The Receptor Basis of Estradiolâ•Žs

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2014 The Receptor Basis of Estradiol's Anorexigenic Effect Sean B. Ogden

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COLLEGE OF ARTS AND SCIENCES

THE RECEPTOR BASIS OF ESTRADIOL’S

ANOREXIGENIC EFFECT

SEAN B. OGDEN

A Thesis submitted to the Department of Psychology in partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded: Summer Semester, 2014

Sean B. Ogden defended his thesis on July 11, 2014. The members of the supervisory committee were:

Lisa A. Eckel Professor Directing Thesis

Diana L. Williams Committee Member

Thomas E. Joiner Committee Member

The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements.

ACKNOWLEDGMENTS

This work was supported by NIH grant DK073936 and an NIH predoctoral fellowship T32 MH093311.

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TABLE OF CONTENTS

List of Figures ...... v

List of Abbreviations ...... vi

Abstract ...... vii

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 3

RESULTS ...... 6

DISCUSSION ...... 8

FIGURES ...... 11

REFERENCES ...... 16

APPENDIX ...... 19

BIOGRAPHICAL SKETCH ...... 20

LIST OF FIGURES

Figure 1. Activation of ER, but not ER, is sufficient to decrease food intake in OVX rats ...... 11

Figure 2. Assessment of non-cumulative food intake following acute administration of PPT and DPN...... 12

Figure 3. DPN did not modulate PPT’s anorexigenic effect ...... 13

Figure 4. Estradiol benzoate’s (EB’s) anorexigenic effect is not attenuated by the ER antagonist PHTPP ...... 14

Figure 5. The ER agonist DPN produces an anxiolytic effect in the elevated plus maze at 24 h following treatment ...... 15

LIST OF ABBREVIATIONS

DPN Diarylpropionitrile EB Estradiol benzoate ER Estrogen receptor ER Estrogen receptor alpha ER Estrogen receptor beta OVX Ovariectomized PHTPP 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol PPT 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol TAM Tamoxifen citrate siRNA Small interfering RNA SERM Selective estrogen receptor modulator

ABSTRACT

It is well established that the ovarian hormone estradiol exerts its diverse actions primarily via the two classic estrogen receptors (ERs), ER and ER. While available data suggest that ER plays an important role (14, 15, 17, 21), the time course over which selective activation of ER inhibits food intake differs from that observed following similar administration of a non-selective ER agonist (estradiol benzoate) and thus requires further study. At present, the contribution of ER to the estrogenic control of food intake remains equivocal. Thus, the goals of the current research were to investigate the time course over which selective activation of ER decreases food intake in ovariectomized rats and whether ER is sufficient or necessary for estradiol’s anorexigenic effect. Experiment 1 revealed that acute administration of the ER agonist PPT produced a rapid, dose-dependent decrease in food intake that was detected within 2 h and persisted for up to 21-h. In contrast, acute administration of a range of doses of the ER agonist DPN failed to influence food intake. Experiment 2 revealed that DPN failed to modulate PPT’s anorexigenic effect. Experiment 3 revealed that the anorexigenic effect of estradiol benzoate (EB) was not attenuated by co-administration of the ER antagonist PHTPP. Experiment 4 revealed that the ERagonist DPN reliably decreased anxiety-like behavior in the elevated plus maze and that this action of DPN was attenuated by co-administration of the ER antagonist PHTPP. These positive findings confirm the involvement of ER in mediating estradiol’s anxiolytic effect and provide critical evidence that the same dose of these compounds that failed to alter food intake in our feeding tests effectively targeted (activated or blocked) ER. Taken together, these data demonstrate that PPT exerts a rapid anorexigenic effect that appears to occur with minimal or no delay. Because this time course appears to preclude the involvement of nuclear ERs, which function to alter gene transcription following a long (12-24 h) latency, PPTs rapid anorexigenic effect suggests the involvement of membrane ER signaling events. Additionally, our data provide compelling evidence that ER is neither sufficient nor necessary for estradiol’s anorexigenic effect. The null findings associated with DPN and PHTPP are not likely due to insufficient targeting of ER as both drugs altered anxiety-like behavior, suggesting that appropriate doses were used to probe ER’s involvement in the estrogenic control of food intake.

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INTRODUCTION

It is becoming increasingly clear that estradiol produces a myriad of behavioral and physiological effects that extend beyond its well-characterized role in regulating reproduction (1- 13). For example, studies in rodents have demonstrated that estradiol exerts a potent inhibitory effect on feeding that is mediated by a selective decrease in meal size (14, 15). This action of estradiol appears physiological since the lack of circulating estrogens in ovariectomized (OVX) rodents or postmenopausal women promotes sustained hyperphagia, weight gain, and increased adiposity (3, 16).

Our group and others have utilized a pharmacological approach to investigate the relative roles of the two classic estrogen receptors (ERs), ER and ER, in mediating estradiol’s anorexigenic effect. This work has shown that the ER agonist 4,4',4''-(4-Propyl-[1H]-pyrazole- 1,3,5-triyl)trisphenol (PPT) produces a behaviorally-specific, dose-dependent decrease in food intake (14, 17, 18), and the ER antagonist MpRP attenuates the anorexigenic effect of both endogenous and exogenous estradiol in female rats (15). An interesting, but unexpected, finding was that PPT’s inhibitory effect on feeding occurs more rapidly than the non-selective ER agonist estradiol benzoate (EB; i.e., 4-6 h vs. 12-24 h, respectively (14, 18). Because pools of nuclear ERcan translocate to the plasma membrane, where they produce more rapid signaling than that initiated via activation of nuclear ER (19), it is possible that PPT’s more rapid anorexigenic effect may involve activation of membrane-bound ER. An important first step in evaluating this hypothesis is to conduct a more detailed analysis of the time course of PPT’s anorexigenic effect since food intake was first measured at 4-6 h following PPT treatment in previous studies (14, 18).

While activation of ER appears to be both sufficient and necessary for estradiol’s anorexigenic effect, the role of ER is more equivocal. Support for the involvement of ER comes from a study in which blockade of ERsignaling, following central administration of ER-targeted antisense oligodeoxyribonucleotides, attenuated EB’s anorexigenic effect in OVX rats (20). In contrast to these findings, pharmacological studies have shown that food intake is not decreased by the ER agonist diarylpropionitrile (DPN), and EB’s anorexigenic effect is not attenuated in rats pretreated with the ER antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl)-

1 pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) (21). However, a potential caveat of any null finding in a pharmacological study is the lack of an appropriate positive control behavior associated with activation of the receptor in question. In this regard, the ER agonist DPN has been shown to decrease anxiety-like behavior in the elevated plus maze in both male and female rats (22-24). Additionally, the anxiolytic effect of DPN was not observed in ER null mice (25), suggesting that ERis both sufficient and necessary for estradiol’s anxiolytic effect. Notably, the dose of DPN used in these studies of anxiety-like behavior has not been used in previous studies probing the involvement of ER in mediating estradiol’s anorexigenic effect.

The current study was designed to extend previous investigations of the relative involvement of ER and ER in mediating estradiol’s anorexigenic effect. First, a series of feeding tests were conducted in OVX rats following single and combined administration of the ER agonist PPT and the ERagonist DPN. Food intake was assessed at multiple time points to identify the onset of each drug’s behavioral action, and across an extended dose range (lower than that used in previous studies) to identify the threshold anorexigenic dose of PPT and incorporate a dose of DPN that has been shown to decrease anxiety-like behavior in rats. Second, estradiol’s anorexigenic effect was assessed in OVX rats pretreated with an extended dose range of the ER antagonist PHTPP. In a final series of studies, we sought to replicate and extend previous reports of DPN’s anxiolytic effect in the EPM and confirm that the ER antagonist PHTPP, administered at a dose used in our feeding tests, attenuates estradiol’s anxiolytic effect in the EPM.

MATERIALS AND METHODS

Animals and housing

Female Long-Evans rats (Charles River Breeding Laboratory, Raleigh, NC; weighing 225-250 g at study onset) were housed individually in shoebox cages. Throughout the study, rats had free access to chow (Purina 5001, St. Louis, MO) and tap water unless otherwise specified. The colony room was maintained at 20 °C with a 12:12 h light/dark cycle (dark onset = 1300 h). Animal usage and all procedures were approved by the Florida State University Institutional Animal Care and Use Committee.

Surgery

Rats were anesthetized with intraperitoneal (i.p.) injections of a mixture of ketamine (50 mg/kg; Ketaset, Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (4.5 mg/kg; Rompun, Mobay, Shawnee, KS) and then bilaterally OVX using an intra-abdominal approach. Immediately following surgery, each rat received an intraperitoneal (i.p.) injection of butorphanol (0.5 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) to minimize post-surgical pain. Behavioral testing began after 7 days of postoperative recovery.

Experiment 1: Comparison of the acute effects of ER and ER agonists on food intake

At study onset, OVX rats were assigned to one of two weight-matched groups (PPT or DPN, n = 8 per group) and housed in custom-cages that facilitated the accurate assessment of food intake. Following adaptation to the custom cages (4-7 days), a series of feeding tests were conducted in rats following subcutaneous (s.c.) injections of the ER agonist PPT (Tocris, Ellisville, MO) or the ERagonist DPN (Tocris) in 0.1 ml dimethyl sulfoxide vehicle (DMSO, Sigma, St. Louis, MO). Doses of PPT (0, 10, 25, 50, and 75 g) and DPN (0, 10, 25, and 50 g) were chosen to extend the dose-response curves of previous studies (14, 15, 21), and incorporated a dose of DPN (and an analogous dose of PPT) that has been shown to decrease anxiety-like behavior in OVX rats (22, 23). On test days, rats received a single dose of PPT or DPN, administered in random order across each dose range, just prior to dark onset. Food cups were removed from the rats’ cages 3-h prior to PPT/DPN treatment and returned at dark onset.

Food intake was monitored at 2, 4, and 21-h following drug treatment. These time points were chosen to confirm and extend previous reports that PPT’s anorexigenic effect occurs within 4-6 h (8, 14, 15, 21). In this and subsequent feeding experiments, a spill paper was placed under each food cup to collect any food spillage at each time point, and feeding tests were conducted at 3-4 day intervals until rats in their respective groups had received each drug treatment.

Experiment 2: Determine whether the ER agonist DPN modulates the anorexigenic effect of the ER agonist PPT

OVX rats (n = 8) received paired s.c. injections (0.1 mL per injection, 0.2 mL total volume) of DMSO + DMSO, DMSO + 25 g PPT, 10 g DPN + 25 g PPT, 25 g DPN + 25 g PPT, and 50 g DPN + 25 g PPT on 4 separate test days spaced at least 4 days apart. Food cups were removed 3-h prior to dark onset and drugs were co-administered just prior to dark onset. Food cups were returned following drug treatment and food intake was monitored at 2, 4, and 21-h.

Experiment 3: Determine whether activation of ERis necessary for estradiol’s anorexigenic effect

OVX rats (n = 8) received paired s.c. injections (0.1 mL per injection, 0.2 mL total volume) of 10g of the ER antagonist PHTPP (Tocris) + sesame oil vehicle, 4 g of the non- selective ER agonist EB (-estradiol-3-benzoate, Sigma-Aldrich, Boston, MA) + oil, 10 g PHTPP + 4 g EB, and oil vehicle only on 4 separate test days spaced at least 4 days apart. The doses of EB and PHTPP were chosen based on previous studies performed in our lab and others (6, 14, 15). Drug injections were administered prior to dark onset and food intake was monitored for 48-h. Food intake was monitored for 48-h because this dose of EB has been shown to decrease food intake on the day following treatment (i.e., after a 24-h delay) (7, 26).

Experiment 4: Examine the effects of ER activation and blockade on anxiety-like behavior in the elevated plus maze

At study onset, OVX rats (n = 42) were assigned to one of the following drug treatment groups (n = 10-11 per group): DMSO + DMSO, DMSO + 10 g DPN, 10 g PHTPP + DMSO,

4 and 10 g PHTPP + 10 g DPN. As in the previous study by Walf and Frye (22), drugs were co-administered s.c. in a total volume of 0.2 mL and then anxiety-like behavior was assessed 48- h later in the elevated plus maze under dark conditions (22, 23). At the start of the trial, rats were placed in the center hub of the maze facing either of the open arms and allowed to explore freely for 5 min. Measurements of time spent in the open arm and open arm entries were used to assess anxiety-like behavior. Throughout the 5-min test, the animals’ movements were monitored via a camera mounted above the elevated plus maze and connected to a computer running tracking software (Noldus, Leesburg, VA). Upon completion of an individual trial, each animal was returned to their home cage and the apparatus was cleaned with disinfectant thoroughly between trials. After behavioral testing, a subset of recorded trials was manually scored to verify accuracy of the tracking software.

A second experiment was conducted to further assess the role of ER in mediating anxiety-like behavior at 24-h after drug treatment. This time point was chosen to model the time course of PPT’s anorexigenic effect and thus the time course over which we examined DPN’s ability to modulate food intake in Experiments 1-3. At study onset, a new cohort of OVX rats (n = 39) was assigned to one of the following 4 drug treatment groups (n = 7-12 per group): DMSO + DMSO, 10 g DPN + DMSO, 10 g PHTPP + 10 g DPN, DMSO + 10 mg/kg tamoxifen (tamoxifin citrate, Tocris) + 10 g DPN. In this experiment, tamoxifen was used as a non- specific ER antagonist, as it has equal binding affinity for ER and ER (27). Rats were then tested in the elevated plus maze, as described above, but at 24-h after drug treatment.

Data analysis

Repeated-measures ANOVAs were used to assess the effects of drug treatment on cumulative and non-cumulative food intake (Experiments 1-3). One-way ANOVAs were used to assess the effects of pharmacological activation and blockade of ERon anxiety-like behavior (time spent in the open arm and open arm entries) in the elevated plus maze (Experiment 4). Tukey’s post-hoc tests were used to investigate group differences following significant ANOVA effects (p < 0.05).

RESULTS

Experiment 1: Activation of ER, but not ER, produced a dose-dependent decrease in food intake

Cumulative food intake. Administration of the ER agonist PPT produced a dose- dependent decrease in cumulative, 21-h food intake, F(4,28) = 15.805, p < 0.001 (Fig 1a). Post- hoc tests revealed that 25 and 50 g PPT decreased food intake relative to vehicle (p’s < 0.05) and 75 g PPT decreased food intake relative to vehicle, 10 g, and 25 g PPT treatments (ps < 0.01). Similar administration of the ER agonist DPN failed to decrease 21-h food intake, F(3,21) = 0.214, p = 0.886 (Fig 1b).

Non-cumulative food intake. An anorexigenic effect of PPT was detected at each of the three time points examined, F(4,28) = 3.32 – 9.491, ps < 0.05 – 0.005 (Fig 2a-c). All doses of PPT produced about a 50% decrease in food intake at 2-h following treatment (ps < 0.0.1). While multiple doses of PPT continued to decrease food intake during the subsequent 2-h period (i.e., from 2-4 h post treatment), only the highest (75 g) dose of PPT decreased food intake during the final interval of the feeding test (i.e., from 4-21 h post treatment) (ps < 0.05). A similar, time- course analysis failed to reveal any anorexigenic effect of DPN, F(2,21) = 0.065-1.545. n.s. (Figs 2d-f).

Experiment 2: The ERagonist DPN failed to modulate the anorexigenic effect of the ER agonist PPT

Food intake was influenced by our combined regimen of PPT/DPN treatment, F(4,28) = 6.745, p < .001 (Fig 3). Post-hoc tests revealed an anorexigenic effect of 25 g PPT that was not modulated by co-administration of the range of DPN doses (ps < 0.01).

Experiment 3: Estradiol’s anorexigenic effect was not altered following blockade of ER

Treatment with the ERantagonist PHTPP did not attenuate the anorexigenic effect of EB, F(3,21) = 10.368, p < .001 (Fig 4). Post-hoc tests revealed that EB produced similar decreases in 21-h food intake in the presence and absence of PHTPP (ps < 0.05), and that PHTPP treatment alone had no effect on 21-h food intake.

Experiment 4: ER activation decreased anxiety-like behavior in the elevated plus maze

48-h Elevated Plus Maze Test As expected on the basis of previous studies (22, 23), acute administration of DPN increased the amount of time spent in the open arms, F(3,36) = 4.711, p < 0.007, and the number of open arm entries, F(3,36) = 3.126, p < .038, that were recorded in the elevated plus maze 48-h following drug treatment. This anxiolytic effect of DPN was not observed in rats that were co-treated with the ER antagonist PHTPP, and PHTPP treatment alone did not alter behavior in the elevated plus maze (data not shown).

24-h Elevated Plus Maze Test The effects of the ER agonists and antagonists were also examined in a second experiment that was conducted 24 h (rather than 48 h) after drug treatment (i.e., at a time in which selective activation of ER/ER was expected to affect feeding behavior). In this experiment, we also examined the ability of tamoxifen (a non-selective ER antagonist) to attenuate DPN’s anxiolytic effect. Both the amount of time spent in the open arms and the number of open arm entries were influenced by our regimen of drug treatment, F(3,35) = 4.142 and 5.153, respectively, ps < 0.01 (Fig. 5). Post-hoc tests revealed that DPN-treated animals spent more time in the open arms than vehicle-treated animals (ps < 0.05), and that this anxiolytic effect of DPN was blocked by tamoxifen, but not by PHTPP, co-treatment (Fig 5a). While DPN was also found to increase the number of open arm entries, relative to vehicle-treated animals (p < 0.05), this anxiolytic effect of DPN was blocked by tamoxifen and PHTPP treatment (Fig 5b).

DISCUSSION

The present results provide further evidence for the role of ER, and negate an important role for ER in mediating estradiol’s anorexigenic effect. The ER agonist PPT produced a potent decrease in food intake within 2 h of treatment that persisted for up to 21 h at the greatest dose tested. In contrast, doses of ER agonists and antagonists that influenced anxiety-like behavior in the elevated plus maze failed to have any effect on feeding, suggesting that activation of ER is neither sufficient nor necessary for the estrogenic control of food intake.

In the present study, we compared the effects of the ER agonist PPT and the ER agonist DPN on food intake in OVX rats. PPT, but not DPN, produced a dose-dependent decrease in food intake. This finding replicates and extends previous studies (15, 17, 21) by identifying the threshold anorexigenic dose of PPT (25 g), which turned out to be considerably lower than the doses of PPT used in previous feeding studies. Thus, in order to minimize behaviorally non-specific effects, future studies should utilize doses of PPT that approach this minimally effective dose. Our analysis of the time course of PPT’s anorexigenic effect further revealed that PPT robustly inhibits dark-phase feeding within 2 h of treatment. Because female rats typically consume only one spontaneous meal during the first 2 h of the dark phase (6), this finding is particularly important as it suggests that PPT, unlike EB, inhibits food intake with minimal or no latency. How PPT exerts this relatively rapid anorexigenic effect is poorly understood. A significant barrier is that the relative kinetics of PPT versus EB have yet to be elucidated at either the systemic pharmacokinetic or molecular level. Thus, it is unclear whether PPT may either cross the blood brain barrier quicker than EB or trigger the necessary intracellular events that mediate E2’s anorexigenic effect quicker. An intriguing possibility is that PPT may preferentially interact with membrane ER, rather than nuclear ER. In support of this hypothesis, membrane ERhas been localized to brain areas implicated in the estrogenic control of food intake (28) and central administration of estradiol-BSA, a membrane-delimited form of estradiol that does not cross plasma membranes (29), was sufficient to decrease 24-h food intake in OVX rats (30). While the latter finding supports the involvement of membrane- bound ERs in mediating estradiol’s anorexigenic effect, the specific contribution of membrane ER is unclear, as is the time course over which estradiol-BSA inhibits feeding. Thus, additional

8 studies are needed to investigate the possible involvement of membrane ER in the estrogenic control of food intake.

Consistent with previous reports (15, 17), the ER agonist DPN failed to decrease food intake in OVX rats and, when co-administered with PPT, failed to modulate PPT’s anorexigenic effect. Importantly, our current study involved an extended range of DPN doses, including one that previously had been shown to exert an anxiolytic effect in rats (22, 23). We also extended our analysis of feeding behavior beyond a 24-h measurement in order to test whether DPN, like PPT, may inhibit food intake with a minimal latency. Thus, our replication of DPN’s inability to suppress feeding in OVX rats under these test conditions provides compelling evidence that selective activation of ER is not sufficient to decrease feeding in female rats.

Available pharmacological data suggest that EB’s anorexigenic effect is not attenuated by co-administration of the ER antagonist PHTPP (21). Our current findings replicate and extend this previous report by providing validation that the dose and regimen of PHTPP treatment that failed to attenuate EB’s anorexigenic effect were sufficient to attenuate DPN’s anxiolytic effect in the elevated plus maze. While our current results provide strong evidence that ER is not necessary for estradiol’s anorexigenic effect, they are contrary to a previous study in which intracerebroventricular infusion of antisense oligodeoxyribonucleotides targeting ER abolished estradiol’s anorexigenic effect in OVX rats (20). At present, it is difficult to reconcile this sole report with the convergent evidence from pharmacological studies that ER, but not ER, is necessary for the estrogenic control of food intake (14, 15, 17, 18, 21). Thus, additional studies investigating the effects of ER-targeted antisense oligodeoxyribonucleotides, or other methods for silencing ER such as siRNA, are needed to validate the earlier work of Liang et al (20). Until such studies have been conducted, the vast majority of published studies, together with the current findings, provide strong evidence that selective activation of ER is neither sufficient nor necessary for estradiol’s anorexigenic effect.

Finally, as expected, the ER-specific drugs used in the current study produced clear effects on anxiety-like behavior in the elevated plus maze. Our demonstration that the ER agonist DPN increased open arm entries and time spent in the open arms replicates previous findings by Walf et al. (22, 23) examining anxiety-like behavior 48 h after DPN treatment. Our

9 additional demonstration that DPN exerted similar anxiolytic effects at 24 h extends this previous work by providing new insight into the duration of DPN’s anxiolytic effects and critical evidence that our dose and regimen of DPN treatment effectively targets ER at a time in which DPN would be expected to inhibit food intake if it were involved in the estrogenic control of food intake. Our data also address the efficacy of available ER antagonists. While PHTPP blocked the increase in open arm entries induced by DPN, it had a more modest effect in attenuating the DPN-dependent increase in open arm duration. Our experiments also utilized tamoxifen, a selective ER modulator (SERM) with high binding affinity for both ER and ER, and mixed tissue-specific ER agonist/antagonist properties (31). Tamoxifen, unlike PHTPP, fully blocked the anxiolytic effects of DPN (Fig. 5a-b). We suspect, however, that the increased efficacy observed with tamoxifen over PHTPP may be attributed to the single dosage of PHTPP used in our study. This dosage may not have been optimal to fully block DPN’s anxiolytic effect and may have contributed to this difference. Because ER antagonists can function more like SERMs (also having tissue-specific ER agonist effects, e.g. (31)), it will be important for those interested in pursuing ER-dependent behavioral effects to test additional doses of PHTPP to identify the optimal dose range over which it blocks estradiol’s anxiolytic effect.

In summary, we have provided evidence that further differentiates the relative roles of ER and ER in the estrogenic control of food intake. Taken together, our findings provide compelling evidence that ER plays a critical role in mediating estradiol’s anorexigenic effect, whereas ER is neither sufficient nor necessary. Our results also add additional support for the critical involvement of ER in mediating estradiol’s anxiolytic effect. The present study adds to the understanding of the individual actions of ER and ER by exploring lower dose ranges of agonists and discerning their time course of action. We have added to the growing body of literature advancing our understanding of the onset of both ER agonists’ behavioral actions and identifying a threshold anorexigenic dose of the ER agonist PPT. Finally, the time course of PPT’s inhibitory effect on feeding suggests the possible involvement of membrane ER-initiated signaling in the estrogenic control of food intake. This idea is particularly exciting as it challenges the long-standing notion that estradiol’s anorexigenic effect is mediated solely by nuclear ER-initiated changes in gene transcription.

Fig. 1. Activation of ER, but not ER, is sufficient to decrease food intake in OVX rats. (A) Acute administration of the ER agonist PPT produced a dose-dependent decrease in food intake. (B) Acute administration of the ER agonist DPN had no effect on food intake. *Less than vehicle, p < 0.05. **Less than vehicle, 10 g PPT and 25 g PPT, p < 0.05.

Fig 2. Assessment of non-cumulative food intake following acute administration of PPT and DPN. (A) All doses of the ER agonist PPT decreased food intake within 2 h of treatment. (B) Multiple doses of PPT decreased food intake from 2-4 h post treatment. (C) The highest dose of PPT decreased food intake from 4-21 h post treatment. (D-F) All doses of DPN failed to alter food intake at each of the three intervals. *Less than vehicle, p < 0.05. **Less than vehicle and 10 g PPT, p < 0.01.

Fig 3. DPN did not modulate PPT’s anorexigenic effect. Food intake was suppressed in PPT- treated animals, relative to vehicle-treated animals. This action of PPT was not modulated in animals receiving co-administration of a range of DPN doses. **Less than vehicle, p < 0.01.

Fig 4. Estradiol benzoate’s (EB’s) anorexigenic effect is not attenuated by the ER antagonist PHTPP. The non-selective ER agonist EB decreased food intake, relative to vehicle treatment. This anorexigenic action of EB was not attenuated by co-administration of PHTPP. Treatment with PHTPP alone did not alter food intake. *Less than veh/veh and PHTPP/veh, p < 0.05.

Fig 5. The ERagonist DPN produces an anxiolytic effect in the elevated plus maze at 24 h following treatment. (A) Animals administered DPN spent more time in the open arms relative to vehicle- and DPN/tamoxifen (TAM)-treated animals. (B) Animals administered DPN entered the open arm more times than all other treatment groups. *Greater than vehicle and DPN/TAM groups, p < 0.05. **Greater than all other groups, p < 0.05.

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APPENDIX

BIOGRAPHICAL SKETCH Sean Brian Ogden General Information Born: May 24, 1988

Education 2011 B.A. Major: Behavioral Neuroscience Purdue University Minors: Biological Sciences, English

2014 (exp) M.S. Major: Psychobiology Florida State University Faculty Advisor: Lisa A. Eckel, Ph.D. Thesis: The receptor basis of estradiol’s anorexigenic effect.

2011- Ph.D. Major: Neuroscience Florida State University Faculty Advisor: Lisa A. Eckel, Ph.D.

Research Experience 2013- Graduate Research Assistant, Florida State University Mentor: Thomas E. Joiner, Ph.D.

2011- Graduate Research Assistant, Florida State University Mentor: Lisa A. Eckel, Ph.D.

2009-2011 Undergraduate Research Assistant, Purdue University Mentor: Susan E. Swithers, Ph.D.

Research Grants

2013-2015 T32 Training Grant: Integrated clinical neuroscience training for translational research. Funded by the National Institute of Mental Health. 8/1/13 – 8/1/15.

Publications

Swithers, SE, Ogden, SB, Laboy, AF & Davidson, TL. (2012). Saccharin pre-exposure enhances appetitive flavor learning in pre-weanling rats. Developmental Psychobiology, 54:818-824.

Swithers, SE, Ogden, SB & Davidson, TL. (2011). Fat substitutes promote weight gain in rats consuming high-fat diets. Behavioral Neuroscience. 125(4), 512-518.

Ogden, SB, Jameson, B & Eckel, LA. (In prep). The impact of weight cycling-induced weight suppression on energy homeostasis in female rats. Manuscript in preparation for submission to International Journal of Obesity.

Ogden, SB, Loney, GC, Dunne, M, & Eckel, LA. (In prep). The receptor basis of estradiol’s anorexigenic effect. Manuscript in preparation for submission to Hormones and Behavior.

Presentations

Stathopoulos, AM, Ogden, SB, Basista, MJ, Ross, MT, Tabbaa, M, Korshunov, K, Terill, S, & Robsion, CL. (2014, November). Expand your mind! Educational outreach by Florida State University Neuroscience. Annual meeting of the Society for Neuroscience, Washington, DC.

Ogden, SB, Jameson, B & Eckel, LA. (2014, June). The impact of weight cycling on caloric intake and bingeing in female rats. Annual UF/FSU Joint Chemosensory Symposium, Gainesville, FL.

Ogden, SB, Jameson, B & Eckel, LA. (2014, April). The impact of weight cycling on caloric intake and bingeing. Florida State University Psychology Department Graduate Research Day, Tallahassee, FL.

Ogden, SB. (2013, November). Weight cycling: a potential contributor to dysregulated eating. Talk presented at the Florida State University Interdisciplinary Clinical Neuroscience Colloquium, Tallahassee, FL.

Robison, CL, Stathopoulos, AM, Ogden, SB & Lieberwirth, CL. (2013, November). Young (and old) minds want to know: educational outreach efforts by the Florida State University (FSU) neuroscience program. Annual meeting of the Society for Neuroscience, San Diego, CA.

Ogden, SB, Jameson, B & Eckel, LA. (2013, August). The impact of weight cycling-induced weight suppression on non-homeostatic feeding and weight gain in female rats. Annual meeting of the Society for the Study of Ingestive Behavior, New Orleans, LA.

Ogden, SB, Dunne, ME, Loney, GC, Santollo, J, & Eckel, LA. (2013, April). ERα is sufficient and necessary for the estrogenic control of food intake. Florida State University Psychology Department Graduate Research Day, Tallahassee, FL.

Ogden, SB & Eckel, LA. (2012, July). The estrogenic control of food intake: the relative involvement of ER and ER. Talk presented at the Florida State University Summer Seminar Series, Tallahassee, FL.

Ogden, SB & Swithers, SE. (2011, May). Sweet taste pre-exposure influences responding to flavor cues in pre-weanling rats. Annual meeting of the Midwestern Psychological Association, Chicago, IL.

Ogden, SB & Swithers, SE. (2011, April). Sweet taste pre-exposure influences responding to flavor cues in pre-weanling rats. Purdue Undergraduate Research Conference, West Lafayette, IN.

Swithers, SE, Laboy, AF, Ogden, SB & Davidson, TL. (2010, November). Effects of adolescent exposure to sweet tastes on energy balance in rats. Annual meeting of the International Society for Developmental Psychobiology, San Diego, CA.

Teaching

2012-13 Lab instructor, EXP 3202: Sensation and Perception

Service

2014 North Florida Brain Bee Coordinator Planned and coordinated the eighth annual North Florida Brain Bee, a local high school competition that tests knowledge of basic neuroscience with a chance at qualifying for the National Brain Bee.

2013- Neuroscience Graduate Student Advisory Committee Member Involved in the governing body responsible for coordinating Florida State Neuroscience program budgets, the planning of colloquia, and promoting faculty/student relations. Specifically, elected to the coordination of recruitment, planning, organizing, and budgeting of the outreach program.

2013- Florida State University Brain Fair Coordinator Planned and coordinated the annual Brain Fair, a local community event where families from the community learn about basic neuroscience and neuroanatomy through hands on demonstrations and activities.

2012- Florida State University Brain Fair Presenter Provided hands-on demonstrations and explanations of basic neuroscience to children and parents who attended the annual Brain Fair outreach event.

2011- Florida State University Neuroscience Outreach Instructor Visited high schools in Leon County during fall semesters to lecture on basic neuroscience and generate interest in the field. The visits provided practical hands-on demonstrations along with insight for students seeking to gain entry into a graduate level neuroscience program.

2011- North Florida Brain Bee Proctor Graded testing materials, guided participants to testing locations, and administered questions during the annual Brain Bee, a neuroscience competition for high school students.