA: sham ABA, OVX sed/restrict: sham sed/restrict, etc.), OVX rats ran significantly less than intact rats did, thus the severity of ABA differed between surgical treatment groups. Regardless, the lack of ovarian hormones resulted in increased anxiety-like behavior in adulthood, OVX ABA rats spent significantly less time in the open arm of the EPM than controls, suggesting increased anxiety in responses to adolescent ABA despite a lack of ovarian hormones. One possible explanation for these data is that decreased ovarian hormones due to ABA [56] or OVX plays a role in the long-term behavioral consequences. This is only a partial explanation, however. If a lack of or diminished ovarian hormones during adolescence was responsible for increased anxiety-like behavior in adulthood, results for all of the OVX groups would have been similar, regardless of treatment during adolescence. Future research will include hormone replacement during ABA to address how ovarian hormones may play a role in the long-term consequences of adolescent ABA.
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Several studies have shown elevated estrogen decreased both ERα and ERβ mRNA or protein levels in several brain regions, including hypothalamus and lateral, medial and basolateral amygdala [63-65]. On the other hand, OVX resulted in opposite outcomes. Due to the fact that estradiol has similar binding affinity to ERα and ERβ [66], and the antagonized effects on anxiety of these two receptors, it is hard to dissociate the anxiogenic effects from estradiol and estrogen receptor signalings. However, interestingly, a decrease in physical activity after OVX has been found in female animals [67], which is similar to what is found in present study. Considering the effects of OVX on increased ERβ expression and reduced physical activity, one may predict that the ABA development should be attenuated because of lacking estrogen. However, elevated food intake and body weight gain were also found in OVX females [68]. In this condition, the 1 h food access may become more restricted and a greater stressor for OVX rats than sham rats. From this point of view, OVX rats may have a greater chance to develop ABA. This is opposite to what is discussed above and can be one of the possibilities that we do not see significant differences between ABA and anxiety development between OVX and sham rats. Furthermore, due to the fact that 90% of AN occurs in females [5], estrogen signaling has been proposed to play a role in AN development [22]. Here, we provide evidence that the ERβ expression level is significantly reduced in amygdala one week after two bouts of ABA compared to wheel/ad lib group. ERβ is known to be involved in anxiety-like behavior [36] and depression [37-39, 49]. To our knowledge, there are currently no data suggesting that exercise or food restriction alone influences ERβ expression in the amygdala. Unfortunately, our experimental groups only included ABA and wheel/ad lib animals, so the possibility that food restriction during adolescence may have an effect on ERβ expression in the amygdala remains and requires attention in future research. Although previous studies showed that exercise
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increased estrogen level in the hippocampus [69] and food restriction reduced CRH in the ventral hippocampus [70], the relationship between these changes and future anxiety or depressive behaviors have not been examined. Our finding reveals a possibility that during adolescence, an important period of brain development, repeated exposure to ABA causes deficits in ERβ signaling and leads to enduring elevated anxiety and depression. This biochemical change also explains why those psychological disorders persist after the elimination of AN symptomology. However, the change in ERβ expression may only reveal part of the mechanism and more research is required to clarify the role of ERβ expression change in amygdala after ABA exposure.
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Collectively, we demonstrate that two bouts of ABA during adolescence are required to produce enduring behavioral changes in female rats, and expand on our previous research on the relationship between adolescent ABA and adult behavioral and neural responses, and identify a potential role for ERβ signaling in mediating long-term behavioral consequences of adolescent ABA. Because adolescence is a period of developmental vulnerability and marked by changes in the activity of the HPG axis, the ABA paradigm provides a tool to further identify neurobiological and hormonal factors mediating the relationships between AN-like behaviors and their short- and long-term effects on the CNS and behavior.
5. Acknowledgements
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Perspecitves. In closing, I (KPK) wish to acknowledge the contributions of Randall Sakai for the positive impact he made on many scientists, including mine. He was an incredibly supportive mentor who went out of his way to ensure his trainees, whether they were members of his lab or not, were seen and heard as they found their way in graduate school and on to labs where they would receive the best post-doctoral training, and eventually develop their independent careers.
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6. References
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The authors wish to acknowledge the contributions of Meredith Cobb, Sara L. Hargrave, and Mary Ann Honors to the data contained within this manuscript. Each contributed greatly to data collection and analyses for these experiments.
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Figure 1. Scheme of activity-based anorexia (ABA) designs. (A) Female Long Evans rats were assigned to one of the four groups and experienced one bout ABA for four days on P38. Elevated plus maze (EPM) was performed on P90 to evaluate their anxiety-like behaviors in adulthood. Rats were sacrificed after EPM. (B) Female rats in late adolescent ABA study only experienced one bout of four-day ABA on P48. (C) Rats experienced two bouts of ABA during adolescence, starting on P38 and P48. Porsolt forced swim test (FST) were performed to test depressive like behaviors on P82 to P84 followed by sacrifice. (D) Ovariectomy or sham operation (OP) were performed on P25, followed by two bouts of four-day ABA on P37 and P49 in order to test the role of ovarian hormones on adolescent ABA development and adulthood anxiety-like behaviors. (E) Rats were sacrificed one week after two bouts of ABA in order to determine whether ABA affected ERβ expression levels in the amygdala.
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Figure 2. Adulthood anxiety-like behaviors after one-time exposure of adolescent ABA. The percentage of time spent in open arms on the elevated plus maze from rats experienced a single, four-day bout of ABA paradigm during either (A) mid- or (B) late adolescence. Data are shown as mean ± SEM.
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Figure 3. Body weight change in OVX and sham rats during two-bout ABA. Both (A) sham and (B) OVX adolescent female rats experienced ABA during P37-41 and P49-53. Data are shown as mean ± SEM.
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Figure 4. Food intake in OVX and sham rats during two-bout ABA. Both (A) sham and (B) OVX adolescent female rats experienced ABA during P37-41 and P49-53. Data are shown as mean ± SEM.
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Figure 5. Wheel running distance in OVX and sham rats during two-bout ABA. Adolescent female rats experienced ABA during P37-41 and P49-53. Daily running distance was monitored in both (A) sham and (B) OVX rats. (C) Comparison between sham vs. OVX ABA groups. Data are shown as mean ± SEM. *p < 0.05
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Figure 6. Adulthood anxiety like behaviors in OVX and sham rats after two-bout ABA during adolescence. Adolescent female rats experienced ABA during P37-41 and P49-53. The percentage of time spent in the open arms on elevated plus maze of (A) Sham and (B) OVX rats at P90. Data are shown as mean ± SEM. *p < 0.05
Figure 7. ERβ expression level in amygdala. The expression level was measured by qPCR one week after two bouts of ABA. Data are shown as mean ± SEM. *p < 0.05
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Highlights ● Experiencing activity-based anorexia (ABA) twice in adolescent females increases anxiety- like behavior in adulthood.
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● Repeated bouts of the ABA paradigm during adolescence are required for long-term
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● ABA-induced changes in CNS ERβ signaling may play a role in the long-term consequences of adolescent exposure to ABA.