Estradiol Exerts Activational and Organizational Control Over the Expression of Daily and Circadian Activity Rhythms in Mice

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ESTRADIOL EXERTS ACTIVATIONAL AND ORGANIZATIONAL CONTROL OVER THE EXPRESSION OF DAILY AND CIRCADIAN ACTIVITY RHYTHMS IN MICE

SARA E. ROYSTON

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience in the Graduate College of the University of Illinois at Urbana-Champaign, 2014

Urbana, Illinois

Doctoral Committee:

Assistant Professor Megan Mahoney, Chair Assistant Professor Sidonie Lavergne Associate Professor Lori Raetzman Associate Professor Robert Wickesberg

Abstract

Robust sex differences in daily and circadian activity rhythms, as well as the manifestation of circadian disruption-associated pathologies, have been reported in numerous species, including humans and laboratory models. These differences are mediated, at least in part, by sexually dichotomous estrogenic signaling throughout life.

However, the mechanism(s) underlying the impact of estrogens on the formation and expression of circadian activity outputs remains poorly characterized. A more comprehensive understanding of both the full effect(s) of estrogenic signaling on circadian rhythms throughout life and the precise method by which these effects are exacted will expand our understanding of the intersection of endocrinology and chronobiology, provide greater insight regarding disease pathogenesis, and provide novel targets for the development of efficacious therapies aimed at circadian disruption prevention and alleviation.

Accordingly, I have three primary goals for this thesis. First, I will fully characterize the effects of acutely circulating estradiol on the expression of daily and circadian wheel running rhythms in female mice. Simultaneously, I will further elucidate the estrogen receptor-dependent mechanism(s) underlying these functional effects

(Chapter 2). Second, I will examine the impact of developmental estradiol on adult daily and circadian activity rhythms during a neonatal critical period associated with maturation of the suprachiasmatic nucleus (SCN), a hypothalamic structure regarded as the ‘master oscillator’ (Chapter 3). Finally, because the adult timekeeping system is dependent upon transcriptional regulation in the SCN, I will determine the impact of estradiol on gene expression within the SCN (Chapter 4).

In the work presented here, I show that acutely circulating estradiol increases total wheel activity, consolidates this activity to the dark phase, and enhances wheel running amplitude through mechanism(s) dependent upon estrogen receptor subtype 1 (ESR1) in female mice. Additionally, estradiol more uniformly distributes wheel running activity across the dark phase compared to control treatment, delays the time of peak activity

(acrophase), reduces the phase angle of activity onset, and dampens the behavioral response elicited by a pulse of light administered in the early subjective night, though these effects are mediated by estrogen receptor subtype 2 (ESR2). Activation of either

ESR1 or ESR2 shortens the length of the endogenous day. I further discovered that developmental exposure to estradiol plays a distinct role in organizing the adult time keeping system. Lack of estradiol during a neonatal period associated with SCN maturation increases total wheel running in adult male mice, an effect rescued by exogenous estradiol administration during this critical period. Treatment with estradiol during this same period in female mice reduces the quantity and amplitude of adult wheel running, advances activity acrophase, and condenses the majority of activity completed during the dark phase to the early subjective night. Exogenous estrogen given during

SCN organization shortens the length of the active phase in adult male mice, and robustly effects responsiveness to light at varying times of day, though in a highly sex-dependent manner. Finally, activational estradiol strongly alters the transcriptional makeup within the SCN of female mice, primarily through message downregulation. Enriched molecular pathways in which estradiol decreased transcript expression of critical players include circadian rhythms, mitogen activated peptide kinase (MAPK) signaling, neurotrophin signaling, and long-term potentiation, suggesting that estradiol may be

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dampening plasticity within the SCN. These effects may contribute to the behavioral impact of estradiol outlined in Chapter 2. Together, these data significantly increase our understanding of the activational and organizational effects of estradiol on daily and circadian wheel running activity rhythms, and provide foundational targets for future work further elucidating the causative mechanism(s) underlying their expression.

ACKNOWLEDGMENTS

I am extremely grateful to my advisor, Dr. Megan Mahoney, for her incredible patience, kindness, scientific support, and for generally being a baller. None of this work would have been possible without her. More importantly, because of her mentorship my PhD was productive, fun, and has left me excited for future scientific endeavors. I would like to thank my thesis committee members, Dr. Lori Raetzman, Dr.

Robert Wickesberg, and Dr. Sidonie Lavergne, for their time, enthusiasm, and willingness to get excited about science with me. Thank you to the entire Neuroscience

Program at UIUC and the Medical Scholars Program at UICOM for their continued support, especially Dr. Sam Beshers, Dr. Neal Cohen, Dr. Nora Few, Dr. Jim Slauch, Dr.

Jim Hall, and Jenni Crum. The undergraduate research assistants in the Mahoney lab provided excellent animal care throughout all of my mouse work. In particular, I would like to thank Nadia Ayub, Max Crouse, Vicky Gugala, Tom Kondilis, Steve Lord, Arif

Molla, and Karolina Zapal. Dr. John Katzenellenbogen and Norio Yasui very kindly assisted with estradiol, DPN, and PPT dose calculation and helped me to make all of the pellets described throughout this thesis. Thank you to Dr. David Bunick for providing the ArKO mice used in Chapter 3, and to Dr. Matt Conrad and Trisha Gibbons for their assistance analyzing the TLDA Card data described in Chapter 4. I would like to thank

Dr. Leslie McNeil, Dr. Alex Parent, and Dr. Isaac Bourgeois for fruitful scientific discourse and Dr. Molly King for her eternal optimism. I am incredibly grateful to all of my friends for supporting me throughout my PhD. Finally, thank you Gilly, Olive, and

Dave. You are the best. The very very best.

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION TO CIRCADIAN RHYTHMS...... 1

CHAPTER 2: ESR1 AND ESR2 DIFFERENTIALLY REGULATE DAILY AND CIRCADIAN ACTIVITY RHYTHMS IN FEMALE MICE...... 38

CHAPTER 3: ESTRADIOL EXPOSURE EARLY IN LIFE PROGRAMS DAILY AND CIRCADIAN ACTIVITY RHYTHMS IN ADULT MICE...... 81

CHAPTER 4: CIRCULATING ESTRADIOL ALTERS THE EXPRESSION OF RHYTHMIC AND PLASTICITY-ASSOCIATED TRANSCRIPTIONAL NETWORKS IN THE SUPRACHIASMATIC NUCLEUS OF ADULT FEMALE MICE ...... 120

CHAPTER 5: SUMMARY ...... 171

CHAPTER 1: Introduction to circadian rhythms

Authors: Royston SE1,2

1Neuroscience Program, University of Illinois Urbana-Champaign 2Medical Scholars Program, University of Illinois College of Medicine

Introduction to Circadian Rhythms

Understanding Circadian Rhythms

From single cellular organisms to plants to complex mammalian species, the homeostatic temporal patterning of daily biological and behavioral processes is well documented (Roenneberg and Merrow, 2002). These highly conserved daily, or circadian, rhythms exist at subcellular, cellular, tissue, and organismal levels (O’Neill et al., 2013). Distinct biological oscillations, both in accordance with and in spite of environmental variation, are critical for survival. Indeed, disruption of daily rhythms has been associated with numerous pathologies in humans and mammalian models, including sleep disorders, cardiovascular complications, metabolic disease, immune system impairment, cancer, psychiatric disease, and drug/alcohol addiction (Turek et al., 2001;

Wirz-Justice, 2003; Rosenwasser, 2010; Takahashi et al., 2008; Reddy and O’Neil, 2010;

Karatsoreos, 2012; Bailey and Silver, 2013).

The term ‘circadian’ refers to a rhythm that oscillates around an approximately 24 hr day, the duration of which is controlled, at least in part, by a transcriptional- translational feedback loop (TTFL) expressed within the suprachiasmatic nucleus (SCN)

(O’Neill et al., 2013). Accordingly, this bilateral hypothalamic structure is regarded as the ‘master oscillator’. Simplistically, the TTFL that mediates the length of the innate day, or period, begins with the nuclear transcription of both Clock and Bone Muscle Aryl

Hydrocarbon Receptor Nuclear Translocator-Like (Bmal1) message (Figure 1.1). These transcripts are translated in the endoplasmic reticulum, and their protein products dimerize before re-entering the nucleus. Within the nucleus, CLOCK-BMAL1 binds to the genome through interaction with its E-box response element, promoting the

transcription of Period (Per) and Cryptochrome (Cry). Following protein synthesis, PER and CRY also dimerize prior to feeding back and directly inhibiting the transcription of

Clock and Bmal1 (Zhang and Kay, 2010). A continually evolving list of directly and peripherally associated regulatory players, both at the genomic and proteomic levels, precisely fine tune this ‘simple’ TTFL, thereby mediating the duration of the observed period of slightly less than 24 hr in rodents and slightly greater than 24 hr in humans (Jud et al., 2005; Refinetti, 2010; Zhang and Kay, 2010; O’Neill et al., 2013).

In addition to setting the innate oscillatory period, the 10-20,000 neurons within the SCN also integrate highly diverse neurochemical inputs, adapting downstream circadian outputs. Light is the strongest exogenous input to influence the functions of the

SCN (Figure 1.2) (Bailey and Silver 2013). Light activates non-image forming intrinsically photosensitive retinal ganglion cells (Leak et al., 1999; Berson et al., 2002;

Brown and Piggins, 2007), which then transduce photic cues onto the SCN via glutamatergic signaling along the retinohypothalamic tract (RHT). Additional retinal photic input projects onto the SCN via the geniculohypothalamic tract (GHT), which begins in the intergeniculate leaflet (IGL) (Morin et al., 2013).

The heterogeneous neuronal population within the SCN acts in a coordinated fashion through neural and diffusible signaling to exert control over the clock-controlled activities expressed by its efferent targets (Figure 1.2). These regulated activities are numerous and include gene expression, proteomic regulation, hormone release, feeding, and activity. The focus of the remainder of this document is rodent wheel running behavior. Accordingly, unless stated otherwise, all of the explanation of circadian rhythms that follow will be in reference to this behavior. Feedback mechanisms prevent

light- or dark-induced potentiation of clock-controlled outputs. For example, nocturnal rodents such as mice, rats, and hamsters complete more wheel revolutions in the dark compared to the light. If the absence of light and associated downstream signaling onto the SCN contributes to this cyclic rhythm, one could reason that it would be disadvantageous to become increasingly more active as the dark phase progressed.

Indeed, multiple negative feedback loops exist that balance outputs, inputs, and the SCN itself (Figure 1.2) (Bailey and Silver, 2013).

Importantly, the SCN is both required and sufficient for the expression of biological and behavioral circadian rhythms. Circadian oscillations in body temperature, endocrine function, and behavior are lost following surgical SCN ablation in rodents

(Welsh et al., 2010), but are fully restored to donor rhythmicity when fetal SCN grafts are implanted into lesioned hosts (Ralph et al., 1990; King et al., 2003). Interestingly, only

15-20% of the neurons in the SCN graft need to be functional for the functional restoration of rhythmicity. Oscillations in transcript levels of the 10-20% of genes whose expression is innately rhythmic (Reddy et al., 2006; Deery et al., 2009; Doherty and Kay,

2010; Reddy, 2013) continue for several days after dissociated cell culture preparation

(Welsh et al., 2004; Yoo et al., 2004), suggesting that individual SCN cells themselves play a large role in the expression of oscillations. Additionally, these individual SCN neurons are capable of acting in concert to coordinate peripheral clocks, a phenomenon lost following SCN ablation (Welsh et al., 2010; Tahara et al., 2012). These neurons also couple to synchronize circadian outputs, demonstrating that even though circadian rhythms are inherent to each cell, the overarching network control created by overlapping negative and positive feedback loops is greater than the sum of its parts.

Methodology for understanding daily and circadian activity patterns

To assess daily and circadian wheel running behaviors, rodents are singly housed with free access to a running wheel. Wheel rotations are recorded and plotted over time.

The resultant actogram is shown in Figure 1.3A. The white and black bars across the top indicate whether the lights were on (white) or off (black), while each line is representative of a different day. Frequently, actograms are drawn to show 2 consecutive days side by side, a format known as double-plotting (Jud et al., 2005). This style permits simple visualization of rhythmic trends. Total wheel rotations are binned and plotted chronologically over time; the height of the bar for a given bin is indicative of the magnitude of wheel running completed within that time. In addition to denoting the total quantity of wheel running activity exerted by a given animal, these data are useful for determining if the animal’s activity pattern is synchronized, or entrained, to the light:dark

(LD) environment in which it is housed. A nocturnal animal entrained to LD will become active around the time of lights off and will cease to be active when the lights turn back on as long as the artificial length of day is set within entrainable limits.

Further, these data can be used to assess when the animal becomes active relative to lights off, a measure known as the phase angle of activity onset (Jud et al., 2005; Bailey and Silver, 2013).

Time is arbitrary within an LD environment. To account for this, Zeitgeber

(Zeitgeber = “time giver”) time (ZT) is employed to standardize environmental cues to the clock. Within a 12:12 LD environment, ZT0 denotes the time at which the lights turn on, while ZT12 indicates lights off. Rodents, for example the C57Bl/6 mice used in all of

the subsequent chapters, when housed in constant conditions with no entraining cues free run and exhibit a day length or innate free running period (Tau; τ) that is slightly less than 24 hr (23.7 ± 0.1 hr) (Zheng et al., 2001; Jud et al., 2005). Accordingly, when they are placed in constant darkness (DD) and all Zeitgebers are removed, activity patterns emerge that adhere to this endogenous rhythm (Figure 1.3A). The length of the active phase (α) is also an innate property. Notably, τ and α are disrupted by SCN ablation

(Welsh et al., 2010), further reinforcing the hypothesis that circadian behavioral rhythms are governed by the SCN. Period is determined by measuring either peak to peak or trough to trough activity. Because there is typically only one definitive wheel running activity peak per subjective day, but potentially very many equal troughs, the peak to peak definition is most frequently employed (Figure 1.3B). Activity onset to activity onset is also a highly stable method for determining τ. Both τ and α should be quantified over several days after animals have acclimated to constant lighting conditions to avoid both adjustment effects and single day anomalies.

Within a given circadian rhythm there exists an activity peak and activity trough.

The Raw Amplitude describes the magnitude of the rhythm and is defined as the peak minus the trough (Figure 1.3B). To account the differences in the mean level of activity

(mesor) between individuals, the Adjusted Amplitude of the activity rhythm is defined as the peak minus the mesor (Refinetti, 2010). Irrelevant of its magnitude, the time at which the peak bin of wheel running activity occurs is known as the Acrophase (Figure 1.3B).

Acrophase in nocturnal rodents typically occurs in the early subjective night, while humans and diurnal animals exhibit peak activity during the light phase (Duffy et al.,

2011). Similar to τ and α, Amplitude and Acrophase should be calculated from several

consecutive days of behavioral output to most accurately describe an animal’s daily and circadian activity patterns.

To determine the ability of an animal to adjust its timing, or phase, of activity in response to a change in exogenous photic cue, a phase response experimental design is used. The results of such a paradigm are shown in Figure 1.3C. Animals entrained to an

LD environment are permitted to free run in total darkness for at least 24 hr but not long enough to permit recapitulation of the endogenous rhythm. They are then subjected to a pulse of light, which is indicated by a yellow star, at varying times of day. Following the light pulse, animals continue to free run in DD for at least 8 continuous days. The difference between activity onset in LD prior to light pulse administration and activity onset in DD following the pulse is used to determine the magnitude of the phase shift.

Activity onset is defined as the first bin of continuous wheel running whose magnitude is equal to or greater than at least 10% of the peak activity bin. At least 3 days of continuous LD data should be used to determine pre-pulse activity onset, while a regression line fit through the activity onset of at least 5 days of post-pulse DD data should be used. The first day after the animal is subjected to the light pulse should be disregarded to account for after effects (Jud et al., 2005).

A pulse administered at a time when the animal is accustomed to being in the light, for example ZT4 or ZT11, elicits little to no response (Figure 1.3C) (Bailey and

Silver, 2013). Periods of time normally associated with lights on that do not elicit a phase shift in activity are known as dead zones (Jud et al., 2005). Animals receiving a pulse in the early subjective night (ZT16) respond by delaying the start of activity onset the next day, a phenomenon known as phase delay. Conversely, a maximal phase

advance is elicited by a light pulse in the late subjective night (ZT22) (Jud et al., 2005;

Bailey and Silver, 2013). The ability to phase shift in response to environment stimuli is adaptive and demonstrates behavioral plasticity within the SCN, its inputs, and efferent targets. Similarly, introduction of an exogenous drug or behavioral intervention may alter the magnitude or timing of phase shifts compared to those observed in control animals. These effects may be due to treatment effect(s) upstream, downstream, or at the level of the SCN.

The paradigm used to measure phase shifts in response to alterations in environmental photic input described above is known as an Aschoff type II protocol.

Alternatively, an Aschoff type I design dictates that circadian time (CT) be calculated for each individual mouse based on their respective duration of free running rhythm. This is done by first defining the time of activity onset exhibited by a nocturnal animal that has been acclimated to DD free running conditions as CT12. This time is then added to the animal’s measured τ, and 24 hr is subtracted, yielding the formula: CT12(subsequent day) =

(CT12(measured day) + τ – 24 hr). Additionally, the length of each circadian hour for a given animal is defined as τ/24 hr (reviewed in Jud et al., 2005). Accordingly, the magnitude of each unit time can vary from animal to animal because rather than applying time as a unified constraint for all experimental animals, it is standardized to each subject’s free running rhythm. In order to use an Aschoff type I design to measure photic-induced phase shifts, two important variables should be considered. First, in order for this design to elicit consistent and reproducible data, it is critical that all subjects exhibit highly stable circadian activity patterns under constant light conditions. Second, all mice are subjected to the same stimuli uniformly, but the time at which the light pulse was

delivered is retroactively computed for each animal, resulting in pulse administration at varying CTs across subjects. Accordingly, all work describing phase shifting of activity onset in response to light for the remainder of this document was done using the Aschoff type II paradigm.

Sex differences with daily and circadian activity rhythms: a case for estrogen

There are robust sex differences in daily and circadian activity rhythms

(Karatsoreos, 2012; Mong et al., 2011; Bailey and Silver, 2013). Several of these differences were discovered through the exogenous manipulation of gonadal or ovarian hormone levels or availability, suggesting that sex hormones play a critical role in the regulation of circadian behaviors. Despite an increasingly vast body of work outlining the link between sex differences, sex hormones, circadian behavior, and the diseases associated with their disruption, less than 20% of published research at the intersection of chronobiology and endocrinology focuses specifically on females and/or ovarian signaling (Kuljis et al., 2013). Notably, the incidence of circadian disruption is increased during periods characterized by either acute or chronic shifts in estrogen signaling, including the perinatal period, pregnancy, menopause, and the reproductive cycle (Morin and Cummings, 1982; Baker et al., 2007; Mahoney, 2010; Shechter and Boivin, 2010), suggesting that estradiol plays a role in modifying clock controlled outputs (Figure 1.4).

This hypothesis has been repeatedly supported by experimental data. Maximum daily wheel running activity in intact female rats and hamsters is observed during proestrous when serum estradiol levels are at their peak (Wollnik and Turek, 1988), while removal of circulating estrogens via ovariectomy (OVX) reduces total wheel running

activity in female mice, rats, and degus (Albers, 1981; Labyak and Lee, 1995; Ogawa et al., 2003; Brockman et al., 2011). This effect is rescued by exogenous estradiol replacement (Rodier, 1971; Gentry and Wade, 1976). In female rats and hamsters, estradiol shortens τ (Morin et al., 1977a; Zucker et al., 1980; Albers, 1981) and advances the phase angle of activity onset (Morin et al., 1977b; Albers, 1981). Female OVX hamsters have greater phase angle variability compared to intact or estradiol-treated counterparts (Morin and Cumming, 1982). Additionally, asynchronous wheel running rhythms and splitting, a phenomenon observed in hamsters when constant LL leads to the emergence of two distinct daily activity rhythms, are reduced in OVX females following estradiol replacement (Morin and Cumming, 1982). Further, the quantity of wheel running activity completed by female nocturnal rodents during the light phase is increased following OVX, but made more nocturnal by subsequent estradiol replacement

(Ogawa et al., 2003; Brockman et al., 2011; Blattner and Mahoney, 2012). Together, these data suggest that estrogens modulate both the magnitude and timing of wheel running activity, as well as strengthen entrainment to LD transitions (Gorman and Lee,

2002).

Estrogens not only contribute to the expression of the circadian activity rhythms described above, but also to the ability to adjust to unanticipated environmental changes.

For example, intact female degus subjected to a 6 hr phase shift recover more slowly than their male counterparts (Goel and Lee, 1995), suggesting that this sex difference can be attributed, at least partially, to female sex hormones. Further, intact female estrogen receptor knock-out mice (αERKO), which lack estrogen subtype receptor 1 (ESR1; formerly ERα), but not estrogen subtype receptor 2 (ESR2; formerly ERβ) (Dupont et al.,

2000), exhibit a truncated phase delay when pulsed with light in the early subjective night, but a more robust phase advance when the pulse is administered in the late subjective night compared to intact WT female mice (Blattner and Mahoney, 2013).

Together, these data provide evidence that modified or impaired estrogen signaling alters the adaptive response to photic stimuli.

Our understanding of the effects of estradiol on daily and circadian activity rhythms, though insufficiently characterized, is more complete than our knowledge of the mechanism(s) by which these effects are enacted. As is possible with any exogenous stimuli capable of altering daily and circadian activity rhythms, estradiol may be acting by modulating afferent and efferent SCN projections. It may also act directly on the SCN itself (Figure 1.4). In the mature mouse brain, estrogen receptors are expressed in all three of the major inputs onto the SCN, including the RHT, the IGL, as well as the 5-HT- associated medial (MR) and dorsal raphe (DR) nuclei (Figure 1.5A). Input carried on the

RHT and GHT conveys photic retinal information. GHT-mediated input is associated with arousal and processing the length of day (Mintz et al., 2001; Webb et al., 2008; reviewed in Bailey and Silver, 2013), while the DR/MR pathway transmits activity and exercise information (van Esseveldt et al., 2000). The presence of circulating ligand often influences the activational expression of receptors (Vida et al., 2008), suggesting that sex differences in hormonal milieu may, in part, mediate sex differences in receptor expression within these pathways. Importantly, the type(s) of estrogen receptors expressed in each of these locations, which are likely to influence when and how estradiol effects SCN inputs, have not been fully characterized.

The SCN itself also expresses estrogen receptors (Figure 1.5A,B) and does so in a sex-dependent manner. The SCN can be divided into an outer shell and inner core. Shell neurons frequently express arginine vasopressin (AVP) and exhibit much greater oscillatory amplitude and stronger rhythm than core neurons. Vasoactive intestinal polypeptide (VIP) and gastrin releasing peptide (GRP) are primarily expressed in select core neurons. Further, retinal projections directly input onto core, but not shell neurons

(Figure 1.5A) (reviewed in Bailey and Silver, 2013). Importantly, the patterning of ESR1 and ESR2 is distinct with respect to SCN location (Figure 1.5A,B). ESR2 is more robustly expressed in the SCN than ESR1 and is isolated to non-AVP expressing shell neurons, of which ~38% were ESR2 positive (Vida et al., 2008). While only a small number of neurons express ESR1 (male mice: ~3%; female mice: ~4.5%), it is found almost exclusively in non-VIP and non-GRP core neurons (Vida et al., 2008). Like mice, human women have higher basal levels of SCN ESR1 expression (Kruijver et al., 2002).

Interestingly, no differences in ESR2 levels in the human SCN have been detected across the sexes (Kruijver et al., 2002), while female mice have substantially higher ESR2 levels than males (Vida et al., 2008). Further complicating the patterning of these receptors within the substructural regions of the SCN is the fact that exogenous administration of estradiol reduces ESR2, but not ESR1, expression in both males and females (Vida et al.,

2008). Consequently, innate sex differences in estrogen receptor expression within the

SCN and circulating estradiol contribute to acute receptor patterning, though the degree to which each contributes remains poorly characterized.

Finally, estradiol may regulate SCN efferents. Modulation of estrogenic signaling at this level may directly alter clock-controlled activity outputs downstream of, and

therefore, in spite of the SCN. Additionally, the ability of these regions (Figure 1.4,1.5B) to feedback onto the SCN raises the possibility that ligand binding at any of these targets may indirectly modulate the SCN, consequently impacting daily and circadian activity rhythms. The potential estrogenic networks between the SCN and its efferents are complex and extensive. The SCN directly projects onto 15 distinct brain regions (Morin,

2013), all of which--with the exception of the sub paraventricular zone (sPVZ) and the ventrolateral preoptic area (VLPO)--express at least one type of estrogen receptor (Figure

1.5B) (Bailey and Silver, 2013). Further, the SCN receives coincident feedback from at least 35 brain regions. Multisynaptic connections involving these regions yields an estimated 85 distinct SCN innervation patterns (Morin, 2013). It is unlikely that a single pathway is responsible for all of the effects exerted by estradiol on daily and circadian activity rhythms. While speculative and not the direct focus of this thesis, it is far more likely that a number of distinct and overlapping mechanisms together are responsible for these robust effects.

At the molecular level, estrogens canonically activate their cognate receptors, principally estrogen subtype receptor 1 (ESR1; formerly ERα) and estrogen receptor subtype 2 (ESR2; formerly ERβ) in the central nervous system, which then act as transcription factors (Figure 1.6). Specifically, cytosolic ligand-bound intracellular estrogen receptors decouple from heat shock proteins, dimerize, and bind a variety of co- activators and repressors prior to entering the nucleus and binding to their respective

DNA response elements (King and Greene, 1984; DeMarzo et al., 1991; O’Malley and

Tsai, 1992; O’Malley, 2006; Rosenfeld et al., 2006). However, this homodimer-mediated nuclear binding pathway is not the whole story. The discovery of additional estrogenic

mechanisms has expanded our understanding of how estradiol regulates gene transcription and neuronal excitability. For example, estrogen-bound receptors that complex with other transcription factors are capable of binding to DNA sites outside of their respective response elements (Uht et al., 1997). Additionally, estrogen-induced recruitment of co-activators and co-repressors alters DNA methylation, acetylation, and phosphorylation, supporting an epigenetic role for estradiol (O’Malley, 2006; Rosenfeld et al., 2006). Beyond nuclear translocation, there is strong evidence that both membrane- bound and cytosolic estrogen receptors initiate protease and kinase-dependent intracellular signaling cascades that drive genomic plasticity (Figure 1.6) (Gu et al., 1996;

Zhou et al., 1996; Watters et al., 1997; Bi et al., 2001; Abraham et al., 2004; Zadran et al., 2009). Exogenous estradiol also enhances neuronal excitability within the SCN

(Fatehi and Fatehi-Hassanabad, 2008), possibly through the bidirectional potentiation of

NMDA receptors (Tanaka and Sokabe, 2013) and regulation of presynaptic GABA release (Gillespie et al., 1996; Novak and Albers, 2004), thereby mediating neuronal excitability. Importantly, it is possible that activated ESR1 and ESR2 act through one or many of these mentioned pathways.

Activational vs Organizational Estrogenic Signaling

Importantly, when estradiol influences the formation and expression of daily and circadian rhythms is as important as where and how in the brain it acts. Estradiol can act through both organizational and activational mechanisms (Arnold, 2009), and indeed, there is evidence that both contribute to circadian activity rhythms (Lee et al., 2004;

Hagenauer et al., 2011; Brockman et al., 2011; Blattner and Mahoney, 2012; Kujlis et al.,

2013). However, the attribution of different circadian activity variables to organizational and/or activational regulation, as well as defining the underlying molecular mechanism(s) is incomplete.

During early development, sex determination is primarily driven by the testis determining factor-encoding sry gene on the Y chromosome (Figure 1.7). Subsequent sex differentiation is then mediated by developmental temporal patterning of androgens, estrogens, and progestins, which program adult hormone responsiveness. Early postnatal development is characterized by a brief surge in androgen production by the testis in males (Konkle and McCarthy, 2011). In the brain, and especially within the hypothalamus, androgens are rapidly converted to estradiol by the aromatase cytochrome p450 enzyme (George and Ojeda, 1982; Roselli and Resko, 1993; Konkle and McCarthy,

2011). This results in high circulating brain estradiol levels in neonatal males, and substantially lower levels in neonatal females (Figure 1.7). Peri- and neonatal exposure to estrogens and/or androgens exert a masculinizing effect on the brain (Phoenix et al.,

1959; Arnold, 2009), while the lack of these hormones or the sequestration of estrogens by alpha-fetoprotein (Uriel at al., 1976) results in a feminized default. This early life exposure organizes neural circuits and their respective molecular milieu, adjusting hormone responsiveness in the adult. Following puberty, the high circulating estradiol and progesterone levels characteristic of adult females and high testosterone levels associated with males activationally influence sex specific circadian phenotypes. For example, female mice exhibit greater quantities of daily wheel running activity than their male counterparts (Figure 1.7) (Blattner and Mahoney, 2012).

There is experimental evidence supporting a role for organizational estrogenic control over daily and circadian activity rhythms. Female rodents masculinized in the neonatal period fail to shorten τ when administered estradiol as adults (Zucker et al.,

1980; Albers, 1981). Further, sex differences characteristic of certain circadian variables cannot be not altered by the manipulation of adult sex hormone levels. For example, the inability of female hamsters to entrain to varying LD schedules--a characteristic not observed in males--is not affected by modulation of circulating gonadal hormones.

However, the limits of entrainment in these animals is made more plastic if hormonal manipulation is completed during sexual development (Davis et al., 1983). Aromatase knock-out (ArKO) mice lack the aromatase cytochrome p450 enzyme, and are, therefore, unable to convert testosterone to estradiol (Honda et al., 1998). ArKO females, irrespective of OVX status, exhibit less wheel running activity, but a longer τ than OVX

WT females (Brockman et al., 2011), demonstrating innate differences in circadian activity resulting from absent estrogen signaling throughout the organizational period.

Together, these data strongly suggest that ovarian hormones exert organizational actions to shape circadian behaviors.

However, these actions alone cannot explain the profound effects of circulating estradiol on daily activity rhythms. The discovery that total wheel running exhibited by

OVX WT females treated with estradiol complete significantly more total wheel revolutions than estradiol-treated αERKO OVX females (Ogawa et al., 2003) suggests the predominance of activational, rather than organizational, estradiol-associated control over daily activity patterns. However, the use of global knock-out mice and potential for perinatal and/or neonatal effects of absent estrogen signaling further muddle the

understanding of what parameters are and are not mediated by developmental and/or acute regulation.

Conclusions

The critical importance of circadian outputs is well documented. Sex differences in activity biorhythms, the increased prevalence of shifts in biorhythmic activity during life events characterized by altered estrogenic signaling, and the enrichment of estrogen receptors in circadian-dependent brain regions strongly support a role for estradiol in the expression and formation of daily and circadian activity rhythms. However, the robust influence exerted over these biorhythms by estrogenic signaling is poorly understood, including the effects of developmental versus acutely circulating estradiol.

In the work that follows, I will expand our current understanding of the relationship between estradiol and circadian behavior rhythms by testing my central hypothesis that estradiol acts through both organizational and activational mechanisms to impact the expression of daily and circadian activity rhythms in mice. Additionally, the influence of acutely circulating estradiol on the expression of gene regulatory networks within the SCN will be examined in an effort to correlate estradiol-induced genomic regulation with its control over activity biorhythms. Specifically, in Chapter 2, I will test the hypotheses that estradiol exerts control over daily and circadian activity rhythms in female mice, and that these effects are mediated, in part, by ESR1 and/or ESR2- dependent mechanisms. In Chapter 3, I will explore the developmental role that estrogenic signaling plays in the organization of adult biorhythmic activity outputs during a critical period associated with SCN maturation using male and female mice. I will test the hypotheses that exogenous early life estradiol disrupts the expression of daily and

circadian activity rhythms in adult mice, and also that estradiol administration during

SCN maturation will rescue disrupted rhythms in mice lacking estrogenic signaling at all other developmental times. Finally, in Chapter 4, I will use female mice to test the hypothesis that estradiol and light alter the expression of gene regulatory networks in the

SCN at a time of day when estradiol impedes behavioral plasticity. This novel work greatly enhances our understanding of the intersection between circadian rhythms and sex hormone signaling, and serves as a platform for numerous future studies aimed at further elucidating the mechanisms by which estradiol mediates daily and circadian activity rhythms.

References

Abraham IM, Todman MG, Korach KS, Herbison AE. 2004. Critical in vivo roles for

classical estrogen receptors in rapid estrogen actions on intracellular signaling in

mouse brain. Endocrinology 145:3055-3061.

Albers HE. 1981. Gonadal hormones organize and modulate the circadian system of

the rat. Am J Physiol 241:R62-6.

Arnold AP. 2009. The organizational-activational hypothesis as the foundation for a

unified theory of sexual differentiation of all mammalian tissues. Horm Behav

55:570-578.

Bailey M, Silver R. 2014. Sex differences in circadian timing systems: Implications

for disease. Front Neuroendocrinol. In press.

Baker FC, Kahan TL, Trinder J, Colrain IM. 2007. Sleep quality and the sleep

electroencephalogram in women with severe premenstrual syndrome. Sleep

30:1283-1291.

Berson DM, Dunn FA, Takao M. 2002. Phototransduction by retinal ganglion cells

that set the circadian clock. Science 295:1070-1073.

Bi R, Foy MR, Vouimba RM, Thompson RF, Baudry M. 2001. Cyclic changes in

estradiol regulate synaptic plasticity through the MAP kinase pathway. Proc Natl

Acad Sci U S A 98:13391-13395.

Blattner MS, Mahoney MM. 2013. Photic phase-response curve in 2 strains of mice

with impaired responsiveness to estrogens. J Biol Rhythms 28:291-300.

Blattner MS, Mahoney MM. 2012. Circadian parameters are altered in two strains of

mice with transgenic modifications of estrogen receptor subtype 1. Genes Brain

Behav 11:828-836.

Brockman R, Bunick D, Mahoney MM. 2011. Estradiol deficiency during

development modulates the expression of circadian and daily rhythms in male and

female aromatase knockout mice. Horm Behav 60:439-447.

Brown TM, Piggins HD. 2007. Electrophysiology of the suprachiasmatic circadian

clock. Prog Neurobiol 82:229-255.

Davis FC, Darrow JM, Menaker M. 1983. Sex differences in the circadian control of

hamster wheel-running activity. Am J Physiol 244:R93-105.

Deery MJ, Maywood ES, Chesham JE, Sladek M, Karp NA, Green EW, Charles PD,

Reddy AB, Kyriacou CP, Lilley KS, Hastings MH. 2009. Proteomic analysis

reveals the role of synaptic vesicle cycling in sustaining the suprachiasmatic

circadian clock. Curr Biol 19:2031-2036.

DeMarzo AM, Beck CA, Onate SA, Edwards DP. 1991. Dimerization of mammalian

progesterone receptors occurs in the absence of DNA and is related to the release

of the 90-kDa heat shock protein. Proc Natl Acad Sci U S A 88:72-76.

Doherty CJ, Kay SA. 2010. Circadian control of global gene expression patterns.

Annu Rev Genet 44:419-444.

Duffy JF, Cain SW, Chang AM, Phillips AJ, Munch MY, Gronfier C, Wyatt JK, Dijk

DJ, Wright KP,Jr, Czeisler CA. 2011. Sex difference in the near-24-hour intrinsic

period of the human circadian timing system. Proc Natl Acad Sci U S A 108

Suppl 3:15602-15608.

Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M. 2000. Effect of

single and compound knockouts of estrogen receptors alpha (ERalpha) and beta

(ERbeta) on mouse reproductive phenotypes. Development 127:4277-4291.

Fatehi M, Fatehi-Hassanabad Z. 2008. Effects of 17beta-estradiol on neuronal cell

excitability and neurotransmission in the suprachiasmatic nucleus of rat.

Neuropsychopharmacology 33:1354-1364.

Gentry RT, Wade GN. 1976. Sex differences in sensitivity of food intake, body

weight, and running-wheel activity to ovarian steroids in rats. J Comp Physiol

Psychol 90:747-754.

George FW, Ojeda SR. 1982. Changes in aromatase activity in the rat brain during

embryonic, neonatal, and infantile development. Endocrinology 111:522-529.

Gillespie CF, Huhman KL, Babagbemi TO, Albers HE. 1996. Bicuculline increases

and muscimol reduces the phase-delaying effects of light and VIP/PHI/GRP in the

suprachiasmatic region. J Biol Rhythms 11:137-144.

Goel N, Lee TM. 1995. Sex differences and effects of social cues on daily rhythms

following phase advances in Octodon degus. Physiol Behav 58:205-213.

Gorman M, Lee TM. 2002. Hormones and Biological Rhythms. In: Becker J,

Breedlove S, Crews D McCarthy M, eds. Behavioral Endocrinology. 2nd ed.

Cambridge, MA: MIT Press; 451.

Gu G, Rojo AA, Zee MC, Yu J, Simerly RB. 1996. Hormonal regulation of CREB

phosphorylation in the anteroventral periventricular nucleus. J Neurosci 16:3035-

3044.

Hagenauer MH, King AF, Possidente B, McGinnis MY, Lumia AR, Peckham EM,

Lee TM. 2011. Changes in circadian rhythms during puberty in Rattus

norvegicus: developmental time course and gonadal dependency. Horm Behav

60:46-57.

Honda S, Harada N, Ito S, Takagi Y, Maeda S. 1998. Disruption of sexual behavior in

male aromatase-deficient mice lacking exons 1 and 2 of the cyp19 gene. Biochem

Biophys Res Commun 252:445-449.

Jud C, Schmutz I, Hampp G, Oster H, Albrecht U. 2005. A guideline for analyzing

circadian wheel-running behavior in rodents under different lighting conditions.

Biol Proced Online 7:101-116.

Karatsoreos IN. 2012. Effects of circadian disruption on mental and physical health.

Curr Neurol Neurosci Rep 12:218-225.

King VM, Chahad-Ehlers S, Shen S, Harmar AJ, Maywood ES, Hastings MH. 2003.

A hVIPR transgene as a novel tool for the analysis of circadian function in the

mouse suprachiasmatic nucleus. Eur J Neurosci 17:822-832.

King WJ, Greene GL. 1984. Monoclonal antibodies localize oestrogen receptor in the

nuclei of target cells. Nature 307:745-747.

Konkle AT, McCarthy MM. 2011. Developmental time course of estradiol,

testosterone, and dihydrotestosterone levels in discrete regions of male and female

rat brain. Endocrinology 152:223-235.

Kruijver FP, Swaab DF. 2002. Sex hormone receptors are present in the human

suprachiasmatic nucleus. Neuroendocrinology 75:296-305.

Kuljis DA, Loh DH, Truong D, Vosko AM, Ong ML, McClusky R, Arnold AP,

Colwell CS. 2013. Gonadal- and sex-chromosome-dependent sex differences in

the circadian system. Endocrinology 154:1501-1512.

Labyak SE, Lee TM. 1995. Estrus- and steroid-induced changes in circadian rhythms

in a diurnal rodent, Octodon degus. Physiol Behav 58:573-585.

Leak RK, Card JP, Moore RY. 1999. Suprachiasmatic pacemaker organization

analyzed by viral transynaptic transport. Brain Res 819:23-32.

Lee TM, Hummer DL, Jechura TJ, Mahoney MM. 2004. Pubertal development of sex

differences in circadian function: an animal model. Ann N Y Acad Sci 1021:262-

275.

Lenz K, Nugent B, McCarthy M. 2012. Sexual differentiation of the rodent brain:

dogma and beyond. Front Neurosci 6:1-13.

Mahoney MM. 2010. Shift work, jet lag, and female reproduction. Int J Endocrinol

2010:813764.

Mintz EM, van den Pol AN, Casano AA, Albers HE. 2001. Distribution of

hypocretin-(orexin) immunoreactivity in the central nervous system of Syrian

hamsters (Mesocricetus auratus). J Chem Neuroanat 21:225-238.

Mong JA, Baker FC, Mahoney MM, Paul KN, Schwartz MD, Semba K, Silver R.

2011. Sleep, rhythms, and the endocrine brain: influence of sex and gonadal

hormones. J Neurosci 31:16107-16116.

Morin LP. 2013. Neuroanatomy of the extended circadian rhythm system. Exp Neurol

243:4-20.

Morin LP, Cummings LA. 1982. Splitting of wheelrunning rhythms by castrated or

steroid treated male and female hamsters. Physiol Behav 29:665-675.

Morin LP, Fitzgerald KM, Rusak B, Zucker I. 1977. Circadian organization and

neural mediation of hamster reproductive rhythms. Psychoneuroendocrinology

2:73-98.

Morin LP, Fitzgerald KM, Zucker I. 1977. Estradiol shortens the period of hamster

circadian rhythms. Science 196:305-307.

Novak CM, Albers HE. 2004. Circadian phase alteration by GABA and light differs

in diurnal and nocturnal rodents during the day. Behav Neurosci 118:498-504.

Ogawa S, Chan J, Gustafsson JA, Korach KS, Pfaff DW. 2003. Estrogen increases

locomotor activity in mice through estrogen receptor alpha: specificity for the

type of activity. Endocrinology 144:230-239.

O'Malley BW. 2006. Molecular biology. Little molecules with big goals. Science

313:1749-1750.

O'Malley BW, Tsai MJ. 1992. Molecular pathways of steroid receptor action. Biol

Reprod 46:163-167.

O'Neill JS, Maywood ES, Hastings MH. 2013. Cellular mechanisms of circadian

pacemaking: beyond transcriptional loops. Handb Exp Pharmacol (217):67-103.

doi:67-103.

Phoenix CH, Goy RW, Gerall AA, Young WC. 1959. Organizing action of prenatally

administered testosterone propionate on the tissues mediating mating behavior in

the female guinea pig. Endocrinology 65:369-382.

Pinaud R, Tremere LA. 2012. Control of central auditory processing by a brain-

generated oestrogen. Nat Rev Neurosci 13:521-527.

Ralph MR, Foster RG, Davis FC, Menaker M. 1990. Transplanted suprachiasmatic

nucleus determines circadian period. Science 247:975-978.

Reddy AB. 2013. Genome-wide analyses of circadian systems. Handb Exp

Pharmacol 217:379-88.

Reddy AB, Karp NA, Maywood ES, Sage EA, Deery M, O'Neill JS, Wong GK,

Chesham J, Odell M, Lilley KS, Kyriacou CP, Hastings MH. 2006. Circadian

orchestration of the hepatic proteome. Curr Biol 16:1107-1115.

Reddy AB, O'Neill JS. 2010. Healthy clocks, healthy body, healthy mind. Trends Cell

Biol 20:36-44.

Refinetti R. 2010. The circadian rhythm of body temperature. Front Biosci

(Landmark Ed) 15:564-594.

Rodier WI. 1971. Progesterone-estrogen interactions in the control of activity-wheel

running in the female rat. J Comp Physiol Psychol 74:365-373.

Roenneberg T, Merrow M. 2002. Life before the clock: modeling circadian evolution.

J Biol Rhythms 17:495-505.

Roselli CE, Resko JA. 1993. Aromatase activity in the rat brain: hormonal regulation

and sex differences. J Steroid Biochem Mol Biol 44:499-508.

Rosenfeld MG, Lunyak VV, Glass CK. 2006. Sensors and signals: a

coactivator/corepressor/epigenetic code for integrating signal-dependent programs

of transcriptional response. Genes Dev 20:1405-1428.

Rosenwasser AM. 2010. Circadian clock genes: non-circadian roles in sleep,

addiction, and psychiatric disorders? Neurosci Biobehav Rev 34:1249-1255.

Shechter A, Boivin DB. 2010. Sleep, Hormones, and Circadian Rhythms throughout

the Menstrual Cycle in Healthy Women and Women with Premenstrual

Dysphoric Disorder. Int J Endocrinol 2010:259-345.

Tahara Y, Kuroda H, Saito K, Nakajima Y, Kubo Y, Ohnishi N, Seo Y, Otsuka M,

Fuse Y, Ohura Y, Komatsu T, Moriya Y, Okada S, Furutani N, Hirao A,

Horikawa K, Kudo T, Shibata S. 2012. In vivo monitoring of peripheral circadian

clocks in the mouse. Curr Biol 22:1029-1034.

Takahashi JS, Hong HK, Ko CH, McDearmon EL. 2008. The genetics of mammalian

circadian order and disorder: implications for physiology and disease. Nat Rev

Genet 9:764-775.

Tanaka M, Sokabe M. 2013. Bidirectional modulatory effect of 17beta-estradiol on

NMDA receptors via ERalpha and ERbeta in the dentate gyrus of juvenile male

rats. Neuropharmacology 75:262-273.

Turek FW, Dugovic C, Zee PC. 2001. Current understanding of the circadian clock

and the clinical implications for neurological disorders. Arch Neurol 58:1781-

1787.

Uht RM, Anderson CM, Webb P, Kushner PJ. 1997. Transcriptional activities of

estrogen and glucocorticoid receptors are functionally integrated at the AP-1

response element. Endocrinology 138:2900-2908.

Uriel J, Bouillon D, Aussel C, Dupiers M. 1976. Alpha-fetoprotein: the major high-

affinity estrogen binder in rat uterine cytosols. Proc Natl Acad Sci U S A

73:1452-1456.

van Esseveldt KE, Lehman MN, Boer GJ. 2000. The suprachiasmatic nucleus and the

circadian time-keeping system revisited. Brain Res Brain Res Rev 33:34-77.

Vida B, Hrabovszky E, Kalamatianos T, Coen CW, Liposits Z, Kallo I. 2008.

Oestrogen receptor alpha and beta immunoreactive cells in the suprachiasmatic

nucleus of mice: distribution, sex differences and regulation by gonadal

hormones. J Neuroendocrinol 20:1270-1277.

Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM. 1997. Rapid

membrane effects of steroids in neuroblastoma cells: effects of estrogen on

mitogen activated protein kinase signaling cascade and c-fos immediate early

gene transcription. Endocrinology 138:4030-4033.

Webb IC, Patton DF, Hamson DK, Mistlberger RE. 2008. Neural correlates of

arousal-induced circadian clock resetting: hypocretin/orexin and the

intergeniculate leaflet. Eur J Neurosci 27:828-835.

Welsh DK, Takahashi JS, Kay SA. 2010. Suprachiasmatic nucleus: cell autonomy

and network properties. Annu Rev Physiol 72:551-577.

Welsh DK, Yoo SH, Liu AC, Takahashi JS, Kay SA. 2004. Bioluminescence imaging

of individual fibroblasts reveals persistent, independently phased circadian

rhythms of clock gene expression. Curr Biol 14:2289-2295.

Wirz-Justice A. 2003. Chronobiology and mood disorders. Dialogues Clin Neurosci

5:315-325.

Wollnik F, Turek FW. 1988. Estrous correlated modulations of circadian and

ultradian wheel-running activity rhythms in LEW/Ztm rats. Physiol Behav

43:389-396.

Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM,

Hong HK, Oh WJ, Yoo OJ, Menaker M, Takahashi JS. 2004.

PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals

persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U

S A 101:5339-5346.

Zadran S, Qin Q, Bi X, Zadran H, Kim Y, Foy MR, Thompson R, Baudry M. 2009.

17-Beta-estradiol increases neuronal excitability through MAP kinase-induced

calpain activation. Proc Natl Acad Sci U S A 106:21936-21941.

Zhang EE, Kay SA. 2010. Clocks not winding down: unraveling circadian networks.

Nat Rev Mol Cell Biol 11:764-776.

Zheng B, Albrecht U, Kaasik K, Sage M, Lu W, Vaishnav S, Li Q, Sun ZS, Eichele

G, Bradley A, Lee CC. 2001. Nonredundant roles of the mPer1 and mPer2 genes

in the mammalian circadian clock. Cell 105:683-694.

Zhou Y, Watters JJ, Dorsa DM. 1996. Estrogen rapidly induces the phosphorylation

of the cAMP response element binding protein in rat brain. Endocrinology

137:2163-2166.

Zucker I, Fitzgerald KM, Morin LP. 1980. Sex differentiation of t-e circadian system

in the golden hamster. Am J Physiol 238:R97-101.

Figure 1.1.

Figure 1.1. Simplified Transcription-Translation Feedback Loop (TTFL) within the SCN that encodes the length of the innate day. Input such as daily light cues onto the central oscillator, the suprachiasmatic nucleus (SCN), drives the transcription of the core clock genes clock and bmal1. The subsequently translated protein products then induce the transcription of per and cry, whose protein products then inhibit clock and bmal1 transcription through a continuous negative feedback loop. The known players in this

TTFL are numerous and continually evolving. This loops takes approximately 24 hr to complete a full cycle and is hypothesized to modulate circadian outputs, including include gene rhythmicity, metabolism, total activity, and others (modified from Zhang and Kay, 2010; Bailey and Silver, 2013).

Figure 1.2.

Figure 1.2. Circadian feedback paradigm. Photic input is projected onto the suprachiasmatic nucleus (SCN) from the retina. The SCN responds by transmitting neural and diffusible outputs onto its efferents, which then mediate clock controlled activity. Negative feedback from these efferents onto circadian system inputs regulates network potentiation (modified from Bailey and Silver, 2013).

Figure 1.3.

Figure 1.3. Assessment of daily and circadian activity rhythms. (A) Representative actogram depicting entrainment, phase angle of activity onset, and magnitude of wheel running rhythms over several days in both total darkness (DD) and 12:12 light:dark (LD) housing conditions. Black bars across the top represent lights off during LD, while white indicates the lights were on. (B) Circadian parameters assessed based on activity rhythm include the mean activity observed per unit time across the 24 hr day (Mesor), the innate length of day (Period), the difference between the activity peak and trough (Raw

Amplitude), the difference between the activity peak and the Mesor (Adjusted

Amplitude), and the time at which the activity peak occurs (Acrophase). (C) Shifts in phase of activity onset in response to a photic cue can be measured by subjecting animals

entrained to LD to a light pulse (indicated by the yellow star) at various times of day and measuring the change in phase response. A pulse administered when the animal is accustomed to being in the light (Time: 4hr, 11hr) elicits little response, while a pulse in the early subjective night (Time: 14-16hr) will elicit a maximal phase delay. A phase advance is observed when the pulse is administered in the late subjective night (Time:

22hr) (modified from Bailey and Silver, 2013).

Figure 1.4.

Figure 1.4. Estrogen influenced clock controlled activities. Estrogens are capable of modulating daily and circadian activity rhythms, though our functional understanding of the impact of estradiol on these outputs is incomplete. Further, whether estrogens exert these effects by modifying circadian input pathways, the central oscillator itself, output pathways from the suprachiasmatic nucleus (SCN), and/or SCN efferent targets has not been fully elucidated (modified from Bailey and Silver, 2013).

Figure 1.5.

Figure 1.5. Estrogen receptors are expressed upstream, within, and downstream of the suprachiasmatic nucleus (SCN). (A) The three main inputs onto the SCN, the retinohypothalamic tract (RHT), the geniculohypothalamic tract (GHT) from the intergeniculate leaflet (IGL), and the dorsal and medial raphe nuclei (DR and MR) all express estrogen receptors. Red indicates that estrogen receptors are expressed in this brain region while blue indicates that androgen receptors are expressed. The specific type(s) of receptors present are known for some, but not all, regions shown. (B) The

SCN itself expresses estrogen receptors. Similarly, estrogen receptors are expressed by most SCN efferent targets, including the anteroventral periventricular zone (AVPV), medial preoptic area (mPOA), median preoptic area (MnPO), bed nucleus of the stria

terminalis (BNST), lateral septum (LS), amygdala (AMY), paraventricular nucleus of the hypothalamus (PVN), anterior paraventricular thalamic nuclei (PVT), habenula (HB),

IGL, dorsomedial hypothalamus (DMH), ventromedial nucleus of the hypothalamus

(VMH), retrochiasmatic area (RCh), and the arcuate nucleus (ARC). Hypothalamic efferents are indicted by black arrows. Grey arrows indicate extrahypothalamic targets

(Bailey and Silver, 2013).

Figure 1.6.

Figure 1.6. Estradiol induces genomic responses in neurons expressing estrogen receptors. Ligand-bound estrogen receptors can act directly as nuclear transcription factors either by dimerizing and binding their respective estrogen response element

(ERE) or an alternative TF (Ex: Fos). Non-nuclear mechanisms that also result in a genomic response include initiation of kinase and protease-dependent signaling cascades following activation of membrane-bound estrogen receptors, potentiation of estrogen- sensitive ion channels, and interaction with integral membrane proteins (modified from

Pinaud and Tremere, 2012).

Figure 1.7.

Figure 1.7. Organizational and activational influence(s) of estradiol on the expression of daily and circadian activity rhythms. Sex determination is governed by chromosomal sex and, primarily by the SRY gene on the Y chromosome. Resultant testes development leads to high perinatal testosterone levels in males, which is converted to estradiol in the brain by aromatase. Consequently, neonatal males have high brain estradiol levels compared to age-matched females. These early life events organize adult estradiol responsiveness. Following puberty, females have high circulating estradiol and progesterone levels, while males have high testosterone. These circulating hormones exert activational effects. Together, organizational and activational hormone signaling drives the mature sex-dependent circadian phenotype. For example, female mice exhibit higher levels of daily wheel running than their male counterparts (modified from Lenz et al., 2012).

CHAPTER 2: ESR1 and ESR2 differentially regulate daily and circadian activity rhythms in female mice

Authors: Royston SE1,2, Yasui N3, Kondilis AG*4, Lord SV*4, Katzenellenbogen JA3, Mahoney MM1,4

1Neuroscience Program, University of Illinois Urbana-Champaign 2Medical Scholars Program, University of Illinois College of Medicine 3Department of Chemistry, University of Illinois Urbana-Champaign 4Department of Comparative Biosciences, University of Illinois Urbana-Champaign

Keywords: Circadian, Estrogen, Activational

Acknowledgements The authors are grateful to members of the Mahoney laboratory for assistance with animal husbandry and experimental support, as well as Dr. A. Parent and Mr. I. J. Bourgeois for fruitful discussion and critical reading of this manuscript.

All experiments were designed by SER and MMM. JAK aided in dose calculation. SER and NY made the pellets used throughout. SER, AGK, and SVL completed all vaginal cytology, breeding, weaning, genotyping, ovariectomy, and pellet implantation. SER performed data analyses, and, with MMM, wrote this manuscript.

Endocrinology, 155(7):2613-23.

Abstract

Estrogenic signaling shapes and modifies daily and circadian rhythms, the disruption of which have been implicated in psychiatric, neurologic, cardiovascular, and metabolic disease, among others. However, the activational mechanisms contributing to these effects remain poorly characterized. To determine the activational impact of estrogen on daily behavior patterns and differentiate between the contributions of the estrogen receptors ESR1 and ESR2, ovariectomized adult female mice were administered estradiol, the ESR1 agonist propylpyrazole triol (PPT), the ESR2 agonist diarylpropionitrile (DPN), or cholesterol (CTL). Animals were singly housed with running wheels in a 12:12 Light:Dark cycle or total darkness. Estradiol increased total activity and amplitude, consolidated activity to the dark phase, delayed the time of peak activity (acrophase of wheel running), advanced the time of activity onset, and shortened the free running period (τ), but did not alter the duration of activity (α). Importantly, activation of ESR1 or ESR2 differentially impacted daily and circadian rhythms. ESR1 stimulation increased total wheel running and amplitude, and reduced the proportion of activity in the light vs the dark. Conversely, ESR2 activation modified the distribution of activity across the day, delayed acrophase of wheel running, and advanced the time of activity onset. Interestingly, τ was shortened by estradiol or either estrogen receptor agonist. Finally, estradiol-treated animals administered a light pulse in the early subjective night, but no other time, had an attenuated response compared to CTL. This decreased phase response was mirrored by animals treated with DPN, but not PPT. To conclude, estradiol has strong activational effects on the temporal patterning and

expression of daily and circadian behavior, and these effects are due to distinct mechanisms elicited by ESR1 and ESR2 activation.

Introduction

Circadian timekeeping systems utilize approximately 24 hr endogenous rhythms to adapt and respond to cyclically changing environments. Disruptions of these rhythms are associated with increased incidence of chronic and acute disease, including cancer, obesity, metabolic disorder, compromised immunity, cardiovascular disease, sleep disorders, and a variety of psychiatric conditions (1-7). There are sex differences in the manifestation of these conditions. For example, cardiovascular disease and hypertension are more prevalent among women subjected to circadian disruptions than in men (8).

This is perhaps not surprising considering that sex differences in circadian rhythms are found across a variety of species (7, 9-12). Importantly, daily and circadian rhythms are modulated in part by shifts in hormone signaling during the perinatal period, pregnancy, menopause, and throughout the reproductive cycle (13-16), suggesting ovarian hormones such as estradiol play a role in modifying circadian rhythms.

Indeed, the maximum daily activity in intact female rats and hamsters is observed during proestrus, corresponding to peak serum estradiol levels (17). Removal of circulating estrogens via ovariectomy (OVX) reduces total wheel running activity in female mice, rats, and degus (18-21), an effect reversed by exogenous estradiol replacement (22, 23). Estradiol shortens the length of the free running period (tau; τ) (18,

24-26) and advances the onset of wheel running activity in mice, rats, and hamsters (18,

27). It also consolidates activity to the dark phase of the LD cycle in mice (20, 21, 28).

Female OVX hamsters have greater variability in the timing of activity onset compared to intact or estradiol-treated counterparts, while the incidence of asynchronous or split wheel running rhythms in female OVX hamsters is reduced by estradiol replacement

(13). Further, intact female estrogen receptor knock-out mice (αERKO) in which ESR1

(formerly ERα), but not ESR2 (formerly ERβ) has been removed (29), exhibit a truncated response to a light pulse given in the early subjective night, but a more robust behavioral shift when the pulse is administered in the late subjective night compared to intact WT female mice (30). Taken together, these data suggest that estrogens regulate both the magnitude and timing of wheel running activity, strengthen entrainment to LD transitions

(31), and modulate behavioral shifts in response to a photic cue.

The mechanisms underlying estradiol’s effects on circadian and daily activity rhythms, including the roles of ESR1 and ESR2, have yet to be determined. Ogawa et al.

(20) utilized αERKO and βERKO mice to demonstrate that ESR1, but not ESR2, regulates the estradiol-induced increase in activity. The difference in total wheel running between OVX WT and αERKO, but not βERKO females treated with estradiol (20) suggests the predominance of an activational, rather than organizational mechanism that is dependent on ESR1. However, the use of global knockout mice and the potential for developmental effects of absent estrogen signaling confound the interpretation of these previous studies. Moreover, total daily activity is the only variable that has been shown to be under the control of a specific estrogen receptor. To study the activational effects of estrogenic signaling on the expression of daily and circadian activity rhythms, I pharmacologically manipulated the activity of ESR1 and/or ESR2 in OVX WT female mice. I show here that receptor activation differentially modulates behavioral rhythmicity, suggesting unique roles for ESR1 and ESR2.

Methods

Animals and Housing

Circadian activity patterns in ESR1 knockout (αERKO; -/-) and wild-type (WT;

+/+) littermates have been described previously (30). To ensure this work would be comparable to previous studies, αERKO heterozygotes (+/-) on the C57Bl6/J background were crossed, and the resulting WT female mice were used. Mice were maintained in accordance with the University of Illinois Urbana-Champaign Institutional Animal Care and Use Committee guidelines and the NIH guide for the Care and Use of Laboratory

Animals. Animals were maintained at a constant temperature (22○C) in 12:12 hr light:dark (LD) or constant darkness (DD), and were given food and water ad libitum.

Animals housed in DD were exposed to a dim red light briefly each day (<5 min) to assess animal health but otherwise were not exposed to any light.

Animals were fed Teklad 2016 rodent diet throughout the study because it contains <12 ppm soy estrogen isoflavones, including daidzein and genistein. These levels are subthreshold for eliciting biological activity (per Harlan Laboratories, 2014).

Animals were group-housed with same sex littermates from weaning (p21) until post sexual maturation (p50), at which time they were singly housed in cages (28 cm L x 16 cm W x 12 cm H) outfitted with a metal running wheel (11 cm diameter) affixed to the lid. During the light phase, the light intensity measured at the top of the cage ranged from 220-360 lx (average 290 lx).

The experimental timeline is shown in Figure 2.1. Beginning on p50, and continuing through two estrous cycles, daily vaginal cytology was performed on each animal to confirm cyclicity prior to ovariectomy. Throughout the study, all vaginal

cytology samples were collected at varying times of day to avoid the introduction of a confounding time cue. On p60, running wheels were removed, and mice were anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively; i.p.) and maintained with isoflurane gas as necessary. Ovaries were removed through bilateral incisions, and the muscle/fascia and overlying skin were closed with surgical silk and staples. Vaginal cytology was examined daily for the next 8-10 days (Figure 2.1) to verify that animals were no longer exhibiting signs of cyclicity. Wheels were returned after 5 days of recovery (Figure 2.1).

Drug Pellets and Administration

To manipulate estrogen receptor activity, I used specific ESR1 and ESR2 agonists. The ESR1 agonist 4,4′,4″,-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) has a higher relative binding affinity (RBA) for ESR1 over ESR2 and an estimated 1000 times higher relative potency for ESR1 (32, 33). Conversely, the RBA and relative potent selectivity for ESR2 exhibited by 2,3-bis[4-hydroxyphenyl]-propionitrile (DPN) are 70 and 170 fold greater than those observed for ESR1 (34). DPN (rac-DPN (1)) was synthesized in the Katzenellenbogen laboratory at the University of Illinois Urbana-

Champaign as described (34, 35). PPT was purchased from Orbiter Research

(Champaign, IL). Administered doses were as follows: 25 µg estradiol (LowE), 50 µg estradiol (HighE), 2 mg PPT (LowPPT), 5 mg PPT (HighPPT), 2 mg DPN (LowDPN), and 5 mg DPN (HighDPN). The estradiol concentrations used were selected based on those that increased wheel-running activity, uterine weight, and the expression of corneated vaginal epithelial cells; these changes occur predominantly through the

activation of ESR1 (30, 36-38). The physiologic pattern of activated ERS1 and ESR2 throughout the estrous cycle is unknown. Accordingly, doses of PPT or DPN 100-fold greater were chosen to align with previously reported efficacious doses (37, 39-42) and to account for the reduced transcriptional activity of these compounds compared to estradiol

(33, 34). Specifically, my doses were selected based on studies that found PPT treatment caused estrus-like epithelial cytology (43, 44) and uterine hypertrophy (37). DPN doses were selected based on studies that found it reduced G6PDH and increased the expression of progesterone receptor (PR) and androgen receptor (AR) message and protein without altering uterine size or the proliferation of luminal epithelial cells (37, 43, 44). Each drug was compounded with enough ≥99% cholesterol (Sigma Aldrich; St Louis, MO) to produce a cholesterol pellet with a total weight of 20 mg (45, 46). Control (CTL) pellets contained 20 mg cholesterol alone. Approximately ten days after ovariectomy (~p70) experimental mice were anesthetized under isoflurane gas, and pellets were implanted subcutaneously (Figure 2.1). Mice were randomly assigned to treatment groups prior to pellet implantation.

To compare the efficacy of my doses with previously published work, vaginal smears were examined for 8-10 consecutive days following pellet implantation. Mice implanted with LowE, HighE, or HighPPT pellets exhibited vaginal cytology containing exclusively, or nearly exclusively, corneated cells, while cytology from CTL, LowDPN, or HighDPN-treated animals had zero to very few corneated cells. Animals treated with

LowPPT pellets persistently exhibited a mixture of corneated cells, nucleated cells, and leukocytes. Daily wheel running activity was quantified from 10 days to 4 weeks after pellet implantation (Figure 2.1), as previously described (20). During this experimental

phase, vaginal cytology was collected every 2 days (dieb. alt.) at varying times during the day to avoid providing a non-photic cue. Cytology for each mouse remained consistent throughout the study. Uterine size was examined at the end of the study (Figure 2.1) to further confirm sustained drug pellet efficacy. Consistent with previous findings (37, 43,

44), mice administered either dose of estradiol or PPT had increased uterine size compared to CTL, LowDPN, or HighDPN mice.

Assessment of daily activity patterns and circadian variables

Wheel revolutions were detected via a magnetic switch affixed to each cage. All data were recorded in 10-min bins using VitalView and visualized with ActiView (Mini

Mitter, Bend, OR, USA). An individual bin was considered active if the total amount of activity during that period was equal to or exceeded at least 10% of the animal’s maximum daily activity. The following parameters were assessed in LD: average daily activity, the proportion of activity in light vs dark (LD proportion), the distribution of wheel running across the total 24 hr day as well as the 12 hr dark phase, acrophase of wheel running activity, amplitude, and phase angle of activity onset. The duration of the free running period (τ) and the active phase (α) were determined in animals housed in

DD. The first 3 days of DD data were ignored to account for potential acute after-effects.

To evaluate the effect of estrogenic modulation on the responsiveness to light at different times of the subjective day, a modified Aschoff’s Type II method (described below) was completed with light pulses at times corresponding to the former Zeitgeber time (ZT) 4,

16, and 22 (ZT 0 and 12 = lights on and off, respectively). Where possible, animals were

used for multiple analyses, and were allowed at least one week to re-entrain following a transition from DD to LD.

For parameters assessed in LD, animals were housed in 12:12 LD conditions for at least one week prior to data collection. Consistent phase angle during the last 4 days of this period was required to confirm entrainment (data not shown). Mean daily wheel running activity was determined by averaging the number of wheel revolution per 10 min bin for each animal over 3 consecutive days in LD. Data from each individual animal were normalized to pre-pellet baseline, which was similarly calculated by taking the mean activity for 3 days in LD post ovariectomy and pre-pellet implantation. Total wheel running activity for each treatment group was then divided by the CTL group value to determine fold change.

To analyze temporal patterning of daily activity across the dark phase, as well as in the light compared to dark phase, each 10-min activity bin was averaged over 3 consecutive days, summed into 1 hr bins, and plotted over time. To determine the distribution of activity within the first and second halves of the dark phase, the average wheel revolutions per 10-min bin over 3 days were grouped by treatment, and then summed into ZT12-18 and ZT18-24 bins. The LD proportion describes the number of wheel revolutions completed during the light phase (L) compared to the total amount in the light and dark (D), and is defined as L/(L+D).

Acrophase of wheel running activity in LD corresponds to the time (10 min bin) of maximum activity. It was determined using a cosine fitting function (Actiview) using

3 days of activity. Amplitude reflects the difference between the largest activity peak (10 min bin) and mesor (47) observed from averaging 3 days of wheel running in LD.

Subtracting the mesor from the peak enabled me to compare the magnitude of the rhythm in relation to each animal’s baseline activity. Phase angle of activity onset was determined by measuring the difference in minutes between lights off and the onset of activity across 5 consecutive days in LD. Onset was defined as the first 10-min bin of activity that equaled or exceeded 10% of the maximum activity peak that was not followed by more than 2 consecutive 10-min bins of inactivity (28).

The length of the free running period (τ) was measured by placing a line of best fit through activity onset for the last 5 consecutive days in DD as previously described

(48, 49). Minimally, data from the first 3 days of DD were omitted from the analysis to limit the contribution of transition effects. The length of the active period (α) was determined by averaging the duration between the time of activity onset and when activity stopped over 5 days in DD. Activity cessation was defined as the last 10-min bin with wheel revolutions equal to or greater than 10% of peak activity before a break in activity lasting 2 or more hours.

I determined the effect of estrogenic signaling on the behavioral shift in response to light pulses at varying times of day (21). Briefly, animals entrained to 12:12 LD were placed in DD for at least 24 hr. A pulse of light (~290 lx) was given for one hour at times corresponding to ZT 4, 16, or 22 followed by 4-6 days in DD. Shams were treated the same, but were not subjected to a light pulse. The phase shift was calculated as the difference between the pre and post pulse onset of wheel running activity. This experimental design allowed me to utilize the uniformity of the entrained animals’ subjective day before their free running rhythms drifted (50).

Statistical Analyses

Results are reported as mean ± S.E.M. For total daily activity, LD proportion, α,

τ, phase angle, acrophase of wheel running activity, and amplitude, between group differences were assessed using one way ANOVAs and/or non-parametric Dunn’s tests with treatment group (CTL, LowE, HighE, LowPPT, HighPPT, LowDPN, HighDPN) as the independent variable, followed by Tukey post-hoc tests as appropriate. For phase response to light pulses, I used ANOVAs to analyze differences across treatment groups within each time point, and to identify differences between non-pulsed and pulsed animals receiving the same treatment. Distribution of activity across the 24 hr day or the

12 hr dark phase was assessed using 2-way repeated measures ANOVAs with treatment and time as independent variables. All analyses were performed with SigmaPlot 12.0

(Systat Software, Inc). Comparisons resulting in a priori value (p) < 0.05 were considered significantly different.

Results

Estradiol increases total wheel running activity through activation of ESR1, but not ESR2

The effect of estradiol and selective estrogen receptor stimulation on wheel running activity is shown in Figure 2.2A, while total wheel running activity across all treatment groups is quantified in Figure 2.2B. Standardized post pellet activity is expressed as fold change compared to pre-pellet CTL. ESR1 activation played a predominant role in the increase in daily activity that occurred following activational estradiol treatment. Both the Low (fold change compared to CTL: 4.233 ± 1.106; p<0.001) and HighE doses (fold change compared to CTL: 5.798 ± 1.061; p<0.001)

increased relative wheel running compared to CTL (1.00 ± 0.123). Like estradiol, PPT administration increased relative wheel running (fold change compared to CTL: 3.012 ±

0.459; p<0.001). HighPPT animals did not differ from LowE or HighE animals (p>0.05 for all comparisons between HighE, LowE, and HighPPT). The magnitude of wheel running observed in the LowPPT, LowDPN, and HighDPN treatment groups was not different from CTL, and was significantly lower than estradiol-treated animals (p<0.05).

ESR1 stimulation mimics the estradiol-induced consolidation of activity to the dark

All animals housed in LD were more active during the dark phase than the light phase (Figure 2.2A,C,E). To determine whether activational estradiol is sufficient to consolidate activity to the dark phase as reported previously (20, 21, 28), I calculated the

LD proportion. Both LowE (0.006 ± 0.002; p = 0.02) and HighE (0.004 ± 0.002; p =

0.008) significantly reduced the LD proportion compared to CTL (0.05 ± 0.01), indicating that nearly all wheel running took place during the dark phase in these mice.

This effect was mirrored by administration of HighPPT (0.006 ± 0.002; p = 0.025). The

LowDPN dose had no effect compared to CTL, and nominal reductions observed following either LowPPT or HighDPN administration were not significant (Figure 2.2C).

ESR2 activation results in a greater distribution of activity across the dark phase

Wheel running increased rapidly following lights out in all treatment groups (Fig

2A,E). To further assess the effect of estrogen receptor activation throughout the dark phase, mean wheel revolutions per treatment group were binned in 1 hr increments and plotted over time (Fig 2E). Two-way repeated measures ANOVA revealed a significant

effect of time (F23 = 107.457, p<0.001) and drug treatment (F6 = 19.022, p<0.001), as well as a significant interaction (F138 = 8.706, p<0.001). Overall, more wheel revolutions were completed in the first 6 hours of the dark phase compared to the later night.

Additionally, mice receiving LowE, HighE, or HighPPT pellets were more active than

CTL mice throughout the majority of the dark phase. In contrast, LowPPT animals were only more active than CTL animals from ZT14-17. HighDPN mice were not more active than CTL animals overall, but did complete more wheel revolutions at ZT14, 15, and 20 compared to CTL mice. At no time were LowDPN mice more active than their CTL counterparts.

Animals treated with estradiol had sustained activity over a greater length of time than CTL animals, which completed their maximum amount of wheel running within the first 2 hr of darkness. Further, while HighPPT treatment statistically recapitulated the estradiol-induced increase in total wheel running (Figure 2.2B), activity was consolidated to the early (ZT12-18), rather than later (ZT18-24), subjective night (Figure 2.2A). To further assess this interaction, I investigated whether ESR1 or ESR2 stimulation contribute to when activity occurs during the dark phase. The total number of wheel revolutions completed during the dark phase for each treatment group was summed and set at 1.0 (100%). The fraction of total activity completed during the first (ZT12-18) and second (ZT18-24) half of the dark phase was determined and are shown in Figure 2.2D.

In all treatment groups, except LowDPN, two-way repeated measures ANOVA revealed that a greater amount of the total activity was completed from ZT12-18 than from ZT18-

24. Interestingly, compared to the amount observed in the CTL group (ZT12-18: 0.77 ±

0.03; ZT18-24: 0.23 ± 0.03), both estradiol doses ([LowE: ZT12-18: 0.64 ± 0.03, p =

0.007; ZT18-24: 0.36 ± 0.03, p = 0.007], [HighE: ZT12-18: 0.63 ± 0.04, p = 0.007;

ZT18-24: 0.37 ± 0.04, p = 0.007]) and DPN-treated animals ([LowDPN: ZT12-18: 0.56 ±

0.07, p = 0.006; ZT18-24: 0.44 ± 0.07, p = 0.006], [HighDPN: ZT12-18: 0.64 ± 0.05, p =

0.031; ZT18-24: 0.37 ± 0.05, p = 0.031]) completed a smaller fraction of their total wheel running in the first half of the night. Despite the difference in magnitude of activity between PPT and CTL (Figure 2.2B), there was no difference in the temporal distribution of activity across the dark phase between these two groups (Figure 2.2D).

Estradiol increases amplitude and delays acrophase of wheel running activity through

ESR1 and ESR2 activation, respectively

All of the mice tested demonstrated behavioral rhythmicity with clearly evident active and inactive periods (Figure 2.2A). To assess whether the timing and magnitude of these activity patterns were manipulated by selective estrogen receptor stimulation, I compared both the phase peak of activity (acrophase of wheel running activity) (Figure

2.3A,C) and amplitude (Figure 2.3B,C) across treatments. I found that ESR2 activation via DPN shifted the acrophase of wheel running activity, while ESR1 activation via PPT increased amplitude. There was a significant drug effect on acrophase of wheel running activity (p = 0.002). The acrophase for wheel running behavior (Figure 2.3A,C) was delayed in animals treated with estradiol (LowE: ZT15.95 ± 0.20; HighE: ZT15.91 ±

0.21) compared to CTL (ZT14.63 ± 0.16; p<0.05 for both comparisons). Post-hoc analysis revealed a similar effect in animals treated with HighDPN (ZT16.15 ± 0.49; p<0.05).

Amplitude (Figure 2.3B,C) was higher in estradiol-treated animals compared to

CTL animals (CTL: 263.14 ± 31.73 wheel revolutions; Low E: 449.06 ± 30.91 wheel revolutions, p<0.001; High E: 438.57 ± 21.57 wheel revolutions, p<0.001). This effect was recapitulated by treatment with HighPPT (486.52 ± 37.44 wheel revolutions; p<0.001). Administration of LowPPT (349.63 ± 28.76 wheel revolutions) or either dose of DPN (LowDPN: 276.61 ± 32.34 wheel revolutions; High DPN: 350.69 ± 16.81 wheel revolutions) failed to modify amplitude compared to CTL. However, the amplitudes observed following these treatments were significantly different from those induced by

HighPPT (p<0.05 for all comparisons) (Figure 2.3B). Similarly, the amplitude observed in estradiol-treated animals was greater than that observed in LowDPN animals (p<0.05).

Overlapping the observed amplitude and acrophase of wheel running activity for each treatment group (Figure 2.3C) demonstrates the similarity between estradiol treatment and ESR2 activation with respect to acrophase of wheel running activity, and, conversely, the role of ESR1 in the increased amplitude induced by Low or HighE treatment.

Estradiol or DPN advances activity onset

I calculated the mean phase angle of activity onset for each treatment group to determine the effects of selective estrogen receptor stimulation (Figure 2.4). Compared to CTL animals, in which activity began 20 ± 4 min after lights off, HighE mice became active significantly earlier (9 ± 1 min after lights off; p = 0.0254). Similarly, both Low (6

± 3 min) and HighDPN pellets (2 ± 2 min) significantly advanced the phase of activity onset (p = 0.029 and 0.002, respectively). In contrast, selective activation of ESR1 did

not alter the phase angle compared to CTL. Similarly, the low dose of estradiol had no effect on phase angle.

Estrogen receptor activation shortens the total period without altering the length of the active period

Under free running conditions (DD), modulation of estrogen receptor activation altered period length (Figure 2.5A). A one-way ANOVA revealed a significant treatment effect (p<0.001). The average period length of animals treated with either dose of E was shorter than that observed in CTL animals (CTL: 23.85 ± 0.07 hr; LowE: 23.50 ± .06 hr in Low E; HighE: 23.53 ± 0.04 hr; p<0.001 for both comparisons). A similar effect was observed in animals treated with either dose of PPT (LowPPT: 23.62 ± 0.05 hr; p = 0.047 compared to CTL; HighPPT: 23.55 ± 0.06 hr; p = 0.004 compared to CTL). HighDPN

(23.43 ± 0.04 hr), but not the low dose, also decreased τ compared to CTL (p<0.001).

LowDPN administration had no effect on τ. However, these mice differed significantly from mice treated with LowE (p = 0.021), HighE (p = 0.04), and HighDPN (p = 0.002).

One-way ANOVA revealed a significant drug effect on the length of the active phase (α, p = 0.01) (Figure 2.5B). However, Tukey post-hoc analysis found no difference between

CTLs and any other group. The only difference observed across α was that HighPPT animals had a shorter α (482 ± 38 min) than animals treated with the lower dose of estrogen (702 ± 17 min; p<0.05).

Estradiol or ESR2 activation attenuate the phase delay associated with a light pulse at

ZT16

I sought to determine the impact of estradiol on behavioral phase response in response to photic stimuli. Mice housed in DD were subjected to a 1 hr pulse of light at

ZT4, ZT16, or ZT22, or remained non-pulsed (Figure 2.6). A pulse delivered in the early subjective night at ZT16 caused a phase delay of -81 ± 8 min in CTL animals. Relative to CTL mice, there was a significantly dampened delay in animals administered LowE (-

21 ± 12 min; p = 0.002), HighE (-25 ± 13 min; p = 0.005), or HighDPN (-17 ± 8 min; p<0.001). Though PPT at either dose or LowDPN reduced the phase delay compared to

CTL treatment, the differences were not significant. Comparisons within each treatment group revealed that CTL mice pulsed with light at ZT 16 had a significantly delayed activity onset compared to non-pulsed CTL animals (p<0.001). Similarly, LowPPT

(p<0.001), HighPPT (p<0.001), LowDPN (p = 0.01), and HighE (p = 0.02) animals pulsed at ZT16 exhibited a significant phase delay compared to their non-pulsed counterparts. In contrast, the phase shift observed in LowE or High DPN animals was not different from that observed in non-pulsed animals given the same treatment.

I did not find that estradiol, PPT, or DPN resulted in a behavioral shift in response to a 1 hr light pulse administered at other times. A light pulse during the subjective day

(ZT4) induced phase advances that ranged between 26 and 63 minutes, but there was no treatment effect nor did pulsed animals differ from non-pulsed counterparts. A 1 hr pulse at ZT22, corresponding to the late subjective night, resulted in a relatively large phase advance across all groups of animals. Group means varied from 108 min to 141 min, but there were no significant differences. Within each treatment group, the phase advance was significant relative to non-pulsed counterparts (p<0.001 for all comparisons).

Because the innate free running period in mice is less than the 24 hr day they are

entrained to in 12:12 LD housing conditions (51), the non-pulsed sham animals all slightly phase advanced (range: 11-25 min across groups). No difference was observed between the treatment groups that did not receive a light pulse.

Discussion

I tested the hypothesis that estradiol exerts activational control over daily and circadian rhythms in female mice. Further, I sought to differentiate between the roles of

ESR1 and ESR2 in these behaviors. I found that ESR1 primarily regulates estradiol- induced increases in activity magnitude, while ESR2 controls phase and temporal distribution of activity. The total amount and amplitude of wheel running, as well as the

LD proportion were modulated by estradiol through an ESR1-dependent mechanism.

Conversely, ESR2 activation delayed acrophase of wheel running activity, distributed activity across the dark phase, and advanced phase angle of activity onset. To the best of my knowledge, this is first time estrogenic effects on the acrophase of wheel running activity have been demonstrated at all, and certainly the first time that a receptor- dependent mechanism has been suggested. Interestingly, mice exhibited a shorter τ when either ESR1 or ESR2 were stimulated. Further, behavioral phase shifts in response to a light pulse in the early subjective night, but at no other time, was muted by activational estrogen signaling through a mechanism involving ESR2. These results provide novel insight into the activational effects of estradiol, increase our knowledge regarding their underlying mechanisms, and provide evidence of the differential roles played by ESR1 and ESR2.

Ogawa et al. (20) demonstrated that ESR1, but not ESR2, loss of function prevents the estradiol-induced increase in wheel running activity in both males and females. Here I expand upon this work and demonstrate that ESR1 stimulation is sufficient to increase total daily wheel running. Further, I observed an increased activity amplitude in mice administered estradiol or HighPPT. Because entrained mice exhibit clear peaks and troughs of activity across a single day in LD, elevations in amplitude are expected to coincide with increased total activity assuming the animals remain rhythmic as they did here. It is possible that ESR1 stimulation increases activity levels overall, but due to masking effects, the increase is only observed in the dark phase, thus altering LD proportion. I also cannot rule out that increased wheel running itself is enough to drive activity-mediated feedback that alters outputs of the master circadian clock, located in the suprachiasmatic nucleus (SCN) (52). Regardless of the locus for controlling activity in the current study, these effects can still be considered under the control of ESR1 because the only difference across all mice tested was the drug they received.

The phase angle of activity onset relative to the time of lights-off is advanced in intact female rats on the day of estrus when serum estradiol levels peak and progesterone levels are at their minimum (17, 53). It is similarly advanced in female hamsters administered estradiol (27), but is unaffected in female Octodon degus treated with progesterone (54). While these data support the hypothesis that increased estrogenic signaling strengthens entrainment to light (31), they do not rule out the possibility that changes in progesterone signaling may also modulate the phase angle of activity onset.

Although changes in activity corresponding to the stage of the estrus cycle are well documented in rats and hamsters (17, 18, 24, 55), these rhythms have not been observed

in mice. Accordingly, I did not anticipate that selective estrogen receptor stimulation would alter phase angle of activity onset in my mice. However, I found that estradiol advanced activity onset by approximately 50% in female mice. Treatment with DPN was sufficient to recapitulate this result, suggesting this is an ESR2-dependent mechanism.

To my knowledge, no previous report has outlined the relationship between ESR2 and phase angle. Blattner and Mahoney (28) showed that selective removal of classical estrogen receptor signaling (mutation of the ESR1 ERE-binding domain in NERKI mice) or complete loss of ESR1 failed to alter phase angle, leading to the conclusion that phase angle is not regulated by an ESR1-dependent mechanism. My study also points to a role for ESR2-dependent distribution of activity across the dark phase, perhaps accounting for the delayed acrophase of wheel running activity observed when this receptor subtype was stimulated.

The length of the free running period under constant lighting conditions is shorter in female rats, hamsters, and humans than in males, and is shorter still in females administered exogenous estradiol (10, 18, 24, 56, 57). Recent work using intact WT,

αERKO, and NERKI female mice found no differences in τ, demonstrating that removal of ESR1 signaling does not alter period length (28). Interestingly, I show here that estradiol, both doses of PPT, and HighDPN all shorten τ in a similar manner. My data suggest that stimulation of either ESR1 or ESR2 is sufficient to shorten period, and that this effect is activational. While it is possible that the shortening of τ induced by estradiol is ESR1-dependent only and that the dose of DPN administered was high enough to act on both receptors, at no time did I observe physiologic evidence of ESR1 activation in either LowDPN or HighDPN mice. For example, I did not detect corneated

vaginal cells or uterine hypertrophy in mice treated with DPN. These data allude to a common regulatory mechanism for ESR1 and ESR2’s control of period, but not other tested variables in which differential results were produced. Though unlikely, there also exists the possibility that a different estrogen-dependent pathway capable of utilizing all 3 compounds as ligands, but not directly tested here, is responsible.

I also measured the length of the active period under constant darkness. Though estradiol had no effect, the higher dose of PPT shortened α compared to LowE animals.

Interestingly, αERKO mice have a longer α (28), suggesting that the length of the active phase is at least partially controlled by estrogenic signaling. However, in the current study α was not affected by circulating DPN alone, a treatment that should have been similar to αERKO animals in that only ESR2 is acted on by estradiol. Because the critical difference in these studies is the use of transgenic mice compared to acute hormone receptor activation, these date seem to support an organizational, but not activational role for estradiol in the regulation of this circadian variable, but warrants future developmental research.

I sought to determine whether estradiol exerts an activational effect on the magnitude of light-induced behavioral phase shifting across the subjective day. I found that a light pulse in the late subjective night (ZT 22) resulted in a large phase advance in all treatment groups, while light pulses administered in the subjective day (ZT 4) had no effect (Figure 2.6). However, a light pulse in the early subjective evening (ZT 16) resulted in a differential response dependent upon treatment. Because removal of endogenous hormone signaling truncates the behavioral phase delay in response to light in WT OVX (21) and intact αERKO females (30), I anticipated that mice treated with

PPT or estradiol would have a more robust phase delay than CTL animals. Rather than a larger phase delay, I found that estradiol-treated mice had a significantly truncated response to a 1 hr light pulse at ZT 16 relative to CTL mice. Animals treated with DPN also had a reduced phase delay when pulsed at ZT16 compared to CTL mice. These data suggest that estradiol exerts an activational effect on phase response to light when the pulse is administered in the early subjective night, but no other time, and that this response is likely mediated by an ESR2-dependent mechanism. While these current findings are somewhat incongruous with previous studies, they do not take into account organizational effects that may also be impacting photic phase responsiveness. Future study is needed to determine the exclusively organizational regulation.

It remains unknown how estradiol mechanistically enacts these effects on daily and circadian activity measures, including whether estradiol acts directly or indirectly on central oscillators. Multiple types of estrogen receptors are expressed within structures that input directly or indirectly onto the SCN (7, 58-61) as well as within the SCN itself.

ESR1 and ESR2 are localized to the SCN in rats and humans (62, 63), though their expression is relatively small in mice (64). There is evidence that GABAergic neurons within the SCN express estrogen receptors, especially ESR2 (65), and that estradiol administration enhances SCN neuronal excitability (58). Further, pharmacologic

GABAA or GABAB receptor agonists injected into the SCN of nocturnal or diurnal rodents dampens phase shifting in response to a light pulse in the early subjective night

(67-69). Though not directly tested here, these data combined with my current findings support the possibility that estradiol blocks the phase delay induced by a ZT16 light pulse through an ESR2-mediated mechanism that drives excitability, resulting in greater

GABA release, and subsequent GABAA and GABAB receptor binding. Further work is needed to assess the validity of this model. Importantly, the mechanism and the locus of action are likely dependent on the specific measure being examined. For example, previous work suggests that estradiol’s effect on total wheel running involves the medial preoptic area (mPOA) (20, 63-65), but the involvement of additional structures and downstream pathway(s) in this and the other measures reported upon here remain work for future study.

Conclusions

I demonstrate here that estradiol increases total activity and amplitude, reduces the LD proportion, delays acrophase of wheel running activity, shortens τ, and dampens the light pulse-induced phase shift in wheel running in the early subjective night.

Importantly, activational stimulation of ESR1 or ESR2 differentially impacts daily and circadian activity outputs. ESR1 regulates estradiol-associated elevations in total wheel running activity and amplitude, while also consolidating activity to the dark phase.

Conversely, ESR2 stimulation more significantly mediates the timing of activity, including sustaining more constant levels of activity across the active period, delaying acrophase of wheel running activity, advancing phase angle, and occluding the phase response associated with a pulse of light in the early subjective night. Together, my data contribute to a more complete understanding of the activational influence of estradiol on the expression of daily and circadian rhythms in female mice. Further, I suggest, for the first time, distinct mechanisms by which ESR1 and ESR2 activity shape biorhythmic behavior.

References

1. Turek FW, Dugovic C, Zee PC. Current understanding of the circadian clock and

the clinical implications for neurological disorders. Arch Neurol.

2001;58(11):1781-7.

2. Wirz-Justice A. Chronobiology and mood disorders. Dialogues Clin Neurosci.

2003;5(4):315-25.

3. Rosenwasser AM. Circadian clock genes: non-circadian roles in sleep, addiction,

and psychiatric disorders? Neurosci Biobehav Rev. 2010;34(8):1249-55.

4. Takahashi JS, Hong HK, Ko CH, McDearmon EL. The genetics of mammalian

circadian order and disorder: implications for physiology and disease. Nat Rev

Genet. 2008;9(10):764-75.

5. Reddy AB, O'Neill JS. Healthy clocks, healthy body, healthy mind. Trends Cell

Biol. 2010;20(1):36-44.

6. Karatsoreos IN. Effects of circadian disruption on mental and physical health.

Curr Neurol Neurosci Rep. 2012;12(2):218-25

7. Bailey M, Silver R. Sex differences in circadian timing systems: Implications for

disease. Front Neuroendocrinol. 2013. doi:pii: S0091-3022(13)00070-8.

8. Kivimäki M, Virtanen M, Elovainio M, Väänänen A, Keltikangas-Järvinen L,

Vahtera J. Prevalent cardiovascular disease, risk factors and selection out of shift

work. Scand J Work Environ Health. 2006;32(3):204-8.

9. Karatsoreos IN, Silver R. Minireview: The neuroendocrinology of the

suprachiasmatic nucleus as a conductor of body time in mammals. Endocrinology.

2007;148(12):5640-7.

10. Duffy JF, Cain SW, Chang AM, Phillips AJ, Münch MY, Gronfier C, Wyatt JK,

Dijk DJ, Wright KP Jr, Czeisler CA. Sex difference in the near-24-hour intrinsic

period of the human circadian timing system. Proc Natl Acad Sci U S A.

2011;108 Suppl 3:15602-8.

11. Mong JA, Baker FC, Mahoney MM, Paul KN, Schwartz MD, Semba K, Silver R.

Sleep, rhythms, and the endocrine brain: influence of sex and gonadal hormones.

J Neurosci. 2011;31(45):16107-16.

12. Kuljis DA, Loh DH, Truong D, Vosko AM, Ong ML, McClusky R, Arnold AP,

Colwell CS. Gonadal- and sex-chromosome-dependent sex differences in the

circadian system. Endocrinology. 2013;154(4):1501-12.

13. Morin LP, Cummings LA. Splitting of wheelrunning rhythms by castrated or

steroid treated male and female hamsters. Physiol Behav. 1982;29(4):665-75.

14. Baker FC, Kahan TL, Trinder J, Colrain IM. Sleep quality and the sleep

electroencephalogram in women with severe premenstrual syndrome. Sleep.

2007;30(10):1283-91.

15. Mahoney MM. Shift work, jet lag, and female reproduction. Int J Endocrinol.

2010;2010:813764.

16. Shechter A, Boivin DB. Sleep, Hormones, and Circadian Rhythms throughout the

Menstrual Cycle in Healthy Women and Women with Premenstrual Dysphoric

Disorder. Int J Endocrinol. 2010;2010:259345.

17. Wollnik F, Turek FW. Estrous correlated modulations of circadian and ultradian

wheel-running activity rhythms in LEW/Ztm rats. Physiol Behav.

1988;43(3):389-96.

18. Albers HE. Gonadal hormones organize and modulate the circadian system of the

rat. Am J Physiol. 1981;241(1):R62-6.

19. Labyak SE, Lee TM. Estrus- and steroid-induced changes in circadian rhythms in

a diurnal rodent, Octodon degus. Physiol Behav. 1995;58(3):573-85.

20. Ogawa S, Chan J, Gustafsson JA, Korach KS, Pfaff DW. Estrogen increases

locomotor activity in mice through estrogen receptor alpha: specificity for the

type of activity. Endocrinology. 2003;144(1):230-9.

21. Brockman R, Bunick D, Mahoney MM. Estradiol deficiency during development

modulates the expression of circadian and daily rhythms in male and female

aromatase knockout mice. Horm Behav. 2011;60(4):439-47.

22. Rodier WI. Progesterone-estrogen interactions in the control of activity-wheel

running in the female rat. J Comp Physiol Psychol. 1971;74(3):365-73.

23. Gentry RT, Wade GN. Sex differences in sensitivity of food intake, body weight,

and running-wheel activity to ovarian steroids in rats. J Comp Physiol Psychol.

1976;90(8):747-54.

24. Morin LP, Fitzgerald KM, Zucker I. Estradiol shortens the period of hamster

circadian rhythms. Science. 1977;196(4287):305-7.

25. Zucker I, Fitzgerald KM, Morin LP. Sex differentiation of t-e circadian system in

the golden hamster. Am J Physiol. 1980;238(1):R97-101.

26. Blattner MS, Mahoney MM. Estrogen receptor 1 modulates circadian rhythms in

adult female mice. Chronobio Int. 2014; in press.

27. Morin LP, Fitzgerald KM, Rusak B, Zucker I. Circadian organization and neural

mediation of hamster reproductive rhythms. Psychoneuroendocrinology.

1977;2(1):73-98.

28. Blattner MS, Mahoney MM. Circadian parameters are altered in two strains of

mice with transgenic modifications of estrogen receptor subtype 1. Genes Brain

Behav. 2012;11(7):828-36.

29. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M. Effect of

single and compound knockouts of estrogen receptors alpha (ERalpha) and beta

(ERbeta) on mouse reproductive phenotypes. Development. 2000;127(19):4277-

91.

30. Blattner MS, Mahoney MM. Photic phase-response curve in 2 strains of mice

with impaired responsiveness to estrogens. J Biol Rhythms. 2013;28(4):291-300.

31. Gorman MR, Lee TM 2002 Hormones and Biological Rhythms. In: Becker JB,

Breedlove SM, Crews D, McCarthy MM, eds. Behavioral Endocrinology. 2nd ed.

Cambridge: Massachusetts Institute of Technology; 451-494.

32. Sun J, Meyers MJ, Fink BE, Rajendran R, Katzenellenbogen JA,

Katzenellenbogen BS. Novel ligands that function as selective estrogens or

antiestrogens for estrogen receptor-alpha or estrogen receptor-beta.

Endocrinology. 1999;140(2):800-4.

33. Stauffer SR, Coletta CJ, Tedesco R, Nishiguchi G, Carlson K, Sun J,

Katzenellenbogen BS, Katzenellenbogen JA. Pyrazole ligands: structure-

affinity/activity relationships and estrogen receptor-alpha-selective agonists. J

Med Chem. 2000;43(26):4934-47.

34. Meyers MJ, Sun J, Carlson KE, Marriner GA, Katzenellenbogen BS,

Katzenellenbogen JA. Estrogen receptor-beta potency-selective ligands: structure-

activity relationship studies of diarylpropionitriles and their acetylene and polar

analogues. J Med Chem. 2001;44(24):4230-51.

35. Carroll VM, Jeyakumar M, Carlson KE, Katzenellenbogen JA. Diarylpropionitrile

(DPN) enantiomers: synthesis and evaluation of estrogen receptor beta-selective

ligands. J Med Chem. 2012;55:528-537.

36. Carroll JC, Pike CJ. Selective estrogen receptor modulators differentially regulate

Alzheimer-like changes in female 3xTg-AD mice. Endocrinology.

2008;149(5):2607-11.

37. Frasor J, Barnett DH, Danes JM, Hess R, Parlow AF, Katzenellenbogen BS.

Response-specific and ligand dose-dependent modulation of estrogen receptor

(ER) α activity by ERβ in the uterus. Endocrinology. 2003;144(7):3159-66.

38. Couse JF, Korach KS. Estrogen receptor null mice: what have we learned and

where will they lead us? Endocr Rev. 1999 Jun;20(3):358-417. Review. Erratum

in: Endocr Rev 1999 Aug;20(4):459.

39. Harris HA, Katzenellenbogen JA, Katzenellenbogen BS. Characterization of the

biological roles of the estrogen receptors, ERalpha and ERbeta, in estrogen target

tissues in vivo through the use of an ERalpha-selective ligand. Endocrinology.

2002;143(11):4172-7.

40. Lund TD, Rovis T, Chung WC, Handa RJ. Novel actions of estrogen receptor-

beta on anxiety-related behaviors. Endocrinology. 2005;146(2):797-807.

41. Walf AA, Koonce CJ, Frye CA. Estradiol or diarylpropionitrile administration to

wild type, but not estrogen receptor beta knockout, mice enhances performance in

the object recognition and object placement tasks. Neurobiol Learn Mem.

2008;89(4):513-21.

42. Walf AA, Koonce CJ, Frye CA. Estradiol or diarylpropionitrile decrease anxiety-

like behavior of wildtype, but not estrogen receptor beta knockout, mice. Behav

Neurosci. 2008;122(5):974-81.

43. Sánchez-Criado JE, Martín De Las Mulas J, Bellido C, Tena-Sempere M, Aguilar

R, Blanco A. Biological role of pituitary estrogen receptors ERalpha and ERbeta

on progesterone receptor expression and action and on gonadotropin and prolactin

secretion in the rat. Neuroendocrinology. 2004;79(5):247-58..

44. Sánchez-Criado JE, Trudgen K, Millán Y, Blanco A, Monterde J, Garrido-Gracia

JC, Gordon A, Aguilar R, de Las Mulas JM, Ko C. Estrogen receptor (ESR) 2

partially offsets the absence of ESR1 in gonadotropes of pituitary-specific Esr1

knockout female mice. Reproduction. 2012;143(4):549-58.

45. Ogura Y, Ishii K, Kanda H, Kanai M, Arima K, Wang Y, Sugimura Y. Bisphenol

A induces permanent squamous change in mouse prostatic epithelium.

Differentiation. 2007;75(8):745-56.

46. Risbridger GP, Wang H, Frydenberg M, Cunha G. The metaplastic effects of

estrogen on mouse prostate epithelium: proliferation of cells with basal cell

phenotype. Endocrinology. 2001;142(6):2443-50.

47. Refinetti R. The circadian rhythm of body temperature. Front Biosci (Landmark

Ed). 2010 Jan 1;15:564-94.

48. Agostino PV, Plano SA, Golombek DA. 2007. Sildenafil accelerates

reentrainment of circadian rhythms after advancing light schedules. Proc Natl

Acad Sci U S A 104:9834-9839.

49. Ogeil RP, Rajaratnam SM, Redman JR, Broadbear JH. 2010. Acute MDMA

administration alters the distribution and circadian rhythm of wheel running

activity in the rat. Brain Res 1359:128-136.

50. Mrosovsky N. Methods of measuring phase shifts: why I continue to use an

Aschoff type II procedure despite skepticism of referees. Chronobiol Int.

1996;13(5):387-92.

51. Refinetti R. Daily activity patterns of a nocturnal and a diurnal rodent in a

seminatural environment. Physiol Behav. 2004;82(2-3):285-94.

52. van Oosterhout F, Lucassen EA, Houben T, vanderLeest HT, Antle MC, Meijer

JH. Amplitude of the SCN clock enhanced by the behavioral activity rhythm.

PLoS One. 2012;7:e39693.

53. Wood GA, Fata JE, Watson KL, Khokha R. Circulating hormone and estrous

stage predict cellular and stromal remodeling in murine uterus. Reproduction.

2007;133:1035-1044.

54. Labyak SE, Lee TM. Estrus- and steroid-induced changes in circadian rhythms in

a diurnal rodent, Octogon degus. Phys Behav. 1995;58(3):573-85.

55. Fitzgerald K, Zucker I. Circadian organization of the estrous cycle of the golden

hamster. Proc Natl Acad Sci U S A. 1976;73:2923-2927.

56. Davis FC, Darrow JM, Menaker M. Sex differences in the circadian control of

hamster wheel-running activity. Am J Physiol. 1983;244(1):R93-105.

57. Schull J, Walker J, Fitzgerald K, Hiilivirta L, Ruckdeschel J, Schumacher D,

Stanger D, McEachron DL. Effects of sex, thyro-parathyroidectomy, and light

regime on levels and circadian rhythms of wheel-running in rats. Physiol Behav.

1989 Sep;46(3):341-6.

58. King JM. Effects of lesions of the amygdala, preoptic area, and hypothalamus on

estradiol-induced activity in the female rat. J Comp Physiol Psychol.

1979;93(2):360-7.

59. Fahrbach SE, Meisel RL, Pfaff DW. Preoptic implants of estradiol increase wheel

running but not the open field activity of female rats. Physiol Behav.

1985;35(6):985-92.

60. Takeo T, Sakuma Y. Diametrically opposite effects of estrogen on the excitability

of female rat medial and lateral preoptic neurons with axons to the midbrain

locomotor region. Neurosci Res. 1995;22(1):73-80.

61. Abizaid A, Mezei G, Thanarajasingam G, Horvath TL. Estrogen enhances light-

induced activation of dorsal raphe serotonergic neurons. Eur J Neurosci.

2005;21(6):1536-46.

62. Su JD, Qiu J, Zhong YP, Chen YZ. Expression of estrogen receptor -alpha and -

beta immunoreactivity in the cultured neonatal suprachiasmatic nucleus: with

special attention to GABAergic neurons. Neuroreport. 2001;12(9):1955-9.

63. Kruijver FP, Swaab DF. Sex hormone receptors are present in the human

suprachiasmatic nucleus. Neuroendocrinology. 2002;75(5):296-305.

64. Vida B, Hrabovszky E, Kalamatianos T, Coen CW, Liposits Z, Kalló I. Oestrogen

receptor alpha and beta immunoreactive cells in the suprachiasmatic nucleus of

mice: distribution, sex differences and regulation by gonadal hormones. J

Neuroendocrinol. 2008;20(11):1270-7.

65. Su JD, Qiu J, Zhong YP, Chen YZ. Expression of estrogen receptor -alpha and -

beta immunoreactivity in the cultured neonatal suprachiasmatic nucleus: with

special attention to GABAergic neurons. Neuroreport. 2001;12(9):1955-9.

66. Fatehi M, Fatehi-Hassanabad Z. Effects of 17beta-estradiol on neuronal cell

excitability and neurotransmission in the suprachiasmatic nucleus of rat.

Neuropsychopharmacology. 2008;33(6):1354-64.

67. Gillespie CF, Huhman KL, Babagbemi TO, Albers HE. Bicuculline increases and

muscimol reduces the phase-delaying effects of light and VIP/PHI/GRP in the

suprachiasmatic region. J Biol Rhythms. 1996;11(2):137-44.

68. Gillespie CF, Mintz EM, Marvel CL, Huhman KL, Albers HE. GABA(A) and

GABA(B) agonists and antagonists alter the phase-shifting effects of light when

microinjected into the suprachiasmatic region. Brain Res. 1997;759(2):181-9.

69. Novak CM, Albers HE. Circadian phase alteration by GABA and light differs in

diurnal and nocturnal rodents during the day. Behav Neurosci. 2004;118(3):498-

504.

Figure 2.1.

Figure 2.1. Methodological timeline. Postnatal day indicated by p.

Figure 2.2.

Figure 2.2. Effects of estrogenic signaling modification on circadian wheel running activity. A, Representative double-plotted actograms depicting the activational effect of differential estrogen receptor stimulation on daily wheel running activity in OVX female

mice measured for 5 consecutive days in 12:12 light:dark (LD, lights off at Zeitgeber,

ZT, 12). White and black bars at the top indicate whether the lights were on or off respectively. Consecutive days are plotted on the y-axis, while the ZT over 48 hr is shown on the x-axis. Each black bar represents a 10-min bin, and the height indicates the amount of wheel running occurring during the period. B-D, Quantified treatment effects on circadian activity (mean ± S.E.M.), including total wheel revolutions per day in LD

(B), LD proportion (C), and fractional breakdown of total activity occurring during the first versus second half of the dark phase (relative activity ZT12-24) (D). Sample size (n) is indicated below each bar. ♦, ○, ●, and ✚ denote significantly different comparisons made to CTL, LowE, HighE, and, HighPPT, respectively. In panel D, within group differences in relative activity ZT12-24 are indicated by *. One symbol p<0.05, and 2 indicate p<0.001. E, Wheel running activity from 3 consecutive days in LD was averaged into 1 hr bins. ♦, ♦♦ p<0.05 and 0.001, respectively, compared to CTL (2-way repeated measures ANOVA). Sample sizes per time points are as follows: CTL (n = 10-

11), LowE (n = 9-10), HighE (n = 11-12), LowPPT (n = 8-9), HighPPT (n = 8), LowDPN

(n = 7-8), HighDPN (n = 7-9).

Figure 2.3.

Figure 2.3. Estradiol increases amplitude through ESR1 and delays acrophase of wheel running activity via ESR2. A, Acrophase of wheel running activity averaged over 3 days in LD across CTL, LowE, HighE, LowPPT, HighPPT, LowDPN, and HighDPN-treated

OVX female mice. Plotted between ZT12 and 18 (mean ± S.E.M.), the sample sizes per group are as follows: CTL (n = 10), LowE (n = 10), HighE (n = 11), LowPPT (n = 8),

HighPPT (n = 8), LowDPN (n = 8), HighDPN (n = 9). ♦♦ denote p<0.001 compared to

CTL. B, Amplitude plotted as the difference between the daily wheel running peak and mesor. Values represent the mean ± S.E.M. The n per treatment is shown below the corresponding bar. ♦, ○, ●, and ✚denote significant comparisons made to CTL, LowE,

HighE, HighPPT, respectively. For one symbol p<0.05, and p<0.001 for two. C,

Polargram representation of the effects of E, PPT, and DPN administration on acrophase of wheel running activity and amplitude. The radial axis value intersected by a given treatment group’s vector corresponds to the acrophase of wheel running activity. Both degrees and ZT are shown. Vector length is indicative of amplitude magnitude.

Variance shown in A and B.

Figure 2.4.

Figure 2.4. Phase angle of activity onset (mean min ± S.E.M.) for CTL, LowE, HighE,

LowPPT, HighPPT, LowDPN, and HighDPN-treated animals. Sample size per treatment group is indicated to the right. ♦ denotes comparisons made to CTL. For one symbol p<0.05, and p<0.001 for two.

Figure 2.5.

Figure 2.5. Effects of estradiol, PPT, and DPN on τ and α. A, The mean free running period (τ) (± S.E.M.), and B, the average duration of the active period (α) (± S.E.M.).

Both parameters were determined from 5 days of data from animals housed in DD.

Samples sizes for each group are indicated below the corresponding bar. ♦, ○, ●, and ∞

denote comparisons made to CTL, LowE, HighE, and Low DPN, respectively. For one symbol p<0.05, and p<0.001 for two.

Figure 2.6.

Figure 2.6. Photic phase response of OVX female mice treated with estradiol, PPT, DPN or CTL. Entrained animals were housed in DD for 24 hr and were either maintained in the dark (No Pulse) or pulsed with light for 1 hr at a time corresponding to the previous

LD schedule’s ZT4, ZT16, or ZT22. Values represent the average (± S.E.M.) relative difference between the pre and post-pulse onset of wheel running activity. Negative values indicate a phase delay, while positive values signify a phase advance. Sample sizes are shown for each treatment group at each time point. ♦ denotes comparisons made to CTL. For one symbol p<0.05, and p<0.001 for two.

79 ESR1 and ESR2 Regulate Activity 80

Table 2.1.

Table 2.1. Effects of treatment on the presence of corneated vaginal epithelial cells and uterine hypertrophy. Categorical analyses of these two measures based on treatment.

Depending on the measure, an assessment of No indicates that at no time did any of the animals in this treatment group present with corneated vaginal epithelial cells or uterine hypertrophy, respectively. Conversely, Yes indicates that corneated cells were always present in all animals within this treatment group or that all animals exhibited uterine hypertrophy. Vaginal cytology from some, but not all, of the LowPPT animals contained exclusively corneated cells throughout the entire study, while others did not.

Consequently, these animals were categorically assessed as Inconsistent.

CHAPTER 3: Estradiol exposure early in life programs daily and circadian activity rhythms in adult mice

Authors: Royston SE1,2, Bunick D3, Mahoney MM1,3.

1Neuroscience Program, University of Illinois Urbana-Champaign 2Medical Scholars Program, University of Illinois College of Medicine 3Department of Comparative Biosciences, University of Illinois Urbana-Champaign

Keywords: Circadian, Estrogen, Organizational

Acknowledgements The authors are grateful to members of the Mahoney laboratory for assistance with animal husbandry and experimental support. We thank Dr. John Katzenellenbogen and Norio Yasui for their assistance making the estradiol pellets.

All experiments were designed by SER and MMM. DB provided the ArKO mice used throughout. Dr. John Katzenellenbogen (JAK) provided the estradiol-containing pellets, which were made by SER and Norio Yasui (NY). SER, Athanasios Kondilis (AGK), Steven Lord (SVL), Victoria Gugala (VG), Nadia Ayub (NA), and Arif Molla (AM) performed all anogenital distance measurements, breeding, weaning, genotyping, gonadectomy, and pellet implantation. SER performed data analyses, and, with MMM, wrote this manuscript.

Physiology and Behavior, in preparation

Abstract

Developmental hormone signaling during discrete critical periods organizes the adult circadian timekeeping system by altering adult hormone sensitivity and shaping fundamental properties of circadian rhythmicity. However, the timing of when developmental estrogens modify the timekeeping system is poorly understood. To test the hypothesis that alterations in perinatal estrogenic signaling organizes the adult daily and circadian activity rhythms, I utilized aromatase knock-out mice (ArKO), which lack the enzyme required for estradiol synthesis. ArKO and wild-type (WT) males and females were administered either estradiol (E) or oil daily for the first 5 days postnatally

(p1-5E and p1-5OIL, respectively) because this treatment window encompasses the emergence of transcriptional rhythmicity and light responsiveness in the suprachiasmatic nucleus, a bilateral hypothalamic structure regarded as the ‘master oscillator’. Following sexual maturation, gonadectomy, and exogenous estradiol supplementation, circadian parameters were assessed. I discovered that altered perinatal estrogenic signaling exerts organizational control over the expression of daily and circadian activity rhythms in adult mice and that these effects are sex-dependent. Specifically, p1-5E reduced total wheel running activity only in mice with low brain estradiol during this developmental period, but had no effect on WT male activity levels. In females, wheel running was consolidated by p1-5E to the early versus late evening, a phenomenon characteristic of male mice. The maximum activity peak was reduced by p1-5E in ArKO females only, while this treatment advanced the time at which peak activity occurred in females, but not males. P1-5E shortened the length of the active phase in WT males, but had no effect on

ArKO males or females of either genotypes. Finally, p1-5E altered the magnitude of

photic-induced shifts in the phase of activity onset, suggesting that neonatal estrogenic signaling impacts circadian behavioral plasticity in adult mice. Importantly, this work expands our knowledge regarding both the critical periods of development of the adult timekeeping system and the role that estrogenic signaling plays in the expression of daily and circadian activity rhythms throughout life.

Introduction

A circadian rhythm describes a 24 hr period under constant conditions. While the circadian timekeeping system is critical to homeostasis, it does not operate autonomously from its environment. Rather, it is plastic within physiologic limits, allowing for acute responsiveness to subsequently integrated cues, including alterations in the length of day, light intensity, and peripheral clock-associated feedback. One such influence on the expression of rhythmicity is gonadal hormone signaling. Substantial sex differences in chronobiology (Mong et al., 2011), higher rates of sleep and circadian disruption- associated pathologies in women (Bailey and Silver, 2013), and altered daily and circadian rhythm activity following circulating estrogen modification (Blattner and

Mahoney, 2014; Royston et al., 2014) strongly suggest a role for estrogenic signaling in the regulation of daily rhythms.

In addition to the effects of circulating estrogens (Royston et al., 2014), developmental estradiol exposure permanently shapes the expression of circadian rhythms. In rodents, peri- and neonatal exposure to estrogens and/or androgens has a masculinizing effect on the brain (Phoenix et al., 1959; Arnold, 2009), while the lack of these hormones or the sequestration of estrogens by alpha-fetoprotein (Uriel at al., 1976) results in a feminized phenotype. This early life hormonal exposure organizes neural circuits and their respective molecular milieu, adjusting hormone responsiveness in the adult. For example, in adult female rats and hamsters, circulating estradiol shortens the duration of the free running period. However, female rodents masculinized by treatment with testosterone in the neonatal period fail to shorten τ when administered estradiol as adults (Zucker et al., 1980; Albers, 1981). Further, female mice incapable of

synthesizing estrogen exhibit less wheel running activity, but a longer τ than ovariectomized (OVX) wildtype (WT) females (Brockman et al., 2011). While these data clearly demonstrate innate differences in circadian activity resulting from aberrant estrogenic signaling during development, it remains unknown when the timekeeping system is sensitive to or can be disrupted by gonadal hormones.

The cytochrome P450 enzyme aromatase is required for the final step in the conversion of C19 steroid hormones to estradiol (Nelson et al., 1993). This enzyme is encoded by the cyp19 gene. To investigate the organizational role of estradiol during a distinct developmental period on the expression of adult circadian patterns, I utilized mice lacking the proximal promoter region and the first 2 exons of the cyp19 gene.

These mice are unable to produce aromatase, and are therefore incapable of synthesizing estradiol, though their ability to respond to exogenous estradiol remains intact (Honda et al., 1998). Aromatase knock-out (ArKO) mice and WT littermates were administered either estradiol (E) or peanut oil daily for the first five days of life (p1-5E or p1-5OIL, respectively). This critical period was selected because it encompasses the emergence of clock gene rhythmicity (p3-5) (Munoz et al., 2000) and light responsiveness (p4) (Huang et al., 2010) within the suprachiasmatic nucleus (SCN), a bilateral hypothalamic structure that is both regarded as the ‘master oscillator’ and required for normal circadian activity rhythms (reviewed in Welsh et al., 2010). Following sexual maturation, both male and female mice were gonadectomized (GDX) and treated with exogenous estradiol.

Timekeeping activity variables were then measured to determine the effect of perinatal modulation of estrogenic signaling on the adult expression of daily and circadian rhythms. Through this experimental design, I demonstrate here that 1) p1-5E perturbs

specific daily and circadian activity rhythm parameters in adult WT animals, 2) developmental estradiol administration to ArKO animals rescues certain WT-like adult activity patterns, and 3) that select circadian timekeeping parameters undergo estrogen- sensitive organization during the first 5 days of life.

Methods

Animals and Housing

Circadian activity patterns in intact and GDX male and female ArKO mice have been reported previously (Brockman et al., 2011). The ArKO breeding colony at the

University of Illinois Urbana-Champaign was originally derived by crossing male and female C57Bl/6 mice obtained from Dr. Honda (Fujita Health University, Japan) that were heterozygous (+/-) for deletions in the cyp19 1st and 2nd exons, as well as the proximal promoter region as described previously (Honda et al., 1998). All mice used for the studies described here were generated by +/- ArKO breeding pairs provided by Dr.

Bunick (UIUC), and were maintained in accordance with the University of Illinois

Urbana-Champaign Institutional Animal Care and Use Committee guidelines and the

NIH guide for the care and use of laboratory animals.

Pregnant dams were observed daily for the presence of litters. The day of birth was designated as postnatal day 1 (p1). Dams were arbitrarily assigned to have their litters treated with E or peanut oil (OIL) when they gave birth to their first litter. All pups were injected subcutaneously (s.c.) at the same time each day for the first 5 days of life

(p1-5) with 20 µL of either OIL or E in peanut oil (1 mg/mL). Peanut oil was selected to minimize infanticide (Hisasue et al., 2010). Entire litters received uniform treatments to

minimize maternal preference. All subsequent litters born to each dam received the same treatment as the first litter. Mice used in this study were generated from 6 breeding pairs whose litters received OIL and 6 breeding pairs whose litters received E. Pups remained with their dam until they were weaned at p21.

At weaning, ear tissue was collected from each animal and genotyping was performed via PCR as described (Bakker et al., 2002). Briefly, tissue was combined with

50 mM NaOH and denaturated at 95 C for 10 minutes, vortexed, and quenched with 1 M

Tris (pH 8). Two distinct sets of primers were used to interrogate the extracted DNA for either the wildtype (WT) aromatase gene (375 bp; ARK-1/ARK-2) or the mutant gene

(231 bp; ARK-1/ARK-3). The sequences used were as follows (5’ to 3’): ARK-1 CGT

GGG CAG GTG ATC AGT TTA CCA TGT CCT AAT CTT CAC; ARK-2 TCT TCT

GAG GCC AAA TAG CGC AAG ATG TTC; ARK-3 CTG CTA AAG CGC ATG CTC

CAG ACT GCC TTG). WT and homozygous ArKO animals were selected for daily and circadian activity analysis.

Animals were maintained at a constant temperature (22 C) in 12:12 hr light:dark

(LD) (unless otherwise noted). Light provided a consistent Zeitgeber across the 24 hr day, with Zeitgeber time (ZT) 0 signifying the time of lights on and ZT12 indicating lights off. Food and water were provided ad libitum. Animals housed within the breeding colony were group-housed with same sex littermates from weaning (p21) until experimental use. Breeding animals were provided with Teklad 8604 rodent diet, which contains 600 µg/g phytoestrogens (Weber at al., 2001). Experimental animals were fed

Teklad 2016 rodent diet because it contains levels of soy estrogen isoflavones, including

daidzein and genistein aglycone equivalents that are subthreshold for eliciting biological activity (<12 ppm; Harlan Laboratories, 2014).

Gonadectomy and estradiol replacement

To assess whether estradiol administration during the first 5 days of life alters the adult timekeeping system, endogenous sex hormone signaling was disrupted by GDX in all experimental animals following sexual maturation (p50). On the day of surgery, mice were deeply anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively; i.p.) and maintained with isoflurane gas as necessary. In females, ovaries were removed through bilateral flank incisions, and the muscle/fascia and overlying skin were closed with surgical silk and staples. In males, the testes, epididymis, and epididymal fat were bilaterally removed via a vertical incision through the linea alba. Following GDX, animals were singly housed in plastic cages (28 cm L x 16 cm W x 12 cm H). To assess gross sexual maturation effects of p1-5E, anogenital distance (AGD) was measured on the day of GDX in all animals. Weights did not vary significantly (data not shown).

Accordingly, only raw AGD is reported here. AGD was defined as the distance between the anus and the vaginal opening or penis.

Approximately ten days after ovariectomy, on p60, experimental mice were anesthetized under isoflurane gas and pellets containing 50 µg estradiol compounded with

20 mg ≥99% cholesterol (Sigma Aldrich; St Louis, MO) were implanted subcutaneously as described previously (Ogura et al., 2007; Risbridger et al., 2001; Royston et al., 2014).

During early pilot work, it was discovered that GDX animals receiving no exogenous hormone supplementation did not run on their wheels, preventing any between group

comparisons. Consequently, I elected to treat all animals with an equal dose of estradiol.

Activational estradiol robustly increases the magnitude of total daily wheel running

(Blattner and Mahoney, 2014; Royston et al., 2014). This experimental approach ensured that all adult animals had standardized levels of circulating hormone, allowing me to differentiate between the effects of perinatal estradiol signaling on daily and circadian activity outputs. The use of pellets removed the need for daily hormone injections, which may have served as a non-photic cue. The estradiol dose selected increases uterine weight, induces estrous-associated vaginal cytology, and alters daily and circadian wheel running activity rhythms (Blattner and Mahoney, 2013; Carroll and Pike, 2008; Royston et al., 2014).

Five days after pellet implantation (p65), cages were outfitted with a metal running wheel (11 cm diameter) affixed to the cage top. During the light phase, the intensity of light measured at the top of the cage ranged from 220-360 lx (average 290 lx). Daily wheel running activity was quantified beginning 10 days after pellet implantation, as previously described (Ogawa et al., 2003; Royston et al., 2014). Wheel running data was collected between 10 days and 4 weeks following pellet implantation.

Assessment of daily activity patterns and circadian variables

A magnetic switch affixed to each cage detected wheel revolutions, which were recorded in 10-min bins using VitalView (MiniMitter, Bend, OR, USA). Data was visualized with ActiView (Mini Mitter, Bend, OR, USA). A bin was considered active if the total amount of activity during the 10 min was equal to or exceeded at least 10% of the animal’s maximum daily activity. The following parameters were assessed in LD:

average daily activity, the proportion of activity completed in the light vs the dark (LD proportion), temporal patterning of activity across the total 24 hr day, acrophase, amplitude, and phase angle. The duration of the free running period (τ) and the active phase (α) were determined when animals were housed in total darkness (DD).

For parameters assessed in LD, experimental animals were housed in 12:12 LD conditions for at least one week prior to data collection. Consistent phase angle during the last 4 days of this period was required to confirm entrainment (data not shown). All animals that ran on the provided wheels exhibited evidence of photic entrainment, but mice that did not run at all were omitted from the study. Mean daily wheel running activity was determined by averaging the number of wheel revolution per 10 min bin for each animal over 3 consecutive days in LD. The mean values per 10 min bin were summed to yield the total wheel revolutions completed during the 24 hr day. To compare the patterning of daily activity over time across treatment groups, each 10 min activity bin was averaged over 3 consecutive days and plotted hourly. Statistical comparisons were performed each hour on the hour. The LD proportion describes the number of wheel revolutions completed during the light phase (L) compared to those completed during the dark phase (D). Defined by the formula L/(L+D), an LD proportion close to 1 is associated with an animal active equally in the light and dark, while a value nearing 0 is indicative of an entirely nocturnal animal. Average LD activity over 3 days was used to calculate LD proportion.

Acrophase, the time of peak activity, was calculated with ActiView using the observed 3-day LD average of each animal’s wheel running rhythm. Amplitude reflects the difference between the activity peak and mesor observed when daily wheel running

was averaged over 3 days in LD. Phase angle of activity onset was determined by measuring the difference in minutes between lights off and the onset of activity across 5 consecutive days in LD. Activity onset was defined as the first 10 min bin of activity that equaled or exceeded 10% of the maximum activity peak that was not followed by more than 2 consecutive 10 min bins of inactivity.

Behavioral assessments requiring constant darkness (DD) were conducted over at least 8 consecutive days. The first 3 days were omitted from analysis to limit the contribution of acute transition effects, and the last 5 days were used for analyses. The length of the free running period (τ) was measured by fitting a line through activity onset for 5 consecutive days as previously described (Agostino et al., 2007; Ogeil et al., 2010).

The length of the active period (α) was determined by averaging the duration between the time of activity onset and activity cessation over 5 days in DD. Activity onset was defined as stated above. Activity cessation was defined as the last 10 min bin with wheel revolutions equal to or greater than 10% of peak activity before a break in activity lasting

2 or more hours.

To determine the effects of p1-5E on the magnitude and direction of shifts in the phase of activity onset in observed animals subjected to a light pulse at varying times of day, I employed a previously described Aschoff type II phase response paradigm

(Royston et al., 2014). Briefly, animals were entrained to LD, and released into DD for at least 24-hr prior to administration of a 1 hr light pulse (~290 lx). Pulses were administered at times corresponding to ZT4, 16, or 22. Entrained animals that were released into DD, but not pulsed with light, served as shams. All animals then continued in DD for at least 5 days. The phase shift was defined as the difference between the pre-

and post-pulse activity onset. The average from 3 days prior to and 5 days following the pulse were used. The day immediately following pulse administration was discarded to account for acute after effects.

Statistical Analyses

Results are reported as mean ± S.E.M. For total daily activity, LD proportion, α,

τ, phase angle of activity onset, acrophase, and amplitude, between group differences were assessed using three way ANOVAs and/or non-parametric Dunn’s test with sex, genotype, and drug as independent variables, followed by Tukey post-hoc tests as appropriate. To determine differences in the patterning of wheel running activity across the 24 hr day, two-way repeated measures ANOVA with treatment (drug/genotype) and time as independent variables were used, followed by Tukey post-hoc test as appropriate.

Males and females were analyzed separately. An a priori interest in the effect(s) of drug and genotype within each time point negated the need for multiple comparison analyses.

Sex, genotype, and/or drug-induced effects on phase shifts in activity onset in response to light pulse administration were assessed within each time point using three-way

ANOVAs with the above stated independent variables. This was followed by Tukey post-hoc tests as appropriate. Two-way ANOVAs followed by similar post-hoc analyses were used to identify significant effects of genotype and drug within sex. Differences in the phase response elicited by a light pulse at a given time and non-pulsed counterparts within a given sex/drug/genotype combination were assessed with one-way ANOVAs, followed by Tukey post-hoc testing as appropriate. All analyses were performed with

SigmaPlot 12.0 (Systat Software, Inc). Comparisons resulting in a priori value (p) < 0.05 were considered significantly different.

Results

Neonatal estradiol administration alters anogenital distance length in male and female mice

Increased AGD is characteristic of the masculine phenotype (reviewed in Golub et al., 2010). Males in the current study had longer AGD than females (F1 = 162.69, p<0.001) (Figure 3.1). Administration of estradiol from p1-5 lengthened anogenital distance in WT (6.57 ± 0.17 mm) and ArKO females (6.93 ± 0.32 mm) compared to p1-

5OIL counterparts (WTF+Oil: 6.21 ± 0.28 mm; ArKOF+Oil: 5.65 ± 0.63 mm; p<0.05). A similar and more robust effect was observed in WT males (WTM+Oil: 11.35 ± 0.71 mm;

WTM+E: 14.27 ± 0.68 mm; p = 0.007). ArKO males treated with oil (13.76 ± 0.80 mm) had longer AGD than oil treated WT males (p = 0.036), an effect potentially accounted for by the higher levels of circulating androgen and testosterone present in these mice compared to WTs (Bianco et al., 2006). Administration of p1-5E to ArKO males (10.52 ±

0.66 mm) restored AGD to levels nearly the same as p1-5OIL WT males. These findings, while not the direct focus of this study, confirm that p1-5E exerted the predicted masculinization effect by lengthening AGD in WT males and females of both genotypes.

Further, the increased AGD observed in ArKO males was rescued to WT levels by p1-5E.

Manipulation of developmental estradiol exposure modulates daily activity patterns in

WT and ArKO mice

Representative activity patterns (actograms) for each treatment group are shown in Figure 3.2A. Female mice were significantly more active than males (Figure 3.2B) (F1

= 6.88, p = 0.012). Irrespective of sex, mice treated with estradiol from p1-5 completed fewer wheel revolutions per day than mice administered oil (F1 = 31.02, p<0.001).

ArKO males administered oil (24,580.57 ± 4,739.56 wheel revolutions per day) from p1-

5 were more active than oil-treated WT males (16,405.57 ± 3,628.88 wheel revolutions per day, p = 0.026). However, while ArKO males that received p1-5E (8,279.9 ±

1,663.98 wheel revolutions per day) completed significantly fewer wheel revolutions per day than their oil-treated counterparts (p<0.001), they were as active as WT males

(p>0.05). Further, within WT males, p1-5E (12,925.75 ± 3,523.12 wheel revolutions per day) did not alter the magnitude of wheel running compared to p1-5OIL (p>0.05).

Female mice treated with p1-5E ran less than those treated with oil (F1 = 22.758, p<0.001) (Figure 3.2A,B). This pattern was observed in both WT and ArKO mice.

Specifically, WT female mice treated with oil (27,391.273 ± 3,176.84 wheel revolutions per day) were significantly more active than WT female mice administered perinatal estradiol (15,949.80 ± 2,507.93 wheel revolutions per day, p = 0.035). Similarly, within

ArKO females, those administered oil from p1-5 (36,250 ± 6,784.72 wheel revolutions per day) ran more than those treated with estradiol (9,771.833 ± 2,423.79 wheel revolutions per day, p<0.001). No differences were observed between ArKO or WT females within either the oil or estradiol treatment groups (p>0.05). An additional pattern evident is that ArKO males resemble both female ArKO and WT mice. Specifically, in these three groups of animals, treatment with estradiol early in development results in a

decrease in activity levels in adult animals. In contrast, WT males do not exhibit differences in total activity following estradiol treatment.

The proportion of wheel running revolutions completed in the light compared to the dark (LD proportion) was calculated to determine if modulation of perinatal estrogenic signaling affects nocturnal activity consolidation in adult mice (Figure 3.2C).

The presence or absence of estrogenic signaling during the first 5 postnatal days had no effect on LD proportion. Estradiol did not perturb this parameter in WT animals or alter the LD proportion in estrogen deficient ArKO animals. There was a main effect for sex

(F1 = 4.979, p = 0.031), and a significant interaction between sex and genotype (F1 =

4.408, p = 0.041). Further analyses revealed that wheel running activity was more consolidated to the dark phase in female ArKO mice compared to male ArKOs (Figure

3.2C) (p = 0.006). Similarly, ArKO males, irrespective of whether they were administered oil (0.017 ± 0.007 wheel revolutions) or estradiol (0.024 ± 0.008 wheel revolutions) from p1-5 had a higher LD proportion than male WT mice (p = 0.024).

Further, female mice of both genotypes and drug treatment groups were equally nocturnal, and no differences were detected across WT mice, irrespective of drug or sex.

To determine the effect(s) of p1-5 estrogenic signaling modulation on the distribution of activity across the day within males and females, mean wheel revolutions observed over 3 days in LD were organized into 1 hr bins and plotted over the 24 hr day

(Figure 3.2D). All mice became active following lights off and inactive following lights on (Figure 3.2A,D). Within males (Figure 3.2D), two-way repeated measures ANOVA revealed a significant effect of treatment (F3 = 4.454, p = 0.011) and time (F23 = 49.958, p<0.001), as well as an interaction between them (F69 = 3.073, p<0.001). Similar effects

were observed within females with respect to treatment (F3 = 9.651, p<0.001), time (F23 =

64.496, p<0.001), and their interaction (F69 = 5.123, p<0.001). While the magnitude of wheel running across time varied by treatment within males, the general trend of temporal distribution was consistent; wheel running was consolidated to the early versus later evening. Within the females, the same trend was observed only within p1-5E animals. Conversely, p1-5OIL animals of both genotypes continued to exhibit a robust quantity of activity across a greater amount of the dark phase. Within both males and females, differences in the amount of wheel running activity across time were greater between p1-5E and p1-5OIL ArKO animals than between p1-5E and p1-5OILWT animals.

Influence of perinatal estrogenic signaling modification on adult acrophase and amplitude

I calculated acrophase (Figure 3.3A) to determine if perinatal estradiol signaling regulates the time at which peak wheel running activity occurs in adult mice. Similar to the effects observed with respect to total activity, I found that within WT males acrophase was not affected by early exposure to estradiol. Though a strong trend was observed between p1-5OIL and p1-5E male ArKO mice, the difference in acrophase between them was not significant. However, within WT and ArKO females, estradiol exposure in the first five days of life had a dramatic effect on the timing (phase) of the peak in activity. Specifically, even allowing for the effects of sex and genotype, estradiol advanced acrophase compared to oil-treated mice F1 = 12.591, p<0.001). When WT and

ArKO females were combined, estradiol administration from p1-5 significantly advanced the timing of peak activity compared to oil treatment (F1 = 17.077, p<0.001). This effect

was observed when comparisons were made between WT females treated with estradiol

(ZT 15.90 ± 0.36) or oil (ZT 16.99 ± 0.22, p = 0.009), as well as between ArKO females administered estradiol (ZT 16.01 ± 0.29) or oil (ZT 17.38 ± 0.21, p = 0.007). No effect of genotype was noted within female mice. Conversely, within males, no statistically significant differences were observed across genotype or drug treatment, despite the strong trending difference between oil and estradiol-treated ArKO males. Further, no sex differences were observed between males and females.

The difference between the daily activity peak and mesor was calculated to determine treatment-induced effects on wheel running amplitude (Figure 3.3B). Female mice exhibited a greater amplitude than male mice (F1 = 4.251, p = 0.044). Irrespective of sex, animals administered estradiol had a smaller amplitude than those administered oil (F1 = 7.956, p = 0.007). Further analyses revealed that the greatest effect of early life estrogenic signaling manipulation on amplitude was observed between ArKO female mice treated with estradiol or oil. ArKO females treated with oil exhibited a significantly higher amplitude of wheel running activity (549.967 ± 51.979 wheel revolutions) than their estradiol-treated counterparts (382.473 ± 47.012 wheel revolutions, p = 0.023).

Interestingly, neither group differed from their WT female counterparts, which, in turn, did not differ from each other. No differences were detected within males, regardless of drug treatment or genotype.

Estradiol levels during p1-5 do not influence the phase angle of activity onset in adult mice

The difference between the time of lights off and the onset of activity was determined for all treatment groups to assess whether estrogenic signaling during p1-5 contributes to the strength of photic entrainment (Gorman and Lee, 2002) in adult mice

(Figure 3.4). While this parameter is robustly regulated by circulating estradiol and activation of ESR2 (Royston et al., 2014; Blattner and Mahoney, 2014), manipulation of p1-5 estradiol levels had no effect on the phase angle of activity onset in adult mice. This finding held true across sexes, genotypes, and drug treatments.

Estrogenic signaling between p1-5 modifies circadian rhythm variables measured in constant darkness

I sought to determine the effects of developmental estradiol on endogenous circadian parameters. I measured both the length of the inherent period (τ) (Figure 3.5A) and the active phase (α) in mice housed in DD (Figure 3.5B). Three-way ANOVA revealed a significant interaction between sex and genotype (F1 = 10.263, p = 0.002), as well as an interaction between sex and drug (F1 = 5.597, p = 0.022) on the regulation of τ

(Figure 3.5A). Within males, τ was longer in estradiol-treated mice compared to those administered p1-5OIL (F1 = 4.666, p = 0.040). Further, ArKO males exhibited a longer τ than WT males (F1 = 7.833, p = 0.010). ArKO males administered p1-5E (23.753 ± 0.078 hr) had the longest τ, which was significantly greater than that observed in their WT estradiol-treated counterparts (23.552 ± 0.0534 hr). Among males, no perinatal effects of estradiol were found either within WT or within ArKO mice. Similarly, within females, neither genotype nor developmental estradiol administration influenced period length.

However, female ArKO mice, irrespective of drug treatment, exhibited a shorter period than male ArKO mice (p = 0.001).

I then examined variability in the length of the active phase (Figure 3.5B). A three-way ANOVA revealed a drug effect on the length of α (F1 = 8.518, p = 0.005).

However, neither genotype nor sex influenced the length of the active phase. Compared to WT oil-treated male counterparts (697.429 ± 40.46 min), α was shortened by p1-5E in

WT males (438.00 ± 104.99 min, p = 0.007). Within estradiol-treated males, α was shorter in ArKO mice (646.455 ± 48.11 min) than in WT mice (p = 0.015). Manipulation of estrogenic signaling during the first 5 days of life had no effect on the length of the active phase in female WT or ArKO mice.

To determine the effect(s) of sex and manipulation of neonatal estrogenic signaling on the behavioral response to photic stimuli, entrained animals were housed in

DD for 24 hr and then subjected to a 1 hr light pulse at ZT4, ZT16, or ZT22 (Figure 3.6).

Animals placed into DD, but not pulsed with light served as non-pulsed shams. No differences were observed between sham animals of varying sexes. Similarly, animals subjected to a light pulse at ZT4, a time corresponding to the early part of the light phase in the previous LD housing environment, did not exhibit shifts in the phase of activity onset that were different than those elicited in non-pulsed sham animals for any treatment group. A significant interaction between genotype and drug (F1 = 5.577; p = 0.023) was observed in animals pulsed at ZT4. Further, male ArKO mice treated with p1-5OIL (85.6

± 27.82 min) phase advanced more than their WT counterparts (19.5 ± 11.47 min; p =

0.023). No differences between females pulsed at ZT4 were observed.

When a light pulse was administered in the early subjective evening (ZT16), a larger phase delay was observed in females (F1 = 4.933; p = 0.032) and ArKO mice (F1 =

5.293; p = 0.026) compared to males and WT mice, respectively. Male (-63.38 ± 11.35 min) and female (-90.57 ± 13.72 min) ArKOs receiving p1-5E, as well as WT females administered p1-5OIL (-53.82 ± 7.81 min) exhibited a phase shift significantly delayed compared to non-pulsed sham animals of the same treatment group (male ArKO p1-5E:

24.88 ± 12.30 min; female ArKO p1-5E: 0.17 ± 8.40 min; female WT p1-5OIL: 22.00 ±

6.09 min; p<0.001 for each respective comparison). Within males, p1-5E increased the magnitude of phase delay induced by a ZT16 light pulse (F1 = 4.450; p = 0.047). A similar effect was associated with the ArKO phenotype in females (F1 = 7.938; p =

0.010). Further, p1-5E ArKO females exhibited a greater phase delay that their WT counterparts when pulsed with light at ZT16 (p = 0.006).

Administration of 1 hr light pulse at ZT22, a time corresponding to the late subjective night, advanced the phase of activity onset in all animals. However, only male

ArKOs (126.25 ± 16.55 min) and WT females (60.56 ± 5.80 min) treated with p1-5OIL exhibited a phase advance significantly greater than that observed in non-pulsed sham animals (male ArKO p1-5OIL: 23.14 ± 10.10 min, p = 0.013; female WT p1-5OIL: 22.00 ±

6.09 min, p = 0.008). Within animals pulsed at ZT22, I observed a significant interaction between genotype and drug (F1 = 10.891; p = 0.002). There were no treatment effects within males. However, p1-5E (100.50 ± 19.16 min) substantially increased the phase advance induced by a ZT22 light pulse in WT females compared to those treated with p1-

5OIL (60.56 ± 5.80 min; p = 0.032). Interestingly, the opposite effect was observed in female ArKO mice; here, p1-5E (56.00 ± 9.43 min) reduced the phase advance compared

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to those treated with p1-5OIL (111.00 ± 16.42 min; p = 0.030). Within oil-treated females,

ArKO mice exhibited a greater phase advance compared to WT mice (p = 0.017).

Importantly, the administration of p1-5E to ArKO females reduced the magnitude of this phase advance to levels nearly identical to WT females receiving p1-5OIL.

Discussion

Perinatal estrogenic signaling in mice plays a robust organizational role in the development of both sexual differentiation and adult hormone responsiveness (reviewed in Gillies and McArthur, 2010). In the current study, I investigated whether exogenous estradiol administration during a critical period associated with maturation of the SCN disrupts the expression of normal daily and circadian activity rhythms in adult male and female mice. Further, I examined whether similar treatment during the same time was sufficient to alter these rhythms in mice not exposed to estradiol at any other developmental time point. Finally, I determined which adult biological timekeeping variables are sensitive to estrogenic influence during the first 5 days of life.

I discovered that total daily activity in both adult male and female mice is altered by p1-5E, though not uniformly across the sexes (Figure 3.2A). In males administered p1-5OIL, ArKO mice exhibit greater total daily wheel running behavior than WT males.

The magnitude of total activity in male ArKO mice treated with oil is more similar to that observed in female mice, and in accordance with the innate sex differences in total wheel running observed in mice in the current and previous studies (Brockman et al., 2011;

Blattner and Mahoney, 2012). Interestingly, between males administered estradiol early in development, ArKOs have the same amount of average wheel running as WT males,

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suggesting that estradiol exposure exclusively during this period is sufficient to recapitulate the WT masculine phenotype. Adult WT or ArKO females treated with p1-

5E were less active than their oil-treated counterparts. These data suggest that, irrespective of estradiol levels at other developmental time points, enhanced exogenous estradiol exposure during p1-5 in females dampens the previously demonstrated ability of estradiol to increase the magnitude of wheel running during the adult phase in females

(Royston et al., 2014).

ArKO males exhibited wheel running behavior that was less nocturnal than what was observed in WT males. Modulation of circulating estradiol from p1-5 did not alter this effect, suggesting that either estradiol does not exert an organizational effect on adult

LD proportion, or that estradiol does have an effect, but that it is not during the perinatal period tested. Robust regulation of LD proportion by circulating estradiol in adult female mice has been previously reported (Royston et al., 2014). The lack of an effect on this measurement by perinatal estradiol manipulation suggests that LD proportion is mediated by estrogen through an either exclusively activational mechanism or through an organizational pathway that is associated with a different critical period.

Though not responsible for the amount of activity occurring between the L and D phases, estrogenic signaling during p1-5 did, in part, program when wheel running occurred during the dark phase (Figure 3.2D). While activity magnitude varied by genotype and p1-5 treatment, male mice were most active in the early evening. This same trend was observed in female mice administered p1-5E. Conversely, oil-treated females of both genotypes continued to exert relatively consistent levels of wheel running activity across the dark phase. These data suggest that concentration of activity around

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the early evening may be a masculine characteristic, and that p1-5E is sufficient to program this trait in female mice.

The time at which peak activity occurred in adult female mice of either genotype tested was advanced by p1-5E (Figure 3.3A), demonstrating an organizational role for estrogen in the control of acrophase. Interestingly, administration of estradiol to adult

WT female mice delays acrophase (Royston et al., 2014), suggesting that these organizational and activational estrogenic mechanisms may be exerting dichotomous effects. While the effect of p1-5E was not significantly different than p1-5OIL within

ArKO males, a strong trend toward advancing acrophase was observed. Combined with my findings in females, these data suggest that exogenous elevation in estradiol during the first 5 days of life in animals characterized by low brain estradiol levels is sufficient to recapitulate the WT male phenotype. Amplitude (Figure 3.3B), which is increased by circulating estradiol in adult female mice (Royston et al., 2014), was reduced in ArKO females administered p1-5E. Similar to the effect of perinatal estradiol on acrophase in female mice, p1-5E appears to exert an effect directly opposite to the result observed following treatment in adults. Together these findings suggest that perinatal estrogen exposure dampens estradiol responsiveness within these variables in adult female mice.

Phase angle of activity onset is advanced by circulating estradiol (Blattner and

Mahoney 2014; Royston et al., 2014) through an ESR2-dependent mechanism (Royston et al., 2014). Further, this variable is altered in adult male and female WT and ArKO mice following GDX (Brockman et al., 2011). These previous findings support a role for estradiol in the regulation of phase angle. However, combined with the current finding that perinatal estrogenic signaling manipulation does not alter adult phase angle of

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activity onset irrespective of sex, genotype, or drug treatment (Figure 3.4), the role of estradiol is likely entirely activational. Alternatively, it may be exerting an organizational effect during a critical period different than the one tested here.

Period length is an innate property of the SCN, and is lost following SCN ablation

(Welsh et al., 2010). Further SCN-lesioned animals subsequently transplanted with exogenous SCN tissue express the graft donor’s circadian period (King et al., 2003,

Ralph et al., 1990). Period length across both sexes was relatively unaffected by p1-5E

(Figure 3.5A), though ArKO males did exhibit a longer τ than either WT males or their female counterparts. These findings suggest that estrogenic signaling during critical period(s) other than the one tested here programs adult period length, potentially in conjunction with the effects of activational estradiol.

The length of the active phase was shortened by p1-5E administration to WT males (Figure 3.5B), demonstrating that elevated estradiol in animals with already relatively high neonatal levels disrupts normal α patterning. This effect was not observed within ArKO males, potentially indicating that estradiol in excess of, but not below, WT levels in males can alter α in adults. P1-5E had no effect on α in females. Similarly, α was not altered by activational estradiol modulation in adult females (Royston et al.,

2014), suggesting that the length of the active period in females may not be under the control of estrogenic mechanisms. However, there exists the possibility that lack of estrogens during an untested critical period programs adult α; Blattner and Mahoney

(2012) reported that adult intact female mice in which estrogen subtype receptor 1

(ESR1) has been removed (αERKO) exhibited a longer active period compared to WT females.

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I used a photic phase response paradigm to discover that p1-5E levels are sufficient to program the behavioral response induced by the administration of a light pulse at select times of day, and that these effects are sex specific. As demonstrated previously (Brockman et al., 2011; Blattner and Mahoney, 2013; Royston et al., 2014),

LD entrained mice released into DD phase advanced slightly. Introduction of a light pulse at ZT4 induced a similar phase advance in most treatment groups. However, male mice that developed in the absence of estradiol completely exhibited a much larger phase advance at this time than WTs. This effect was rescued by p1-5E treatment (Figure 3.6), suggesting that estrogenic signaling is required during this critical period for the normal behavioral response to a light pulse at ZT4 in male mice.

When the light pulse was administered at ZT16, male mice treated with p1-5E exhibited a larger phase delay than those treated with oil. Because androgens are readily converted to estradiol in the male neonatal hypothalamus (Mong et al., 2011), I predicted that estradiol-treated males would exhibit the most masculinized wheel running behavioral phenotype. However, females exhibited greater phase delays than males when pulsed at this time point, with the largest observed in ArKO females, suggesting that males treated with p1-5E are more similar to females than to males receiving p1-5OIL.

Further complicating the story, elevated levels of circulating estradiol in adult females dampens the phase delay associated with a light pulse at ZT16 (Royston et al., 2014).

Together, these findings may point to an inverted-U where estradiol mediates the magnitude of photic phase responsiveness, and that innate masculine and feminine phenotypes oscillate around this U in an estradiol dose-dependent manner. Future study is needed to verify this model.

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Mice subjected a light pulse in the late subjective night responded by advancing the phase of activity onset. No treatment effect was observed in males. However, the increased advanced observed in p1-5E WT females compared to those receiving oil indicates that elevated estradiol disrupts adult behavioral responsiveness to a stimulus at

ZT22. Interestingly, the opposite effect was observed within ArKO females. Oil-treated

ArKO females phase advanced more robustly than WT females receiving the same treatment. Administration of p1-5E to ArKO females returned their phase advance to one of similar magnitude as exhibited by WTs. These findings demonstrate that within females, p1-5E is capable of both disrupting normal phase shifting and also rescuing altered behavioral responsiveness following a ZT22 light pulse.

Conclusions

The current study demonstrates that estradiol exerts organizational control over select daily and circadian activity rhythms. Several distinct patterns emerged from my data. First, some variables, despite being estrogen-sensitive (Brockman et al., 2011;

Blattner and Mahoney 2012; Blattner and Mahoney, 2013; Blattner and Mahoney, 2014;

Royston et al., 2014), were not altered by p1-5 treatment with estradiol. LD proportion, τ, and phase angle of activity onset were relatively unaffected, suggesting that these variables are either not sensitive to permanent organization by estrogenic signaling, or that estradiol can program them, but not during the critical period tested. The second pattern to emerge was that estradiol during p1-5 in excess of those circulating in WT animals disrupts adult expression of certain daily and circadian activity outputs. For example, within WT females, p1-5E reduces total wheel running, concentrates activity to

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the early evening, advances acrophase, and exaggerates the normal behavioral responsiveness to a light pulse in the late subjective night. Further, WT males treated with p1-5E exhibit a shorter active period than those administered oil. Finally, I show here that modulation of estradiol from p1-5 rescues select daily and circadian activity outputs disrupted in mice lacking estrogens throughout development. Disruptions in the quantity of total wheel running and behavioral response to a ZT4 light pulse in male

ArKO mice are returned to WT levels by p1-5E. Similarly, the increased phase advance observed in female ArKOs following a ZT22 light pulse is eliminated by p1-5E.

Importantly, the work presented here substantially expands our understanding of the developmental effects exerted by estradiol and marks the first time its organizational role has been investigated across the expression of so many daily and circadian behavioral outputs.

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References

Agostino PV, Plano SA, Golombek DA. 2007. Sildenafil accelerates reentrainment of

circadian rhythms after advancing light schedules. Proc Natl Acad Sci U S A

104:9834-9839.

Albers HE. 1981. Gonadal hormones organize and modulate the circadian system of the

rat. Am J Physiol 241:R62-6.

Arnold AP. 2009. The organizational-activational hypothesis as the foundation for a

unified theory of sexual differentiation of all mammalian tissues. Horm Behav

55:570-578.

Bailey M, Silver R. 2013. Sex differences in circadian timing systems: Implications for

disease. Front Neuroendocrinol.

Bakker J, Honda S, Harada N, Balthazart J. 2002. The aromatase knock-out mouse

provides new evidence that estradiol is required during development in the female

for the expression of sociosexual behaviors in adulthood. J Neurosci 22:9104-

9112.

Bianco JJ, McPherson SJ, Wang H, Prins GS, Risbridger GP. 2006. Transient neonatal

estrogen exposure to estrogen-deficient mice (aromatase knockout) reduces

prostate weight and induces inflammation in late life. Am J Pathol 168(8): 1869-

78.

Blattner MS, Mahoney MM. 2013. Photic phase-response curve in 2 strains of mice with

impaired responsiveness to estrogens. J Biol Rhythms 28:291-300.

Blattner MS, Mahoney MM. 2012. Circadian parameters are altered in two strains of

108

mice with transgenic modifications of estrogen receptor subtype 1. Genes Brain

Behav 11:828-836.

Blattner MS, Mahoney MM. 2014. Estrogen receptor 1 modulates circadian rhythms in

adult female mice. Chronobiology International accepted for publication.

Brockman R, Bunick D, Mahoney MM. 2011. Estradiol deficiency during development

modulates the expression of circadian and daily rhythms in male and female

aromatase knockout mice. Horm Behav 60:439-447.

Carroll JC, Pike CJ. 2008. Selective estrogen receptor modulators differentially regulate

Alzheimer-like changes in female 3xTg-AD mice. Endocrinology 149:2607-2611.

Gillies GE, McArthur S. 2010. Estrogen actions in the brain and the basis for differential

action in men and women: a case for sex-specific medicines. Pharmacol Rev

62:155-198.

Golub MS, Wu KL, Kaufman FL, Li LH, Moran-Messen F, Zeise L, Alexeeff GV,

Donald JM. 2010. Bisphenol A: developmental toxicity from early prenatal

exposure. Birth Defects Res B Dev Reprod Toxicol 89(6): 441-66.

Gorman M, Lee TM. 2002. Hormones and Biological Rhythms. In: Becker J, Breedlove

S, Crews D McCarthy M, eds. Behavioral Endocrinology. 2nd ed. ed. Cambridge,

MA: MIT Press; 451.

Hisasue S, Seney ML, Immerman E, Forger NG. 2010. Control of cell number in the bed

nucleus of the stria terminalis of mice: role of testosterone metabolites and

estrogen receptor subtypes. J Sex Med 7:1401-1409.

Honda S, Harada N, Ito S, Takagi Y, Maeda S. 1998. Disruption of sexual behavior in

109

male aromatase-deficient mice lacking exons 1 and 2 of the cyp19 gene. Biochem

Biophys Res Commun 252:445-449.

Huang J, Lu C, Chen S, Hua L, Qian R. 2010. Postnatal ontogenesis of clock genes in

mouse suprachiasmatic nucleus and heart. Lipids Health Dis 9: 22.

King DP, Takahashi JS. 2000. Molecular genetics of circadian rhythms in mammals.

Annu Rev Neurosci 23:713-42.

Mong JA, Baker FC, Mahoney MM, Paul KN, Schwartz MD, Semba K, Silver R. 2011.

Sleep, rhythms, and the endocrine brain: influence of sex and gonadal hormones.

J Neurosci 31:16107-16116.

Muñoz Llamosas M, Huerta JJ, Cernuda-Cernuda R, García-Fernández JM. 2000.

Ontogeny of a photic response in the retina and suprachiasmatic nucleus in the

mouse. Brain Res Dev Brain Res 120(1):1-6.

Nelson DR, Kamataki T, Waxman DJ, Guengerich FP, Eastbrook RW, Feyereisen R,

Gonzales FJ, Coon MJ, Gunsalus IC, Gotoh O. 1993. The P450 superfamily:

update on new sequences, gene mapping, accession numbers, early trivial names

of enzymes and nomenclature. DNA Cell Biol 12(1): 1-51.

Ogawa S, Chan J, Gustafsson JA, Korach KS, Pfaff DW. 2003. Estrogen increases

locomotor activity in mice through estrogen receptor alpha: specificity for the

type of activity. Endocrinology 144:230-239.

Ogeil RP, Rajaratnam SM, Redman JR, Broadbear JH. 2010. Acute MDMA

administration alters the distribution and circadian rhythm of wheel running

activity in the rat. Brain Res 1359:128-136.

Ogura Y, Ishii K, Kanda H, Kanai M, Arima K, Wang Y, Sugimura Y. 2007. Bisphenol

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A induces permanent squamous change in mouse prostatic epithelium.

Differentiation 75:745-756.

Phoenix CH, Goy RW, Gerall AA, Young WC. 1959. Organizing action of

prenatally administered testosterone propionate on the tissues mediating mating

behavior in the female guinea pig. Endocrinology 65:369-382.

Ralph MR, Foster RG, Davis FC, Menaker M. 1990. Transplanted suprachiasmatic

nucleus determines circadian period. Science 247(4945):975-978.

Risbridger GP, Wang H, Frydenberg M, Cunha G. 2001. The metaplastic effects of

estrogen on mouse prostate epithelium: proliferation of cells with basal cell

phenotype. Endocrinology 142:2443-2450.

Royston SE, Yasui N, Kondilis AG*, Lord SV*, Katzenellenbogen JA, Mahoney MM.

2014. ESR1 and ESR2 differentially regulate daily and circadian activity rhythms

in female mice. Endocrinology accepted for publication.

Uriel J, Bouillon D, Aussel C, Dupiers M. 1976. Alpha-fetoprotein: the major high-

affinity estrogen binder in rat uterine cytosols. Proc Natl Acad Sci U S A

73:1452-1456 .

Weber, K.S., Setchell, K.D., Stocco, D.M., Lephart, E.D., 2001. Dietary soy

phytoestrogens decrease testosterone levels and prostate weight without altering

LH, prostate 5alpha-reductase or testicular steroidogenic acute regulatory peptide

levels in adult male Sprague–Dawley rats. J. Endocrinol 170, 591–599.

Welsh DK, Takahashi JS, Kay SA. 2010. Suprachiasmatic nucleus: cell autonomy and

network properties. Annu Rev Physiol 72:551-77.

Zucker I, Fitzgerald KM, Morin LP. 1980. Sex differentiation of t-e circadian system in

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the golden hamster. Am J Physiol 238:R97-101.

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Figure 3.1.

Figure 3.1. Anogenital distance (AGD) measured at p50 following p1-5 estradiol manipulation. Sample sizes (n) are shown below each bar. * and ** are indicative of comparisons where p<0.05 and 0.001, respectively.

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Figure 3.2.

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Figure 3.2. Effects of organizational estrogenic signaling manipulation from p1-5 on circadian wheel running activity. A, Representative double-plotted actograms depicting the organizational effect of estradiol (E) or oil treatment on daily wheel running activity in GDX WT and ArKO mice measured for 3 consecutive days in 12:12 light:dark (LD, lights off at ZT12). White and black bars indicate whether the lights were on or off.

Consecutive days are plotted on the y-axis, while the ZT is shown on the x-axis. Each black bar represents a 10 min bin, and the height indicates the amount of wheel running occurring during the period. B-C, Quantified treatment effects on circadian activity

(mean ± S.E.M.), including total wheel revolutions per day in LD (B), and LD proportion

(C). Sample size (n) is indicated below each bar. *, and ** denote statistical comparisons where p<0.05 or p<0.001, respectively. D, Wheel running activity from 3 consecutive days in LD was averaged into 1 hr bins. Sample sizes are indicated in B,C.

Males and females were separated and differences between treatment groups at each time point were determined using two-way repeated measures ANOVAs. * indicates a significant difference compared to same sex WT+Oil animals; # indicates a significant difference compared to same sex ArKO+Oil animals; + indicates a significant difference compared to same sex WT+E animals.

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Figure 3.3.

Figure 3.3. Acrophase and Amplitude in adult mice following developmental estrogen manipulation. A, Acrophase of wheel running activity averaged over 3 days in LD across male and female WT or ArKO mice administered estradiol (E) or oil from p1-5. Plotted between ZT12 and 18 (mean ± S.E.M.), the sample sizes per group (n) are as follows:

Oil/WT/male (7), E/WT/male (7), Oil/ArKO/male (7), E/ArKO/male (11),

Oil/WT/female (11), E/WT/female (5), Oil/ArKO/female (4), and E/ArKO/female (6).

B, Amplitude plotted as the difference between the daily wheel running peak and mesor.

Values represent the mean ± S.E.M. The n per treatment is shown below the corresponding bar. For both A,B, *, and ** denote statistical comparisons where p<0.05 or p<0.001, respectively.

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Figure 3.4.

Figure 3.4. Phase angle of activity onset (mean min ± S.E.M.) for male and female WT or

ArKO mice treated perinatally with estradiol (E) or oil. Sample size per treatment group is indicated to the right.

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Figure 3.5.

Figure 3.5. Effects of developmental estradiol administration on τ and α. A, The mean free running period (τ) (± S.E.M.), and B, the average duration of the active period (α) (±

S.E.M.). Both parameters were determined from 5 days of data from animals housed in

DD. Samples sizes for each group are indicated below the corresponding bar. *, and ** denote statistical comparisons where p<0.05 or p<0.001, respectively.

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Figure 3.6.

Figure 3.6. Photic phase response of male and female WT or ArKO mice treated with estradiol (E) or oil p1-5. Entrained animals were released into DD for at least 24 hr and pulsed with light for one hr at Zeitgeber time (ZT) 4, 16, or 22. Sample sizes varied from

3-11 animals per treatment group per time point. * and ** denote statistical comparisons between treatment groups within a given time point where p<0.05 or p<0.001, respectively. ★ indicates a statistical comparison between a treatment group pulsed at a specific time and its non-pulsed sham counterpart where p<0.05.

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CHAPTER 4: Circulating estradiol alters the expression of rhythmic and plasticity- associated transcriptional networks in the suprachiasmatic nucleus of adult female mice

Authors: Royston SE1,2, Mahoney MM1,3

Keywords: Circadian, Estrogen, Transcriptional Regulation

Acknowledgements The authors are grateful to members of the Mahoney laboratory for assistance with animal husbandry and experimental support, and thank Athanasios Kondilis in particular for his assistance with the gene expression studies. I thank Norio Yasui and Dr. Katzenellenbogen for their help making the estradiol and control pellets used throughout this chapter, and Dr. Matt Conrad and Trisha Gibbons for assistance with TLDA Card data analyses.

All experiments were designed by SER and MMM. JAK provided the estradiol- and cholesterol-containing pellets, which were made by SER, NY, AK, and Victoria Gugala (VG). SER, AGK, Steven Lord (SVL), VG, Nadia Ayub (NA) and Arif Molla (AM) performed all vaginal cytology, breeding, weaning, genotyping, gonadectomy, and pellet implantation. SER and AK completed all tissue dissection, RNA extraction, cDNA synthesis, and processing of TLDA cards. SER selected genes for interrogation, performed data analyses, and, with MMM, wrote this manuscript.

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Abstract

The adult circadian timekeeping system is governed by a master circadian oscillator located within the suprachiasmatic nucleus (SCN) of the hypothalamus. Light powerfully mediates daily and circadian rhythm outputs and gene expression through inputs onto the SCN. Activity and transcription-associated circadian parameters within the SCN are also robustly affected by circulating estradiol. Further, estradiol interacts with light in the early evening to modify photic responsiveness in female mice. Despite the observed functional relationship between light, estradiol, and circadian activity rhythms, the molecular mechanism(s) underlying these effects in the female central nervous system are poorly understood. In an effort further define these mechanisms, I tested the hypothesis that estradiol alters the expression of transcriptional networks within the SCN. I took advantage of a medium throughput gene expression assay capable of interrogating multiple transcripts simultaneously. To ensure that data generated in this study would be applicable to published work outlining the effects of activational estrogenic signaling on daily and circadian activity rhythms, tissue was collected from adult female mice handled identically to those described in Chapter 2. Specifically, ovariectomized (OVX), mice were implanted with a pellet containing either cholesterol

(CTL) or estradiol (50 µg) and permitted access to running wheels. In the early subjective night, a time when light causes a maximal shift in the timing of rhythmicity, animals of both treatment groups were either pulsed with light (1 hr) or allowed to continue running in the dark. SCN were then collected and estradiol and light-induced changes in gene regulatory networks were quantified and analyzed. In the early evening estradiol exerts much greater control over the SCN transcriptional milieu than light, and

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that estradiol overwhelmingly downregulates gene expression compared to CTL animals.

Further, I identified multiple overlapping gene regulatory networks enriched by estradiol and/or a pulse of light, including pathways not previously implicated in estrogen- dependent message regulation in the SCN. The work presented here substantially advances our understanding of how estradiol and light mediate transcriptional patterning within the SCN, and, importantly, provide a framework for future projects aimed at further elucidating the molecular circadian machinery.

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Introduction

Estrogenic signaling in female mice robustly modifies the expression of daily and circadian activity rhythms through both activational (Blattner and Mahoney, 2014;

Royston et al., 2014a) and organizational mechanisms (Brockman et al., 2011; Blattner and Mahoney, 2012; Royston et al., 2014b). Importantly, circulating estradiol not only alters the strength of photic entrainment (Gorman and Lee, 2002; Blattner and Mahoney,

2014; Royston et al., 2014a) and quantity of daily wheel running (Blattner and Mahoney,

2014; Royston et al., 2014a), but also dampens photic responsiveness in the early subjective night and shortens the length of the endogenous day (τ) (Royston et al.,

2014a). These functions of the timekeeping system are regulated by the suprachiasmatic nucleus (SCN) (Welsh et al, 2010). Widely regarded as the ‘central oscillator’, the SCN is a hypothalamic structure located anterior to the optic chiasm (Klein et al., 1991). This structure is necessary for initiating and mediating critical circadian functions, including the length of τ, the magnitude and patterning of daily activity, and entraining innate rhythms to light signals. Multiple inputs, including photic excitatory glutamatergic signals from the retinohypothalamic tract, converge onto the SCN (Leak et al., 1999;

Brown and Piggins, 2007), where 10-20,000 heterogeneous neurons integrate highly variable neurochemical inputs, and, in turn, innervate an equally diverse set of outputs

(Welsh et al., 2010).

The SCN is the neural locus for the endogenous daily rhythmicity. Circadian patterns of behavior, body temperature, and endocrine secretion are disrupted by surgical

SCN ablation (Welsh et al., 2010). Importantly, SCN-ablated animals receiving transplanted fetal SCN tissue take on the circadian patterns of the graft host (Ralph et al.,

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1990; King et al., 2003), demonstrating a dependence on the SCN for the expression of clock-controlled outputs. Of note, nearly all cells, tissues, and organs express some form of transcriptional daily rhythmicity. Additionally, an estimated 10-20% of all genes are expressed with a 24 hr rhythm in a tissue or cell specific manner (Reddy et al., 2006;

Deery et al., 2009; Doherty and Kay, 2010; Reddy, 2013). Many of these rhythmic functions are not lost following SCN ablation or dissociation (Welsh et al., 2004; Yoo et al., 2004). Rather, peripheral clocks can continue operating for a few days, but critical entrainment between these disjointed rhythms ceases and coordinated oscillation fails

(Tahara et al., 2012). To accomplish the monumental task of integrating and coordinating so many different individual oscillators, the SCN employs numerous timekeeping mechanisms, including powerful control over multiple overlapping transcriptional and gene regulatory networks (Ueda et al., 2005; Yan et al., 2008).

The primary oscillatory mechanism utilized by the SCN involves a core delayed transcriptional negative feedback loop (Sato et al., 2006; Siepka et al., 2007; Takahashi et al., 2008). Circadian Locomotor Output Cycles Kaput (CLOCK)/ Bone Muscle Aryl

Hydrocarbon Receptor Nuclear Translocator-Like (BMAL) dimers drive transcription of the core clock genes Period (Per) and Cryptochrome (Cry) via E-box element binding, resulting in increased PER and CRY translation (Welsh et al., 2010). Following dimerization, PER/CRY returns to the nucleus, and negatively feeds back onto its own transcriptional machinery. The proteins themselves are then degraded by Beta-

Transducin Repeat Containing E3 Ubiquitin (β-TrCP1) and F-box and leucine-rich repeat protein 3 (FBXL3), which form ubiquitin ligase complexes, and permit the cycle to begin anew. The summation of this transcription/translation feedback loop is the basis for the

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~24 hour temporal patterning of CLOCK-associated gene expression within the SCN

(Welsh et al., 2010; O’Neill et al., 2013). Additional overlapping feedback loops further mediate CLOCK-associated gene expression. For example, Nuclear Receptor Rev-ErbA

Beta (REV-ERBα), in concert with retinoic acid receptor-related orphan receptor (ROR), blocks Bmal1 (also known as Arntl) expression (Crumbley and Burris, 2011), while metabolic NAD+/NAMPT (nicotinamide adenine dinucleotide/nicotinamide phosphoribosyltransferase)-mediated mechanisms control Clock salvage, actions hypothesized to mediate the precision of SCN gene rhythms (Nakahata et al., 2009;

Ramsey et al., 2009).

Importantly, canonical CLOCK-associated transcriptional pathways alone cannot account for the robust rhythmic control exerted by the SCN (O’Neill et al., 2013).

Membrane depolarization, Ca2+ signaling, and Ca2+/cAMP regulatory elements (CREs) are additional innate and powerful modulators of mammalian timekeeping (Shibata et al.,

1987; Travnickova-Bendova et al., 2002; Ikeda et al., 2003; Lundkvist et al., 2005; Nahm et al., 2005; Zhang et al., 2005; O’Neill et al., 2008). In addition to acting on the SCN, these effectors are also rhythmic themselves (de Jeu et al., 1998; Ikeda et al., 2003;

O’Neill et al., 2008), suggesting both that they are reciprocally modulated by timekeeping mechanisms and that non-genomic mediators also contribute to circadian function.

While circulating estrogens are capable of substantially influencing the expression daily and circadian activity rhythms (Ogawa et al., 2003, Blattner and Mahoney, 2014;

Royston et al., 2014a), the molecular pathways underlying these effects are unclear.

Recent work has demonstrated that, within hypothalamic and cortical neurons, estrogenic

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signaling and photic stimulation act through non-genomic Ca2+ dependent pathways

(Colwell, 2000; Wang et al., 2013), and mediate neuronal excitability (Fatehi and Fatehi-

Hassanabad, 2008; Han et al., 2011). Further, both estradiol and light act as transcriptional regulators (Takamata et al., 2011) through pathways that include alterations in the SCN expression of the nuclear transcription factor cAMP response element binding protein (CREB) (Ginty et al., 1993; von Gall et al., 1998; Kornhauser et al., 2002; Abizaid et al., 2004; Sakamoto et al., 2013).

Because all of these molecular regulatory actions underlie rhythmic SCN outputs, and estradiol alters light responsiveness, circadian wheel running rhythms (Royston et al.,

2014a), and SCN gene expression (Abizaid et al., 2004; Peterfi et al., 2004), I sought to further elucidate the mechanism(s) underlying the influence of light and estradiol on female mammalian timekeeping in the SCN. To do this, I manipulated circulating estradiol levels and light exposure in the early subjective evening prior to collecting bilateral SCN samples. Following RNA extraction and cDNA synthesis, 96 genes expressed in the SCN were interrogated for light and/or estradiol-induced expression regulation. Probes were selected against a wide variety of Gene Ontology (GO) classes that include genes whose protein products mediate processes including canonical

CLOCK pathways, transcriptional regulation, and metabolism, as well as neuronal excitability and plasticity, allowing me to comprehensively examine the downstream effects of estradiol and light on multiple focused gene regulatory networks.

Methods

Overview

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SCN RNA was extracted from mice administered estradiol or CTL and then either pulsed with light in the early subjective evening or maintained in total darkness. This time of day was selected because it is associated with robust behavioral and neural responses to light cues in numerous species (Colwell 1999, Schwartz 1999).

Additionally, my own work demonstrates that female mice administered estradiol exhibit a reduced shift in the phase of activity onset in response to a photic cue administered at this time (Royston et al., 2014a). The RNA was analyzed for changes in the expression of 96 genes selected based on a literature search. The data was used to generate heat maps. Finally, based on the heat maps, I selected several pathways to use for Kyoto

Encyclopedia of Genes and Genomes (KEGG) analysis. Details are as follows.

Animals and Housing

Past work has described alterations in daily and circadian activity patterns in

OVX WT female mice following estradiol replacement (Royston et al., 2014a). Mice used in the current study were obtained from the same colony and handled nearly identically to ensure that any detected alterations in transcript expression could be correlated to the behavioral changes previously reported (Royston et al., 2014a). Briefly, estrogen subtype receptor type 1 knock-out (αERKO) heterozygotes on the C57Bl6/J background were crossed, and the resulting wild-type (WT) female mice were used. The mice initially used to start the αERKO breeding colony at the University of Illinois

Urbana-Champaign were a kind gift from Dr. Pierre Chambon (IGBMC, France), and the colony has been maintained as described previously (Blattner and Mahoney, 2013) since

2005.

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All mice were maintained in accordance with the University of Illinois Urbana-

Champaign Institutional Animal Care and Use Committee guidelines and the NIH guide for the care and use of laboratory animals. Food and water were provided ad libitum, and animals were maintained at a constant temperature (22 C) in 12:12-h light:dark (LD)

(unless otherwise noted). Animals within the breeding colony were group-housed with same sex littermates from weaning (postnatal, p21) until post sexual maturation (p50), at which time female experimental animals were singly housed in plastic cages (28 cm L x

16 cm W x 12 cm H) outfitted with a metal running wheel (11 cm diameter) affixed to the cage top. During the light phase, the intensity of light measured at the top of the cage ranged from 220-360 lx (average 290 lx). Experimental animals were fed Teklad 2016 rodent diet because it contains <12 ppm soy estrogen isoflavones, including daidzein and genistein aglycone equivalents. Even at maximal doses, these levels are subthreshold for eliciting biological activity based on the average daily mouse diet (per Harlan

Laboratories, 2014).

Daily vaginal cytology was performed on each experimental animal through two estrous cycles beginning on p50 to confirm cyclicity prior to OVX. On the day of OVX

(p60), running wheels were removed. Mice were deeply anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively; i.p.) and maintained with isoflurane gas as necessary. Ovaries were removed through bilateral flank incisions, and the muscle/fascia and overlying skin were closed with surgical silk and staples. Vaginal cytology was examined daily for the next 8-10 days, and verified that experimental animals were no longer cycling or exhibiting signs of estrus. Wheels were returned after

5 days of post-surgical recovery.

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Drug Pellets and Administration

To determine whether circulating estradiol alters gene expression in the SCN, mice received either estradiol or cholesterol pellets. Mice were randomly assigned to treatment groups prior to pellet implantation. Approximately ten days after OVX, on p70, experimental mice were anesthetized under isoflurane gas, and pellets containing either estradiol (50 µg + ≥99% 20 mg cholesterol; Sigma Aldrich, St Louis, MO) or cholesterol alone (CTL; 20 mg) were implanted subcutaneously. Pellets were produced as described previously (Risbridger et al., 2001; Ogura et al., 2007; Royston et al., 2014a;

Royston et al., 2014b). The dose selected increases uterine weight, induces estrus- associated vaginal cytology, and alters wheel running activity and daily and circadian activity patterns (Carroll and Pike, 2008; Blattner and Mahoney, 2014; Royston et al.,

2014a). Vaginal smears were examined for 8-10 consecutive days following pellet implantation. Mice receiving estradiol pellets presented with vaginal cytology containing entirely, or nearly entirely, corneated cells, while smears collected from CTL animals had zero to very few corneated cells. During this experimental phase, vaginal smears were performed diebus alternis. Cytology for each mouse remained consistent throughout the study.

Mice continued in LD with free access to their running wheels for 2 weeks after pellet implantation. Previous work demonstrated that adult exposure to estradiol alters photic phase response in the early subjective night, but no other time (Royston et al.,

2014a). To determine the effects of circulating estradiol and light, as well as the interaction between them on SCN gene expression, mice were put in DD for 28 hr and then either pulsed with light (290 lx) for 1 hr at the time corresponding to Zeitgeber (ZT)

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16 or maintained in the dark (ZT0 = lights-on and ZT12 = lights-off). Therefore, I had four experimental groups: mice were either treated with estradiol or CTL, and either pulsed with light at ZT16 or served as non-pulsed controls. At the end of the 1 hr photic stimulus, all mice were deeply anesthetized under isoflurane gas and rapidly decapitated.

Whole brain tissue was removed, rapidly frozen, and stored at -80 C. At this time, uterine size was visually examined. Mice implanted with estradiol pellets had increased uterine size compared to CTL mice as reported previously (Royston et al., 2014a;

Royston et al., 2014b).

RNA extraction and cDNA synthesis

SCN were bilaterally dissected from each whole frozen brain. Gene expression changes were assessed in a total of 4 samples (n) per treatment group. To ensure that each n contained a sufficient quantity of RNA, and therefore cDNA, to interrogate all of the genes of interest, SCN tissue from 4 animals (8 SCN total) were pooled together per n. Therefore, 16 animals per treatment group were used.

RNA was extracted with RNAqueous-Micro kits (Ambion, AM1931) according to manufacturer instructions. Optional additional micro filter cartridge wash steps were completed with ACS grade 100% EtOH, but no further modifications from the included protocol were made. Briefly, 8 individual SCN were lysed in 100 µL lysis solution and thoroughly homogenized. Fifty µL 100% EtOH was added, and the total lysate volume was bound to a micro filter cartridge through centrifugation. The filter was washed 3 times, and the pure RNA was eluted. Prior to long-term storage at -20 to -80 C, RNA samples were incubated with DNAse at 37 C for 30 min. A total of 45 µL RNA+DNAse

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was obtained. This reaction mix was centrifuged until a DNAse pellet formed, resulting in 40 µL of pure RNA. Nanodrop was used to determine the concentration and purity of all samples.

For these experiments, 1 µg RNA were required for reverse transcription with the

High Capacity cDNA Reverse Transcription kit (Applied Biosystems, 4368814).

According to manufacturer protocol, 100 mM dNTP, random primers, MultiScribe reverse transcriptase, RT buffer, and nuclease-free H2O were combined with RNA at a

1:1 ratio and centrifuged. The following thermocycler program was used to complete cDNA synthesis: 25C for 10 min, 37C for 120 min, 85 C for 5 min, and 4C for ∞. cDNA was stored at -20 C.

TaqMan Low Density Array (TLDA) and analyses

TaqMan Low Density Array (TLDA) plates (Applied Biosystems) were used to assess the expression of 96 transcripts (Table 4.1). These genes were selected from an extensive primary literature search based on the criteria that they are expressed in the

SCN and have been implicated in estrogenic signaling mechanisms and/or circadian rhythms. Actb, the gene encoding β Actin, was selected as a housekeeping gene. The gene 18s was auto-loaded as a secondary housekeeping gene, but because preliminary analyses showed it was regulated by estradiol (data not shown), I chose not to normalize any data to this transcript.

RNAse-free water was combined with 1 µg cDNA to yield 200 uL total volume.

This quantity was combined with 200 uL TaqMan universal master mix II (Applied

Biosystems, 4440038). Plates were loaded, centrifuged, and run with the 7900HT Fast

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Real-Time PCR system (Applied Biosystems) according to manufacturer recommendation. This design resulted in the combination of 10.42 ng cDNA with each probe. Each sample was run in duplicate.

Data was visualized with SDS 2.4 (Applied Biosystems). Threshold cycle (CT) values in excess of 22 indicate insufficient message quantity for proper expression quantification, and were consequently omitted. Of the 96 genes examined, only 6 genes were excluded, including Aanat, Cartpt, Mtnr1b, Opn4, Pax4, and Prkcb. All other genes are listed in Figure 4.1. Relative quantification (RQ) values were calculated

- C according to the equation: ΔΔ CT =Δ CT, experimental - Δ CT, reference; RQ = 2 ΔΔ T. RQ values were then used to generate heat maps using HCL algorithms (TM4-MeV4.4), which enabled visualization of expression differences across treatments. DAVID bioinformatics resources (Huang et al., 2008; http://david.abcc.ncifcrf.gov) were used to complete the gene ontology (GO) and KEGG pathway analyses.

Statistical analyses

To determine the statistical significance of differences in gene expression induced by estradiol and/or exposure to light for 1 hr at ZT16, two way ANOVAs were performed for each interrogated gene. Independent variables were drug treatment (estradiol or oil) and light (pulse or no pulse), while RQ values served as the dependent variable. A priori value (p) <0.05 was considered statistically significant. Statistical analyses were performed with SigmaPlot 12.0 (Systat Software, Inc.). All statistical analyses concerning enrichment of GO terms and KEGG pathway were computed within the

DAVID software suite (Huang et al., 2008; http://david.abcc.ncifcrf.gov). Biological

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functions were considered significantly overrepresented if the enrichment score (p)

<0.05.

Results

I sought to identify transcripts within the SCN whose expression is mediated by circulating estradiol and/or exposure to light in the early subjective night (ZT 16) in female mice. To enable interrogation of related genes while continuing to pursue hypothesis-driven findings and focus the burden of analysis (Brazma et al., 2001), I elected to design a medium throughput PCR assay containing 96 distinct probes, including housekeeping genes. Genes of interest whose protein products are 1) expressed in the SCN and 2) associated with circadian pathways and/or the genomic or non- genomic signaling pathways downstream of estrogenic activity were selected for interrogation following an extensive literature search.

The identities of all genes that met threshold criteria are shown in the heatmap in

Figure 4.1 (all interrogated genes shown in Table 4.1). Because all genes were normalized to CTL animals not pulsed with light at ZT16 (CN) to reflect a standard fold change, only CTL+pulse (CP), estradiol+no pulse (EN), and estradiol+pulse (EP) are shown. Genes were clustered according to Hierarchical Clustering (HCL) parameters

(Eisen et al. 1998), and colors were used to indicate fold change in message expression compared to CN. Green indicates that expression was downregulated, while red indicates upregulation. Black informs no change. Overwhelmingly, gene expression was suppressed by estradiol. In comparison to estradiol, light alone did not induce the same magnitude of regulation. Further, of the less than 15 transcripts that were altered by light,

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the trend was toward upregulation. Gene expression patterns observed in animals treated with estradiol and pulsed with light at ZT16 more closely resembled what was seen in estradiol treated animals that were not pulsed, than oil-treated animals receiving a pulse.

Bioinformatic sorting of genes of interest with DAVID (Huang et al., 2008; http://david.abcc.ncifcrf.gov) revealed significant enrichment of Gene Ontology (GO) terms (Table 4.2). Enriched terms included Rhythmic Processes (p = 3.1 E-33),

Circadian Rhythms (p = 3.6 E-29), Regulation of Metabolic Processes (p = 2.9 E-16), and

Regulation of Transcription (p = 3.3 E-14), among others. I was particularly interested in further clarifying the impact of estradiol and a photic cue on the expression of transcriptional families associated with Rhythmic Processes, Regulation of Transcription, and Metabolic Processes because these are critical responsibilities of the SCN. To do this, I selected for the interrogated genes falling within each of these GO-terms and then sorted by treatment group (EP, EN CP). Following HCL clustering, I generated heat maps to describe treatment-associated expression fold changes (Figure 4.2).

Keeping with the trend observed when all interrogated genes were clustered together (Figure 4.1), estradiol exerted a primarily down regulatory effect on gene expression throughout all 3 GO terms, irrespective of whether light was present or not

(Figure 4.2). Sorting genes of interest by GO term allowed me to focus on transcript- specific expression regulation. For example, the expression of Egr3, the message encoding Early Growth Response 3 protein (EGR3), a C2H2-type zinc-finger protein important for rhythmic gene expression (Porterfield and Mintz, 2009), is not altered by either a light pulse at ZT16 or estradiol, but is robustly increased by both light and estradiol. Additionally, the expression of Estrogen Subtype Receptor 2 (Esr2; formerly

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ERβ) was downregulated by estradiol treatment, but is not affected by light.

Interestingly, Estrogen Subtype Receptor 1 (Esr1; formerly ERα) is similarly downregulated by estradiol, but is stimulated by light.

I was interested in which GO terms were enriched within groups of genes regulated by the same treatment in the same direction. To complete this analysis, I grouped all significantly regulated genes by effective treatment. I then sub-grouped them by direction of change. This created the following groups (Table 4.3): genes whose expression was repressed by estradiol; induced by estradiol; repressed by a light pulse; induced by light pulse, or decreased by a light pulse and estradiol. These groups of genes were subjected to DAVID analysis (http://david.abcc.ncifcrf.gov; Huang et al., 2008), and the most enriched biological functions were identified. Genes associated with

‘Circadian Rhythms’ and ‘Rhythmic Processes’ were overwhelmingly repressed by estradiol administration (p = 5.5 E-15 and 1.5 E-11, respectively) (Table 4.3). Expression of transcripts falling within these categories and regulated by estradiol and light were also reduced (p = 1.7 E-7 and 1.0 E-6, respectively). ‘Regulation of Transcription’ was significantly enriched within genes repressed by estradiol (p = 1.4 E-8), induced by estradiol (p = 2.7 E-2), or reduced by estradiol and a light pulse at ZT16 (p = 2.9 E-3).

‘DNA Binding’ was also enriched in genes whose expression was significantly enhanced by estradiol (p = 1.8 E-2), while genes associated with ‘Regulation of Metabolic

Processes’ (p = 3.3 E-10), ‘Protein and Amino Acid Phosphorylation’ (p = 3.6 E-8), and

‘MAPK Signaling Pathways’ (p = 3.4 E-7) were downregulated by estradiol.

A pulse of light at ZT16 to CTL animals resulted in very different GO term enrichment compared to estradiol treatment (Table 4.3). Rather than mediating

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transcriptional mechanisms, genes downregulated by light were significantly associated with ‘Neurotransmitter Binding’ (p = 1.8 E-2), ‘Neurotransmitter Receptor Activity’ (p =

1.8 E-2), and ‘Cell Membrane’ (p = 1.8 E-2). Transcriptional mechanisms were enriched within genes whose expression was induced by light. These biological functions, unlike those altered by estradiol administration, were closely associated with only early transcriptional regulation, and include ‘Fos Transforming Protein’ (p = 1.4 E-3) and

‘bZIP Transcription Factors’ (p = 9.1 E-3). The biological function ‘Neuropeptide’ was also enriched within genes significantly upregulated by light at ZT16 (p = 5.0 E-3). This

GO term (GO:0007218) is a child term of various cell surface receptor-mediated cascades, and is upstream of nuclear-associated mechanisms

(http://david.abcc.ncifcrf.gov), but whether the regulated transcripts within this term directly mediate early transcriptional activity is not known.

I began this experiment interested in regulatory gene networks, as opposed to the impact of estradiol and light on the expression of individual genes. To identify pathways in which interrogated genes were significantly regulated, as well as the specifically altered components within each pathway, I completed KEGG analysis with DAVID

(http://david.abcc.ncifcrf.gov; Huang et al., 2008). The most enriched pathways (Table

4.4) included ‘Circadian Rhythms’ (p = 2.9 E-9), ‘MAPK Signaling Pathways’ (p = 3.4

E-7), ‘Neurotrophin Signaling Pathways’ (p = 3.7 E-5), and ‘Long Term Potentiation’ (p

= 3.8 E-4). I mapped out each pathway and laid my regulated genes of interest within the functional output (Figures 4.3-4.6). Because the regulated areas within a given pathway

(indicated by a red star) may be in reference to a protein product, downstream target, or upstream effector, a table of significantly regulated transcripts within each KEGG

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pathway was also generated. The effecting treatment, direction of change, and statistical significance for each gene are indicated.

Interrogated genes associated with the KEGG pathway ‘Circadian Rhythm’ were generally repressed by estradiol. No genes were regulated by light alone. However,

Bhlhe41 (p = 0.001), whose protein product, DEC2, acts as a directly competitive

CLOCK gene transcriptional repressor (Honma et al. 2002; Sato et al. 2004), Nr1d1 (p =

0.013), the gene encoding REV-ERBα, and Cry2 (p<0.001) were downregulated by estradiol combined with a light pulse at ZT16. Npas2, a basic helix-loop-helix PAS family transcription factor regarded as one of the most critical circadian transcriptional regulators (DeBruyne et al., 2007), but no other interrogated gene in this pathway, was induced by estradiol (p <0.001).

There was substantial overlap of enriched genes of interest associated with

‘MAPK Signaling Pathways’ (Figure 4.4), ‘Neurotrophin Signaling Pathway’ (Figure

4.5), and ‘Long-Term Potentiation’ (Figure 4.6). All significantly regulated genes associated with these pathways within my model were downregulated. Genes encoding growth factors whose expression was suppressed included Bdnf (p = 0.023), Egf

(p<0.001), Fgf (p<0.001), Tgfa (p<0.001), Tgfb1 (p<0.001), and Vegfa (p<0.001), whose protein products are Brain Derived Neurotrophic Factor (BDNF), Epidermal Growth

Factor (EGF), Fibroblast Growth Factor (FGF), Transforming Growth Factor Alpha

(TGFα), Transforming Growth Factor Beta 1 (TGFβ1), and Vascular Endothelial Growth

Factor A (VEGFA, respectively. Expression of the transcript encoding a key neurotrophin receptor and the receptor showing the highest binding affinity for BDNF,

Ntrk2 (protein: Tyrosine Kinase Receptor B; TrkB) was also repressed (p<0.001).

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Within the mammalian CNS, ligand-neurotrophin receptor binding, including

BDNF-TrkB, induces receptor homodimerization, autophosphorylation within intracellular tyrosine residues, and the subsequent initiation of multiple cell signaling cascades. These varying pathways ultimately lead to increased neuronal structural plasticity via activation of the mitogen-activated protein kinase (MAPK) pathway and the transcription factor CREB, greater synaptic plasticity via activation of the protein kinase

C (PKC) pathway, and enhanced transcriptional plasticity via pathways that increase activation of nuclear factor- κB (NFκB) and early activation genes such as JUN

(comprehensively reviewed in Yoshii and Constantine-Paton, 2010).

I found that estradiol or estradiol combined with a ZT16 light pulse not only suppressed expression of the transcripts encoding the effector neurotrophins and their receptors, but also reduced the message for the several of the pathway end points and critical mediators. For example, transcript expression of Creb1 (p<0.001), Creb3

(p<0.001), Jun (p<0.001), MapK14 (p = 0.018), MapK3 (p<0.001), Nfκb1 (p<0.001), and

Prkaca (p<0.001), whose protein product cAMP-Dependent Protein Kinase Catalytic

Subunit Alpha (PKA C-α) is proportionally reflective of PKC activity, were all reduced by either estradiol or estradiol combined with a ZT16 light pulse (Figures 4, 5, 6).

Further, downstream of neurotrophin receptor activation, the PKC cascade potentiates synaptic plasticity, in part, by enhancing intracellular bioactive stores of Ca2+. In addition to the observed decrease in PKC pathway players already stated, I also found that estradiol reduced the expression of genes encoding multiple neuron-associated Ca2+ regulatory proteins in the SCN of female mice, including Calb1 (p<0.001), Calb2

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(p<0.001), Camk2b (p<0.001), Camk2d (p<0.001), and Camk2g (p<0.001) (Figures

4.5,4.6).

Importantly, stimuli beyond neurotrophin-receptor binding can also initiate these plasticity-associated pathways. Glutamate, for example, induces time and dose- dependent long-term potentiation (LTP) in the SCN (Nisikawa et al., 2002). Similarly,

Tumor Necrosis Factor (TNF) activates NFκB through a mechanism separate from the

TrkB-dependent phosphorylation mechanism previously described. The expression of its message, Tnf, was downregulated by estradiol (p<0.001). Taken together, the results shown here demonstrate that estradiol substantially reduces plasticity-associated molecular mechanisms through, often redundant, repression of transcripts encoding ligands, receptors, mid-pathway regulatory components, and downstream targets.

Discussion and Future Directions

Using a medium throughput gene expression array, I discovered that estradiol exerts greater control over the expression of gene regulatory networks in the

SCN than is exerted by light. Further, estradiol primarily downregulates gene expression, though not uniformly. Estrogenic signaling strongly influences the patterning of daily and circadian activity rhythms (Royston et al., 2014a). However, the impact of circulating estradiol on gene expression in the transcriptionally-dependent SCN, a structure demonstrated to mediate the expression of daily biorhythmic outputs has not been well characterized until now. I focused on a single time point in the early subjective night because the timekeeping system of nearly all species alters its rhythmicity in

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response to photic cues at this time. Further, my previously published work shows that estradiol alters light responsiveness at ZT16 (Royston et al., 2014a).

Two noteworthy findings were the light pulse-induced increase in the canonical estrogen receptor ESR1, but not ESR2, and the robust induction of the immediate early gene Egr3 in mice treated with estradiol and pulsed with light at ZT16. The ability of light to induce changes in the expression of ESR1, but not ESR2, suggests that only ESR1 is photosensitive. It also provides unique insight into the potential network-based relationship between light and estrogenic signaling in the SCN. While ESR1 is not robustly expressed in the SCN, the cells that do express it are found only in the core in mice. Interestingly, photic information inputs onto the SCN core via the RHT (reviewed in Silver and Bailey et al., 2014). Conversely, ESR2 has been detected in shell neurons exclusively (Vida et al, 2008). Future joint electrophysiologic and biochemical studies aimed at differentiating between the effects of direct photic input onto SCN core neurons on the induction of ESR1 expression and indirect light-induced ESR1 transcription via a mechanism reliant on any number of the peripheral SCN inputs described in Chapter 1 will further clarify the underlying pathway. Similar studies may also elucidate the mechanism by which estradiol increases neuronal excitability in the SCN (Fatehi and

Fatehi-Hassanabad, 2008). In the current study, light in the early subjective night suppressed genes involved in the regulation of neuronal excitability and transmission, while increasing ESR1. Future studies utilizing estrogen receptor-specific agonists and photic or photic-like input may aid in deciphering the mechanisms by which both light and estrogenic signaling modify SCN excitability.

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Egr3, an immediate early gene, is believed to act as a transcriptional regulator in the SCN in response to retinal input (Morris et al., 1998). In fact, the overall enrichment of transcription and DNA binding-associated GO terms by estradiol suggests that the hormone exerts fine tuned control over transcriptional machinery in the SCN. Further, that estradiol treatment also mediates the expression of genes affiliated with metabolism, phosphorylation, and MAPK signaling support a role for this hormone in the regulation of post-translational modification and cell signaling within the SCN. Interestingly, recent work has suggested that, while the SCN is both transcriptionally and translationally dependent for the expression of circadian rhythms, the regulated message and protein networks need not fully overlap (reviewed in O’Neill et al., 2013). Future studies should focus on the proteomics induced by light and estradiol throughout the subjective day to differentiate between gene and protein-based requirement for the adult timekeeping system.

When estradiol and/or light-regulated genes were mapped onto KEGG pathways, the trend of transcript suppression by estradiol continued to be prominent. Interestingly, within the ‘Circadian rhythm’ KEGG, estradiol differentially regulated Clock and Npas2, which encode circadian rhythm-affiliated transcription factors. Clock and Npas2 appear to act through initially overlapping mechanisms (DeBruyne et al., 2007), though the complete differentiation of their downstream pathway(s) has yet to be fully elucidated.

Because Npas2 expression was induced, while Clock’s was repressed, it is possible that the current results indicate an estradiol-mediated preferential selection of the Npas2- associated transcriptional mechanism over the downstream effects of Clock. Future studies are necessary to test this hypothesis.

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Other significantly overrepresented KEGG pathways included ‘MAPK signaling’,

‘Neurotrophin signaling’, and ‘Long-term potentiation’. All 3 of these pathways are strongly associated with neuronal and synaptic plasticity, and, accordingly, there was substantial overlap of enriched genes. Importantly, MAPK signaling itself is necessary for both neurotrophin signaling and LTP. Further, in studies using rats and mice, MAPK is also required for photic-induced phase shifts in activity onset and activation of clock genes in the SCN (Butcher at al., 2002; Coogan and Piggins, 2003; Dziema et al., 2003).

Additionally, the MAPK pathway both inputs onto the central oscillator and is a direct timekeeping output (Goldsmith and Bell-Pedersen, 2013), further supporting a role for this signaling mechanism in circadian plasticity. My finding that estradiol downregulates genes encoding feed forward components of the MAPK pathway (Figure 4.4) indicates that increased estrogenic signaling may impede SCN plasticity. However, no work to date has examined the role of estradiol on the expression of the MAPK pathway within the SCN.

Protein Kinase A (PKA) signaling, which contributes strongly to MAPK amplification, and, consequently, neurotrophic signaling and LTP as well, may provide a relatively novel target to further explore the relationship between estradiol, circadian behavioral plasticity, and MAPK. I found that administration of both estradiol and a light pulse reduced the expression of the transcript encoding PKA C-α. PKA C-α is the structural subunit of the powerful cell-signaling mediator PKA, and due to the reciprocal relationship between them, is also indicative of PKC activity. Blockade of PKA in the

SCN inhibits glutamate-induced elevations in Per1 expression and attenuates phase shifting in the early subjective evening in vitro (Tischkau et al., 2000). Glutamate is

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often used in place of light stimuli in vitro. Interestingly, my own published work demonstrates that estradiol dampens behavioral phase shifting following a light pulse administered in the early subjective night (Royston et al., 2014a). Together, these findings suggest that a reduction in PKA, such as that induced by estradiol in the current study, may dampen molecular and behavioral photic responsiveness.

To test the hypothesis that estradiol modifies behavioral plasticity in the early subjective night through a mechanism that involves reduced PKA activity, I would create mice with inducible PKA silencing (null) and overexpression (oe) in an SCN-specific manner. I would then repeat the phase response experiment described in Chapter 2

(Figure 4.6) with the following treatment groups: WT+CTL, WT+E, induciblePKASCNnull+CTL, induciblePKASCNnull+E, induciblePKASCNoe+CTL, and induciblePKASCNoe+CTL. If PKA suppression in the SCN is responsible for the estradiol-induced dampening of behavioral plasticity following a light pulse at ZT16, then estradiol administration to PKASCNoe mice should not recapitulate its effects in WT mice. Similarly, PKASCNnull mice should mimic WT+E mice with respect to this measure regardless of whether they receive CTL or estradiol. This experimental design is highly speculative, however, because an SCN-specific target is currently unknown.

Aside from MAPK signaling contributions, neurotrophin signaling generally mediates neuronal survival. Because of strong evidence that estrogen is neuroprotective

(Scott et al., 2012; Spence and Voskuhl, 2012), I anticipated that estradiol administration would increase the expression of genes encoding neurotrophic factors and their respective receptors. Rather, I found the opposite to be true; estradiol exerted a repressive effect on several of these transcripts and none were enhanced (Figure 4.5). Of note is the

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inhibition of Bdnf expression by estradiol and light. BDNF mediates the magnitude of excitatory N-methyl-D-aspartate (NMDA) receptor-associated glutamatergic currents in the SCN (Kim et al., 2006; Michel et al., 2006). It is possible that the reduction in Bdnf following estradiol and a light pulse (Figure 4.5) results in neuroprotective effects within the SCN by dampening NMDAR-mediated synaptic currents, thereby preventing potentially cytotoxic neuronal hyperexcitability. However, the existence of predominantly GABAergic population within this structure dictates the need for functional electrophysiologic analysis of this hypothesis.

Similar studies may also reveal more detail about the role of LTP in the SCN.

While well documented in cortical, cerebellar, and limbic structures, only 4 published studies focus on LTP in the SCN (Nishikawa et al., 1995; Nishikawa et al., 1998;

Fukunaga et al., 2002; Nishikawa et al., 2002). LTP describes the strengthening of synaptic efficiency by repetitive activity, and is perhaps the most robust demonstration of synaptic plasticity. Both the MAPK signaling pathway, and extracellular signal-regulated kinase (ERK) in particular (Winder et al., 1999; Kelleher et al., 2004; Jin and Feig,

2010), and NMDAR are required for the expression of LTP in the SCN (Nishikawa et al.,

1998). ERK, encoded by the gene Mapk3, is repressed by estradiol in the current study.

Further, though BDNF is not directly affiliated with the LTP KEGG pathway, the implication of its control over NMDAR-mediated synaptic currents (Kim et al., 2006;

Michel et al., 2006), its ability to activate ERK (Shimizu et al., 2007; Wang et al., 2014), and its downregulation following estradiol and light administration (Figure 4.5) warrant further investigation into the role it plays in LTP within the SCN. Recent work demonstrates that BDNF, in addition to inducing ERK activation through a

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phosphorylation-dependent mechanism, simultaneously reduces suprachiasmatic nucleus oscillatory protein (SCOP) through calpain-mediated degradation (Wang et al., 2014).

SCOP inhibits the activation ERK by trapping its precursor in an inactive state (Shimizu et al., 2003). SCOP was not interrogated in the current study. Therefore, whether it is induced by estradiol remains work for future studies. Importantly, the data shown here continue to strongly support an inhibitory role for estradiol with respect to LTP and synaptic plasticity in the SCN in the early subjective evening, and provide evidence for future studies aimed at further mechanistic clarification.

Conclusions

Estradiol reduces behavioral phase shifting in response to a light cue in the early subjective night. I demonstrate here that at this same time estradiol also significantly alters the transcriptional milieu within the SCN, a structure required for normal circadian responsiveness, and does so primarily through downregulation of genes known to contribute to neuronal and synaptic plasticity. By using a hypothesis-driven approach to select genes of interest, I have allowed for targeted identification of potential transcripts whose estradiol-mediated alteration in expression may contribute to the hormone’s ability to alter behavioral plasticity. These targets serve as a springboard for future work aimed and linking genes and behavior.

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References

Abizaid A, Mezei G, Horvath TL. 2004. Estradiol enhances light-induced expression

of transcription factors in the SCN. Brain Res 1010:35-44.

Bailey M, Silver R. 2014. Sex differences in circadian timing systems: implications

for disease. Front Neuroendocrinol 35(1):111-39.

Blattner MS, Mahoney MM. 2014. Estrogen receptor 1 modulates circadian rhythms

in adult female mice. Chronobiol Internat accepted for publication.

Blattner MS, Mahoney MM. 2013. Photic phase-response curve in 2 strains of mice

with impaired responsiveness to estrogens. J Biol Rhythms 28:291-300.

Blattner MS, Mahoney MM. 2012. Circadian parameters are altered in two strains of

mice with transgenic modifications of estrogen receptor subtype 1. Genes Brain

Behav 11:828-836.

Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, Aach J,

Ansorge W, Ball CA, Causton HC, Gaasterland T, Glenisson P, Holstege FC,

Kim IF, Markowitz V, Matese JC, Parkinson H, Robinson A, Sarkans U, Schulze-

Kremer S, Stewart J, Taylor R, Vilo J, Vingron M. 2001. Minimum information

about a microarray experiment (MIAME)-toward standards for microarray data.

Nat Genet 29:365-371.

Brockman R, Bunick D, Mahoney MM. 2011. Estradiol deficiency during

development modulates the expression of circadian and daily rhythms in male and

female aromatase knockout mice. Horm Behav 60:439-447.

Brown TM, Piggins HD. 2007. Electrophysiology of the suprachiasmatic circadian

clock. Prog Neurobiol 82:229-255.

146

Butcher GQ, Doner J, Dziema H, Collamore M, Burgoon PW, Obrietan K. 2002. The

p42/44 mitogen-activated protein kinase pathway couples photic input to

circadian clock entrainment. J Biol Chem 277(33):29519-25.

Carroll JC, Pike CJ. 2008. Selective estrogen receptor modulators differentially

regulate Alzheimer-like changes in female 3xTg-AD mice. Endocrinology

149:2607-2611.

Colwell CS, Foster RG. 1992. Photic regulation of Fos-like immunoreactivity in the

suprachiasmatic nucleus of the mouse. J Comp Neurol 324(2):135-142.

Colwell CS. 2000. Circadian modulation of calcium levels in cells in the

suprachiasmatic nucleus. Eur J Neurosci 12:571-576.

Coogan AN, Piggins HD. 2003. Circadian and photic regulation of phosphorylation

of ERK1/2 and Elk-1 in the suprachiasmatic nuclei of the Syrian Hamster. J

Neurosci 23(7):3085-93.

Crumbley C, Burris TP. 2011. Direct regulation of CLOCK expression by REV-ERB.

PLoS One 6(3):e17290. doi: 10.1371/journal.pone.0017290.

de Jeu M, Hermes M, Pennartz C. 1998. Circadian modulation of membrane

properties in slices of rat suprachiasmatic nucleus. Neuroreport 9:3725-3729.

DeBruyne JP, Weaver DR, Reppert SM. 2007. CLOCK and NPAS2 have overlapping

roles in the suprachiasmatic circadian clock. Nat Neurosci 10:543-545.

Deery MJ, Maywood ES, Chesham JE, Sladek M, Karp NA, Green EW, Charles PD,

Reddy AB, Kyriacou CP, Lilley KS, Hastings MH. 2009. Proteomic analysis

reveals the role of synaptic vesicle cycling in sustaining the suprachiasmatic

circadian clock. Curr Biol 19:2031-2036.

147

Doherty CJ, Kay SA. 2010. Circadian control of global gene expression patterns.

Annu Rev Genet 44:419-444.

Dziema H, Oatis, B, Butcher GQ, Yates R, Hoyt KR, Obrietan K. 2003. The

ERK/MAP kinase pathway couples light to immediate-early gene expression in

the suprachiasmatic nucleus. Eur J Neurosci 17(8):1617-27.

Eisen MB, Spellman PT, Brown PO, Botstein D. 1998. Cluster analysis and display

of genome-wide expression patterns. Proc Natl Acad Sci U S A 95:14863-14868.

Fatehi M, Fatehi-Hassanabad Z. 2008. Effects of 17beta-estradiol on neuronal cell

excitability and neurotransmission in the suprachiasmatic nucleus of rat.

Neuropsychopharmacology 33:1354-1364.

Fukunage K, Horikawa K, Shibata S, Takeuchi Y, Miyamoto E. 2002.

Ca2+/calmoldulin-dependent protein kinase II-dependent long-term potentiation

in the rat suprachiasmatic nucleus and its inhibition by melatonin. J Neurosci Res

70(6):799-807.

Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo KE, Takahashi JS,

Greenberg ME. 1993. Regulation of CREB phosphorylation in the

suprachiasmatic nucleus by light and a circadian clock. Science 260:238-241.

Goldsmith CS, Bell-Pedersen D. 2013. Diverse roles for MAPK signaling in circadian

clocks. Adv Genet 84:1-39.

Gorman M, Lee TM. 2002. Hormones and Biological Rhythms. In: Becker J,

Breedlove S, Crews D McCarthy M, eds. Behavioral Endocrinology. 2nd ed. ed.

Cambridge, MA: MIT Press; 451.

Han S, Yu FH, Schwartz MD, Linton JD, Bosma MM, Hurley JB, Catterall WA, de la

148

Iglesia HO. 2012. Na(V)1.1 channels are critical for intercellular communication

in the suprachiasmatic nucleus and for normal circadian rhythms. Proc Natl Acad

Sci U S A 109:E368-77.

Honma S, Kawamoto T, Takagi Y, Fujimoto K, Sato F, Noshiro M, Kato Y, Honma

K. 2002. Dec1 and Dec2 are regulators of the mammalian molecular clock.

Nature 419(6909):841-4.

Huang DW, Sherman BT, Lempicki RA. 2008. Systematic and integrative analysis of

large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44-57.

Ikeda M, Sugiyama T, Wallace CS, Gompf HS, Yoshioka T, Miyawaki A, Allen CN.

2003. Circadian dynamics of cytosolic and nuclear Ca2+ in single

suprachiasmatic nucleus neurons. Neuron 38:253-263.

Jin SX, Feig LA. 2010. Long-term potentiation in the CA1 hippocampus induced by

NR2A subunit-containing NMDA glutamate receptors is mediated by Ras-

GRF2/Erk map kinase signaling. PLoS One 5(7):e111732.

Kelleher RJ, Govindarajan J, Jung HY, Kang H, Tonegawa S. 2004. Translational

control by MPK signaling in long-tern synaptic plasticity and memory. Cell

116(3):467-79.

Kim YI, Choi HJ, Colwell CS. 2006. Brain-derived neurotrophic factor regulation of

N-methyl-D-aspartate receptor-mediated synaptic currents in suprachiasmatic

nucleus neurons. J Neurosci Res 84:1512-1520.

King VM, Chahad-Ehlers S, Shen S, Harmar AJ, Maywood ES, Hastings MH. 2003.

A hVIPR transgene as a novel tool for the analysis of circadian function in the

mouse suprachiasmatic nucleus. Eur J Neurosci 17:822-832.

149

Klein DC, Moore RY, Reppert SM. 1991. Suprachiasmatic Nucleus: The Mind's

Clock. New York: Oxford University Press.

Kornhauser JM, Cowan CW, Shaywitz AJ, Dolmetsch RE, Griffith EC, Hu LS,

Haddad C, Xia Z, Greenberg ME. 2002. CREB transcriptional activity in neurons

is regulated by multiple, calcium-specific phosphorylation events. Neuron 34:221-

233.

Leak RK, Card JP, Moore RY. 1999. Suprachiasmatic pacemaker organization

analyzed by viral transynaptic transport. Brain Res 819:23-32.

Lundkvist GB, Kwak Y, Davis EK, Tei H, Block GD. 2005. A calcium flux is

required for circadian rhythm generation in mammalian pacemaker neurons. J

Neurosci 25:7682-7686.

Michel S, Clark JP, Ding JM, Colwell CS. 2006. Brain-derived neurotrophic factor

and neurotrophin receptors modulate glutamate-induced phase shifts of the

suprachiasmatic nucleus. Eur J Neurosci 24:1109-1116.

Morris ME, Viswanathan N, Kuhlman S, Davis FC, Weitz CJ. 1998. A screen for

genes induced in the suprachiasmatic nucleus by light. Science 279: 1544-7.

Nahm SS, Farnell YZ, Griffith W, Earnest DJ. 2005. Circadian regulation and

function of voltage-dependent calcium channels in the suprachiasmatic nucleus. J

Neurosci 25:9304-9308.

Nakahata Y, Sahar S, Astarita G, Kaluzova M, Sassone-Corsi P. 2009. Circadian

control of the NAD+ salvage pathway by CLOCK-SIRT1. Science 324:654-657.

Nisikawa Y, Shimazoe T, Shibata S, Watanabe S. 2002. Time-dependent effect of

150

glutamate on long-term potentiation in the suprachiasmatic nucleus of rats. Jpn J

Pharmacol 90:201-204.

Nishikawa Y, Shimazoe T, Shibata S, Watanabe S. 1998. -methyl-D-aspartate

receptors are indispensable for the formation of long-term potentiation in the rat

suprachiasmatic nucleus in vitro. Brain Res 782(1-2):303-5.

Nishikawa Y, Shibata S, Watanabe S. 1995. Circadian changes in long-term

potentiation of rat suprachiasmatic field potentials elicited by optic nerve

stimulation in vitro. Brain Res 695(2):158-62.

Ogura Y, Ishii K, Kanda H, Kanai M, Arima K, Wang Y, Sugimura Y. 2007.

Bisphenol A induces permanent squamous change in mouse prostatic epithelium.

Differentiation 75:745-756.

O'Neill JS, Maywood ES, Chesham JE, Takahashi JS, Hastings MH. 2008. cAMP-

dependent signaling as a core component of the mammalian circadian pacemaker.

Science 320:949-953.

O'Neill JS, Maywood ES, Hastings MH. 2013. Cellular mechanisms of circadian

pacemaking: beyond transcriptional loops. Handb Exp Pharmacol (217):67-103.

doi:67-103.

Peterfi Z, Churchill L, Hajdu I, Obal Jr F, Krueger JM, Parducz A. 2004. Fos-

immunoreactivity in the hypothalamus: dependency on the diurnal rhythm, sleep,

gender, and estrogen. Neuroscience 124:695-707.

Porterfield VM, Mintz EM. 2009. Temporal patterns of light-induced immediate-

early gene expression in the suprachiasmatic nucleus. Neurosci Lett 463:70-73.

Ralph MR, Foster RG, Davis FC, Menaker M. 1990. Transplanted suprachiasmatic

151

nucleus determines circadian period. Science 247:975-978.

Ramsey KM, Yoshino J, Brace CS, Abrassart D, Kobayashi Y, Marcheva B, Hong

HK, Chong JL, Buhr ED, Lee C, Takahashi JS, Imai S, Bass J. 2009. Circadian

clock feedback cycle through NAMPT-mediated NAD+ biosynthesis. Science

324:651-654.

Reddy AB. 2013. Genome-wide analyses of circadian systems. Handb Exp

Pharmacol (217):379-88. doi:379-388.

Reddy AB, Karp NA, Maywood ES, Sage EA, Deery M, O'Neill JS, Wong GK,

Chesham J, Odell M, Lilley KS, Kyriacou CP, Hastings MH. 2006. Circadian

orchestration of the hepatic proteome. Curr Biol 16:1107-1115.

Risbridger GP, Wang H, Frydenberg M, Cunha G. 2001. The metaplastic effects of

estrogen on mouse prostate epithelium: proliferation of cells with basal cell

phenotype. Endocrinology 142:2443-2450.

Royston SE, Yasui N, Kondilis AG*, Lord SV*, Katzenellenbogen JA, Mahoney

MM. 2014a. ESR1 and ESR2 differentially regulate daily and circadian activity

rhythms in female mice. Endocrinology accepted for publication.

Royston SE, Bunick D, Mahoney MM. 2014b. Estradiol exposure early in life

programs daily and circadian rhythms in adult mice. In preparation.

Sakamoto K, Norona FE, Alzate-Correa D, Scarberry D, Hoyt KR, Obrietan K. 2013.

Clock and light regulation of the CREB coactivator CRTC1 in the

suprachiasmatic circadian clock. J Neurosci 33:9021-9027.

Sato F, Kawamoto T, Fujimoto K, Noshiro M, Honda KK, Honma S, Honma K, Mato

152

Y. 2004. Functional analysis of the basic helix-loop-helix transcription factor

DEC1 in circadian regulation. Eur J Biochem 271(22):4409-19.

Sato TK, Yamada RG, Ukai H, Baggs JE, Miraglia LJ, Kobayashi TJ, Welsh DK,

Kay SA, Ueda HR, Hogenesch JB. 2006. Feedback repression is required for

mammalian circadian clock function. Nat Genet 38:312-319.

Schwartz WJ, Takeuchi J, Shannon W, Davis EM, Aronin N. 1994. Temporal

regulation of light-induced fos and fos-like protein expression in the ventrolateral

subdivision of the rat suprachiasmatic nucleus. Neuroscience 58(3):573-583.

Scott E, Zhang QG, Wang R, Vadlamudi R, Brann D. 2012. Estrogen neuroprotection

and the critical period hypothesis. Front Neuroendocrinol 33(1):85-104.

Shibata S, Newman GC, Moore RY. 1987. Effects of calcium ions on glucose

utilization in the rat suprachiasmatic nucleus in vitro. Brain Res 426:332-338.

Shimizu, K., Okada, M., Nagai, K. & Fukada, Y. 2003. Suprachiasmatic

nucleus circadian oscillatory protein, a novel binding partner of K-Ras in the

membrane rafts, negatively regulates MAPK pathway. J Biol Chem 278:14920–

14925.

Shimizu K, Phan T, Mansuy IM, Storm DR. 2007. Proteolytic degradation of

SCOP in the hippocampus contributes to activation of MAP kinase and memory.

Cell 128:1219–1229.

Siepka SM, Yoo SH, Park J, Song W, Kumar V, Hu Y, Lee C, Takahashi JS. 2007.

Circadian mutant Overtime reveals F-box protein FBXL3 regulation of

cryptochrome and period gene expression. Cell 129:1011-1023.

153

Spence RD, Voskuhl RR. 2012. Neuroprotective effects of estrogens and androgens

in CNS inflammation and neurodegeneration. Front Neuroendocrinol 33(1):105-

15.

Tahara Y, Kuroda H, Saito K, Nakajima Y, Kubo Y, Ohnishi N, Seo Y, Otsuka M,

Fuse Y, Ohura Y, Komatsu T, Moriya Y, Okada S, Furutani N, Hirao A,

Horikawa K, Kudo T, Shibata S. 2012. In vivo monitoring of peripheral circadian

clocks in the mouse. Curr Biol 22:1029-1034.

Takahashi JS, Hong HK, Ko CH, McDearmon EL. 2008. The genetics of mammalian

circadian order and disorder: implications for physiology and disease. Nat Rev

Genet 9:764-775.

Takamata A, Torii K, Miyake K, Morimoto K. 2011. Chronic oestrogen replacement

in ovariectomised rats attenuates food intake and augments c-Fos expression in

the suprachiasmatic nucleus specifically during the light phase. Br J Nutr

106:1283-1289.

Tischkau SA, Gallman EA, Buchanan GF, Gillette MU. 2000. Differential cAMP of

glutamatergic signaling regulates long-term state changes in the suprachiasmatic

circadian clock. J Neurosci 20(20):7830-7.

Travnickova-Bendova Z, Cermakian N, Reppert SM, Sassone-Corsi P. 2002. Bimodal

regulation of mPeriod promoters by CREB-dependent signaling and

CLOCK/BMAL1 activity. Proc Natl Acad Sci U S A 99:7728-7733.

Ueda HR, Hayashi S, Chen W, Sano M, Machida M, Shigeyoshi Y, Iino M,

Hashimoto S. 2005. System-level identification of transcriptional circuits

underlying mammalian circadian clocks. Nat Genet 37:187-192.

154

Vida B, Hrabovszky E, Kalamatianos T, Coen CW, Liposits Z, Kallo I. 2008.

Oestrogen receptor alpha and beta immunoreactive cells in the suprachiasmatic

nucleus of mice: distribution, sex differences and regulation by gonadal

hormones. J Neuroendocrinol 20(11):1270-7.

von Gall C, Duffield GE, Hastings MH, Kopp MD, Dehghani F, Korf HW, Stehle JH.

1998. CREB in the mouse SCN: a molecular interface coding the phase-adjusting

stimuli light, glutamate, PACAP, and melatonin for clockwork access. J Neurosci

18:10389-10397.

Wang Q, Ye Q, Lu R, Cao J, Wang J, Ding H, Gao R, Xiao H. 2014. Effects of

estradiol on high-voltage-activated Ca(2+) channels in cultured rat cortical

neurons. Endocr Res 39:44-49.

Wang Y, Zhu G, Briz V, Hsu YT, Bi X, Baudry M. 2014. A molecular brake controls

the magnitude of long-term potentiation. Nat Commun 5:3051.

Welsh DK, Takahashi JS, Kay SA. 2010. Suprachiasmatic nucleus: cell autonomy

and network properties. Annu Rev Physiol 72:551-577 .

Welsh DK, Yoo SH, Liu AC, Takahashi JS, Kay SA. 2004. Bioluminescence imaging

of individual fibroblasts reveals persistent, independently phased circadian

rhythms of clock gene expression. Curr Biol 14:2289-2295.

Winder DG, Martin KC, Muzzio IA, Rohrer D, Chruscinski A, Kobilka B, Kandel

ER. 1999. ERK plays a regulatory role in induction of LTP by theta frequency

stimulation and its modulation by beta-adrenergic receptors. Neuron 24(3):715-

26.

Yan J, Wang H, Liu Y, Shao C. 2008. Analysis of gene regulatory networks in the

155

mammalian circadian rhythm. PLoS Comput Biol 4:e1000193.

Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM,

Hong HK, Oh WJ, Yoo OJ, Menaker M, Takahashi JS. 2004.

PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals

persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U

S A 101:5339-5346.

Yoshii A, Constantine-Paton M. 2010. Postsynaptic BDNF-TrkB signaling in synapse

maturation, plasticity, and disease. Dev Neurobiol 70(5):304-22.

Zhang H, Roubos EW, Jenks BG, Scheenen WJ. 2006. Receptors for neuropeptide Y,

gamma-aminobutyric acid and dopamine differentially regulate Ca2+ currents in

Xenopus melanotrope cells via the G(i) protein beta/gamma-subunit. Gen Comp

Endocrinol 145:140-147.

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Table 4.1. List of genes selected for interrogation.

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Table 4.2. Summary of Gene Ontology (GO) terms enriched within interrogated genes.

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Table 4.3. Summary of Gene Ontology (GO) terms significantly enriched within groups of genes regulated in a specific direction by estradiol and/or a light pulse at ZT16.

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Table 4.4. Summary of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways within regulated interrogated transcripts.

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Figure 4.1.

Figure 4.1. Clustered heat map representation of SCN genes interrogated following administration of CTL or estradiol and a ZT16 light pulse or no pulse. The relative

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quantification (RQ) for each gene of interest per treatment group is colored to indicate fold change compared to the CTL/non-pulsed group (CN) (CTL/pulsed = CP;

Estradiol/non-pulsed = EN; Estradiol/pulsed = EP). Green indicates a reduction in expression, while red indicates induction. Black is associated with no change. An HCL algorithm determined within TM4-MeV4.4 was used to cluster genes based on functional properties and target distance.

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Figure 4.2.

Figure 4.2. Clustered heat map representation of interrogated genes that contribute to

Rhythmic Processes, Regulation of Transcription, or Metabolic Processes across treatment groups. The relative quantification (RQ) for each gene of interest per treatment group is colored to indicate fold change compared to the CTL/non-pulsed group (CN)

(CTL/pulsed = CP; Estradiol/non-pulsed = EN; Estradiol/pulsed = EP). Green indicates a reduction in expression, while red indicates induction. Black is associated with no change. An HCL algorithm determined within TM4-MeV4.4 was used to cluster genes based on functional properties and target distance.

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Figure 4.3.

Figure 4.3. KEGG pathway enrichment analysis of regulated genes identified within the

Circadian Rhythm pathway. Only significantly regulated transcripts of interest (p<0.05)

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are shown. Pathway enrichment is indicated by a red star, while the direction in which the specific transcripts were altered and by which treatment are shown in the table.

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Figure 4.4.

Figure 4.4. KEGG pathway enrichment analysis of regulated genes identified within the

MAPK Signaling pathway. Only significantly regulated transcripts of interest (p<0.05)

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are shown. Pathway enrichment is indicated by a red star, while the direction in which the specific transcripts were altered and by which treatment are shown in the table.

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Figure 4.5.

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Figure 4.5. KEGG pathway enrichment analysis of regulated genes identified within the

Neurotrophin Signaling pathway. Only significantly regulated transcripts of interest

(p<0.05) are shown. Pathway enrichment is indicated by a red star, while the direction in which the specific transcripts were altered and by which treatment are shown in the table.

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Figure 4.6.

Figure 4.6. KEGG pathway enrichment analysis of regulated genes identified within the

Long-Term Potentiation pathway. Only significantly regulated transcripts of interest

(p<0.05) are shown. Pathway enrichment is indicated by a red star, while the direction in which the specific transcripts were altered and by which treatment are shown in the table.

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CHAPTER 5: Summary

Authors: Royston SE1,2

1Neuroscience Program, University of Illinois Urbana-Champaign 2Medical Scholars Program, University of Illinois College of Medicine

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In this thesis, I wanted to test the global hypothesis that estradiol exerts both a developmental and adult role on the expression of rhythmic behavioral and transcriptional outputs. The work shown here demonstrates that estradiol exerts both activational and organizational effects on the expression of daily and circadian activity rhythms in mice. These data are the first to show that acutely circulating estradiol mediates circadian rhythms and that its effects are exerted through the differential activation of estrogen subtype receptor 1 (ESR1) and estrogen subtype receptor 2

(ESR2). ESR1 primarily mediates the magnitude of activity, while activation of ESR2 alters the timing of when this activity occurs. Specifically, I found that estradiol increases total wheel running activity, consolidates activity to the dark phase, and increases the amplitude of the activity rhythm through a mechanism dependent on the activation of ESR1. Conversely, an ESR2-dependent mechanism is responsible for activational estradiol’s ability to distribute activity across the subjective night, delay the acrophase of wheel running activity, reduce the phase angle of activity onset, and dampen the behavioral response elicited by a light cue in the early evening. I also discovered that the endogenous length of day is mediated through activation of either ESR1 or ESR2.

Further, developmental estrogenic signaling during a neonatal critical period associated with maturation of the suprachiasmatic nucleus (SCN), the ‘master clock’, programs both the quantity of total wheel running activity as well as the distribution of this activity across the subjective night. It also organizes the acrophase and the endogenous length of the active period, a measure that was not affected by activational estrogenic signaling. While the effects of activational estradiol were robust and altered nearly all examined parameters, modulation of estradiol during the first 5 days of life

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induces more subtle control over daily and circadian activity variables. Additionally, these effects are often sex specific.

Finally, I show here that estradiol not only modifies functional circadian behavioral outputs, but also acts to regulate the master oscillator itself. The transcriptional milieu within the SCN is substantially altered by estradiol at a time of day when estradiol most strongly opposes photic-induced behavioral plasticity. Estradiol primarily exerts these effects through downregulation of message expression.

Importantly, I show, for the first time, that estradiol significantly dampens the expression of transcripts associated with neuronal and synaptic plasticity mechanisms. Together, the studies presented here greatly expand our understanding of estrogenic signaling within the SCN and its ability to shape and modify daily and circadian behavioral rhythms throughout the lifespan.

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