REGULATION of FOOD ANTICIPATORY ACTIVITY a Dissertation Submitted to Kent State University in Partial Fulfillment of the Requir

REGULATION OF FOOD ANTICIPATORY ACTIVITY

A dissertation submitted

to Kent State University in partial

fulfillment of the requirements for the

degree of Doctor of Philosophy

Jessica A. Krizo

August, 2016

Dissertation written by

Jessica A. Krizo

B.S. Kent State University, 2007

Ph.D., Kent State University, 2016

Approved by

Eric Mintz , Chair, Doctoral Dissertation Committee

Colleen Novak , Members, Doctoral Dissertation Committee

John Johnson

Mary Ann Raghanti

Stephen Fountain

Accepted by

Laura Leff , Chair, Department of Biological Sciences

James Blank , Dean, College of Arts and Sciences

TABLE OF CONTENTS

Page TABLE OF CONTENTS……………………………...………………………………....iii

LIST OF FIGURES………………………...…………………………………..…………v

LIST OF ABBREVIATIONS……………………………..……………………………viii

ACKNOWLEDGEMENTS………………………………………………………………ix

CHAPTERS

I. Introduction………………………………………………...……………………..1

Biological and Circadian Rhythms………………………………………………..1

The Mammalian Suprachiasmatic Nucleus……………………………………….3

SCN Afferents and Efferents ……………………………………………………..4

Molecular Mechanism of the SCN ……………………………………………….9

Entrainment to Photic and Non-photic Stimuli…………………………………..12

Extra-SCN and Peripheral Clocks……………………………………………….13

Food Anticipatory Activity………………………………………………………16

Biological Sex and Circadian Rhythms………………………………………….20

Overall Aims……………………………………………………………………..24

II. Role of tissue plasminogen activator in the locomotor response to food restriction ……...…………………………………………………………………26

Introduction………………………………………………………………………26

Materials and Methods……………………………………………………...……29

Results…………………………………………………………………...……….32

Discussion……………………………………………………………………..…46

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III. Sex Differences in FAA in response to restricted feeding…………….….……..50

Introduction………………………………………………………………………50

Materials and Methods………………...…………………………………………53

Results…………………………...……………………………………………….55

Discussion……………………….……………………………………………….65

IV. Role of gonadal hormones in response to FAA ……….…………..………...…..68

Introduction………………………………………………………………………68

Materials and Methods………………………...…………………………………74

Results……………………………………...…………………………………….79

Discussion……………………………...……………………………………….110

V. Global Discussion………...…………………………………………………….118

Future Directions……………………………………………………………….119

REFERENCES…………………………………………………………………………121

LIST OF FIGURES

Figure 1. The mouse suprachiasmatic nucleus …………………...…..…………………..3

Figure 2. Major projections to the SCN…………………..…………...………………….6

Figure 3. Molecular transcription/translation feedback loop….…………..……..……...10

Figure 4. Phase Response Curves…………………………………………...…..………13

Figure 5. The Fibrinolytic Pathway………………………………………………..……28

Figure 6. Food Restriction Protocol………………………………………………..……30

Figure 7. LD RF: representative actograms; 24 hour baseline activity profile….………33

Figure 8. LD RF: fast-induced 24 hour activity profiles…………………………..……35

Figure 9. LD RF: restricted feeding 24 hour activity profiles…………………….…….36

Figure 10. LDsk RF: representative actograms; 24 hour baseline activity profile………………………………………………………………………………….…38

Figure 11. LDsk RF: fast-induced 24 hour activity profiles…………….……………… 39

Figure 12. LDsk RF: restricted feeding 24 hour profile and weight change…….………40

Figure 13. Phase angle of entrainment representative actograms and period ……...……42

Figure 14. Phase angle of entrainment following RF in LD and LDsk ………….……….43 . Figure 15. Food intake analysis…………………………………………….……………45

Figure 16. Wild-type male vs female baseline locomotor activity………………...…….56

Figure 17. Wild-type male vs female fast-induced activity……………………………...58

Figure 18. Wild-type male vs female food restriction induced activity ………………..59

Figure 19. Knock-out male vs female baseline locomotor activity …………………….62

Figure 20. Knock-out male vs female fast-induced activity……………………...……..63

Figure 21. Knock-out male vs female food restriction induced activity ………..……..64

Figure 22. Liver Clock gene expression ………………………………………..………65

Figure 23. Experimental protocol…………………………………..………………...…79

Figure 24. Male representative actograms ………………..……..…………...…………81

Figure 25. Female representative actograms……………………...………………….…82

Figure 26. Baseline total activity sex comparison………………………………...…….83

Figure 27. Male and female total baseline activity ……………………………………..84

Figure 28. Baseline 24 hour activity profile sex comparison …………………………..85

Figure 29. Baseline 24 hour activity profile male and female ………………..….……..86

Figure 30. Baseline weights in males and females …………………………….….……87

Figure 31. Fast day 1 male and female 24 hour activity profiles ……………..….….….89

Figure 32. Fast day 1 male vs female fasted groups 24 hour activity profiles ……...….90

Figure 33. Fast day 2 male and female 24 hour activity profiles ………………….……91

Figure 34. Fast day 2 male vs female fasted groups 24 hour activity profiles …..……..92

Figure 35. Fast-induced weight loss in males and females ………………………..……93

Figure 36. Food restriction total activity level sex comparison …………………..…….95

Figure 37. Food restriction male vs female food restricted 24 hour activity profiles …..96

Figure 38. Food restriction male and female 24 hour activity profiles…………...……..97

Figure 39. Food restriction total activity in males and females ………………...………98

Figure 40. Food anticipatory activity sex comparison ……………………….…………99

Figure 41. Food restriction-induced weight changes in males and females …………..100

Figure 42. Testosterone replacement baseline 24 hour activity profile ………...……..102

Figure 43. Testosterone replacement overall baseline and weight………………...…..103

Figure 44. Testosterone replacement fast day 1 ……………………………...………..104

Figure 45. Testosterone replacement fast day 2 ……………………………………….105

Figure 46. Testosterone replacement food restriction ………………………..………..106

Figure 47. Estrogen replacement baseline 24 hour activity profile ………………..….108

Figure 48. Estrogen replacement overall baseline and weight………………….……..109

Figure 49. Estrogen replacement fast day 1 ……………………………………….…..110

Figure 50. Estrogen replacement fast day 2 ……………………………………….…..111

Figure 51. Estrogen replacement food restriction ……………………………..………112

vii

List of Abbreviations

AL: ad libitum

AR: androgen receptor

AVP: arginine vasopressin

Bmal: brain and muscle aryl hydrocarbon receptor nuclear translocator

Clock: circadian locomotor output cycles kaput

Cry1: cyrptochrome 1

Cry2: cyrptochrome 2

CT: circadian time

DD: constant dark

DHT: dihydrotestosterone

DMH: dorsomedial hypothalamus

DR: dorsal raphe

ERα: estrogen receptor alpha

ERβ: estrogen receptor beta

FAA: food anticipatory activity

FEO: food entrainable oscillator

GDX: gonadectomy

GHT: geniculohypothalamic tract

IGL: intergeniculate leaflet

KO: knock out

LD: light dark

viii

LL: constant light

MASCO: methamphetamine sensitive circadian oscillator mBDNF: mature brain derived neurotrophic factor

MR: median raphe

NPY: neuropeptide Y

ORCH: orchiectomy

OVX: ovariectomy

Per1: period 1

Per2: period 2

PR: progesterone receptor

PRC: phase response curve proBDNF: pro brain derived neurotropic factor

PVN: paraventricular nucleus qRT-PCR: quantitative realtime polymerase chain reaction

RF: restricted feeding

RHT: retinohypothalamic tract

RN: ralphe nuclei

SCN: suprachiasmatic nucleus

TP: testosterone proprionate tPA: tissue plasminogen activator

WT: wild type

ZT: zeitgeber time

Acknowledgments

Every journey starts with that first step that is nervous and unsteady, but throughout the years it is nourished and rewarded with a gentle hand and a proud word. I have been blessed to be surrounded by a village that has worked both directly and indirectly to allow me the resources and strength to achieve more than I ever could alone.

These people are numerous but I will try to acknowledge those that have made an indelible impact on me both personally and professionally.

First, I would like to thank my Ph.D. advisor, Dr. Eric Mintz, for his unwavering support and guidance through my career at Kent State University. He has helped me renew my joy of learning, even when things got rough, through his passion for learning. I have come to understand and deeply respect his manner of mentorship, and know that through this mentorship I have become the driver of my own career.

I would also like to thank the members of my committee: Drs. Colleen Novak,

John Johnson, and Mary Ann Raghanti for their scientific and personal help and guidance over the years. Additionally, I am forever thankful for the support and guidance from

Drs. Heather Caldwell and Jennifer Marcinkiewicz. It is the breadth of knowledge presented to myself and students like me that allow us to develop a strong investigative background to take onward. I have been incredibly fortunate to work with these scientists in an open and collaborative environment.

To the members of the Mintz lab, both past and present: Dr. Erin Paulus, who taught me the importance of attention to detail and helped me learn to think like a

x scientist. Linley Moreland, who’s friendship and teaching provided and strong basis for my work at Kent and helped me to assist others in making the transition. To those I have had the privilege of working with over the years: Tracey Topacio, Jessica Vespoli, Ghada

Nusair, and Amanda Klein: thanks for good company and companionship. A special thank you to William Huffman, who by virtue of friendship helped get me through many of the rough patches with little damage done. To Dr. Ashutosh Rastogi: thank you for your passion for your work, both academically and socially. I have appreciated your guidance in some big decisions. You have been a pleasure to work with and made me enjoy all the chaos that surrounds the last year of graduate school and have been kind to me through some of my hardest trials.

I would also like to acknowledge the tireless work done by the departmental staff.

It was these people who provided the backbone for all that I was able to do. I do think that our experience rivals the best I have seen. I thank them for all their guidance, patience, and advice in the professional pitfalls and graduate school complications that arise.

I am grateful beyond measure for the love and support of my family. To my

Mom, thank you for giving your all in the face of uncertain circumstances to be the rock and center for two incredibly lucky children. I will consider myself lucky should I become half the woman you are. Your belief in me and your welcoming of my passions was vital to me becoming the woman I am today. To my brother, thank you for your friendship and guidance. To the rest of my family, I thank you for the love and support, the good and the bad that we have shared. It has made me who I am. To Lauren, who though not bound by blood or genetics, has been a constant source of support, advice, and

xi love for nearly two decades. To James Zagray, who took a socially awkward 12 year old with a love of science and life and gave her the room and the guidance to flourish. It was in under your tutelage that I knew this is what I wanted to do.

And last, but certainly not least, I would like to thank my husband, Ted. You have been with me through most of this process, and I am forever indebted to you for your love, your patience, and your unending support. You picked me up when I was down, nursed me to health both physically and emotionally, and you pushed me when I needed a boost. You have taken the brunt of the stress and anxiety with a gentle and reassuring calm-even when I was not the easiest to deal with. I lack the requisite vocabulary to share with you how much your love and support has meant.

xii

This dissertation is dedicated to my Mom, Terri Murphy, who taught me to be strong and persevere.

And to my husband, Ted Krizo who helps me remember my strength.

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CHAPTER I

Introduction

1.1 Biological and Circadian Rhythms The earth rotates, tilted on its own axis, creating a 24 hour day. This rotation drives the timing of sunrise and sunset, establishing alternating periods of light and dark

(photoperiods) creating a need for circadian rhythms. Circadian comes from the Latin circa (about) and dia (day). Circadian rhythms are cycles of biochemical, physiological, and behavioral processes that occur with a period of about 24 hours. Circadian rhythms evolved because they allowed organisms to fill different ecological niches in order to maximize fecundity through the balance of maximum resource utilization and minimal predatory threat. This system for timekeeping is both present in and critical to the function of some bacteria, plants, fungi, and animals.

Organisms use external cues, or zeitgebers (from the German time giver), to entrain their behavior and physiology to a 24-hour cycle. For instance, plants require energy derived from the sun for photosynthesis and utilize broad flat leaves to maximize

1 surface area for exposure. However, there is a cost to this exposure through respiration, so to deal with this conflict plants open their leaves during the day and close them at night (Moore-Ede, Sulzman et al. 1982). This kind of adaptation can be seen in animal behavior as well, with prey animals exhibiting activity at times that minimize exposure to predators. An example of this can be seen in rodents and birds of prey. Many rodent species are nocturnal, allowing them to use the low environmental light to attempt to avoid predation. However, this is exploited by predators who have developed abilities that allow them to overcome the low light and thereby reduce competition for diurnal prey species. In this case, it is the ability of both predator and prey to utilize circadian rhythms that allows for the distribution of activity over a 24 hour period within an ecosystem.

These rhythms are not simply mirrors of the external environment. Both plants and animals demonstrate rhythms in activity while removed from light exposure or other time cues, indicating an internally generated (endogenous) rhythm. Three criteria must be met in order for a rhythm to be circadian. First, the rhythm has to oscillate with a period of about 24 hours and persist in constant conditions, such as constant light or constant dark. A rhythm is endogenous if it is maintained in constant conditions.

Second, the rhythm must be entrainable, meaning the rhythm is capable of being reset

(shifted) by environmental stimuli, such as light/dark cycle and temperature. Third, the rhythm must exhibit temperature compensation, meaning that changes in the ambient temperature should not affect the 24 hour period of the rhythm (Moore-Ede, Sulzman et al. 1982; Dunlap 2004; Refinetti 2006).

1.2 The Mammalian Suprachiasmatic Nucleus

1.2.1 Structure

In mammals, endogenous rhythms are generated and regulated by a master circadian pacemaker, the suprachiasmatic nucleus (SCN) (determined by several laboratories, see (Weaver 1998) for review). The SCN is located in the ventral portion of the anterior hypothalamus, dorsal to the optic chiasm and bilateral to the third ventricle

(Figure 1), and is comprised of approximately 20,000 neurons (Van den Pol 1980;

Guldner 1983). Each nucleus of the bilateral SCN is comprised, in simplest terms, of two functionally and neuro-chemically distinct regions; a rhythmic shell is co-localized with arginine-vasopressin (AVP) releasing cells (Hamada, LeSauter et al. 2001; Yan and

Okamura 2002; Hamada, Antle et al. 2004) with little retinal innervation (Moore, Speh et al. 2002), and a light inducible core (Tanaka, Hayashi et al. 1997; Bryant, LeSauter et al.

2000) which releases vasoactive intestinal peptide and gastrin releasing peptide

(LeSauter, Kriegsfeld et al. 2002; Kawamoto, Nagano et al. 2003). The distribution of neuropeptide expression varies between species, making the distinction of shell and core general terms that are not appropriate across all species but are used by convention. See

(Antle and Silver 2005) for review.

1.2.2 Seat of the master pacemaker

The circadian network is a broad network of central and peripheral oscillators whose rhythms are coordinated by the SCN. The SCN is responsible for entraining the circadian system to photic stimuli. When the SCN is intact, neurons of both the SCN and other brain regions are rhythmic; however, when the SCN is isolated, rhythmicity in

3 firing rate persists whereas other brain regions become arrhythmic (Inouye and

Kawamura 1979). Lesions of the SCN eliminate endogenous rhythms in free running conditions (Moore and Eichler 1972; Stephan and Zucker 1972). The result is an animal with erratic bursts of activity, rather than the consolidated sleep wake cycle seen in intact animals. Transplantation of a donor SCN to an arrhythmic, SCN ablated host restores rhythmicity and activity consolidation (Lehman, Silver et al. 1987; DeCoursey and

Buggy 1989; Silver, Lehman et al. 1990) with circadian rhythms matching that of the donor SCN (Ralph, Foster et al. 1990; Kaufman and Menaker 1993; Sujino, Masumoto et al. 2003). SCN neurons in vitro show daily rhythms in firing rate that correspond to the light dark cycle (Green and Gillette 1982; Shibata, Oomura et al. 1982). Circadian rhythms in glucose utilization in vitro confirm the cycling nature of SCN cells (Schwartz and Gainer 1977).

1.3 SCN Afferents and Efferents

The SCN integrates environmental cues and drives circadian oscillations throughout the body. It is therefore critical to understand the paths of input to the SCN as well as the output that connects the SCN to behavioral and physiological circadian rhythms. There are three major projections to the SCN (Figure 2). While these three are the largest projections to the SCN, it is important to note that reciprocal communications with SCN afferents in the hypothalamus are common, and information such as feeding, temperature, social interaction, and locomotor activity or other arousal can also affect the cycling of the SCN (see section: SCN efferents).

SCN

http://www.mbl.org/atlas170/atlas170_frame.html

SCN SCN MN

Optic Chiasm

Figure 1: The mouse suprachiasmatic nucleus (SCN). (A) Coronal image of the mouse brain with labelled SCN. Figure from Mouse Brain Library. (B) Fos-staining in the SCN, labelled.

IGL

GHT RN

RHT

Photic Input SCN

Figure. 2: Major projections to the SCN. The retino-hypothalamic tract (RHT) transmits light information directly to the SCN and the intergeniculate leaflet (IGL). The IGL innervates the SCN via the geniculo-hypothamamic tract (GHT. Both the SCN and the IGL receive direct serotonergic innervation from the raphe nuclei (RN).

1.3.1 SCN Afferents

The first major projection is the RHT, a direct monosynaptic projection of retinal ganglion cells from the retina (Hendrickson, Wagoner et al. 1972; Moore and Lenn 1972;

Pickard and Silverman 1981; Pickard 1982). The RHT most densely innervates the ventral portion of the SCN (Hendrickson, Wagoner et al. 1972; Moore and Lenn 1972;

Johnson, Morin et al. 1988; Levine, Weiss et al. 1991). Photic information is relayed to the SCN via glutamate, the primary neurotransmitter in the RHT (Ding, Chen et al. 1994;

Mintz, Marvel et al. 1999) though aspartate, substance P, and PACAP also present

(Castel, Belenky et al. 1993; Ebling 1996). Evidence for the role of the RHT in photic entrainment can be demonstrated by electrical activation of the optic nerves, which leads to shifting of rhythms in the SCN consistent with a light pulse (Shibata and Moore 1993).

Lesioning the RHT blocks light-induced phase shifts and prohibits entrainment to light- dark cycles (Johnson, Moore et al. 1988). These studies confirm the need for the RHT in photic entrainment.

The second major afferent projection to the SCN is from the intergeniculate leaflet (IGL) (Ribak and Peters 1975; Swanson and Cowan 1979; Card and Moore 1982;

Card, Brecha et al. 1983; Moore, Gustafson et al. 1984). The IGL is innervated by the retina, thereby creating an indirect retinal projection to the SCN (Frost, So et al. 1979;

Hickey and Guillery 1979; Swanson and Cowan 1979) that can affect photic entrainment

(Harrington and Rusak 1986; Pickard 1989). This projection uses neuropeptide Y (NPY) as its primary neurotransmitter (Card, Brecha et al. 1983; Moore, Gustafson et al. 1984;

Harrington, Nance et al. 1985). The IGL is also involved in non-photic regulation of the

7 circadian system (Janik and Mrosovsky 1994; Wickland and Turek 1994) through the actions of NPY (Biello, Janik et al. 1994).

The third major projection to the SCN is the serotonergic innervation from the raphe nuclei. Many retrograde and anterograde tracing studies have confirmed the projections from both the median raphe (MR) (Meyer-Bernstein and Morin 1996; Meyer-

Bernstein, Blanchard et al. 1997; Meyer-Bernstein and Morin 1999) and the dorsal raphe

(DR) (Hay-Schmidt, Vrang et al. 2003). This projection relays non-photic information via serotonin. Microdialysis studies have shown that stimulation of either the MR or DR cause an increase in serotonin in the SCN (Dudley, Dinardo et al. 1999). Additionally, electrical stimulation of the raphe nuclei can shift behavioral responses and attenuate fos expression following a light pulse (Meyer-Bernstein and Morin 1999). The DR also projects to the IGL, thereby indirectly influencing the SCN (Meyer-Bernstein and Morin

1996; Moga and Moore 1996). Several studies indicate the raphe contribute to non- photic regulation of the SCN (Shibata, Oomura et al. 1982; Mintz, Gillespie et al. 1997;

Ehlen, Grossman et al. 2001).

1.3.2 SCN Efferents

The SCN integrates information from several regions as discussed above. In order to regulate rhythms, the SCN sends out many projections, utilizing both neural projections and diffusible signals. Neural projections typically act upon the following:

A) endocrine neurons, B) pre-autonomic neurons, or C) interneurons. Endocrine neurons are those that release hormones both centrally and peripherally. Pre-autonomic neurons are the origins of neurons that project to the pre-ganglion cells of the sympathetic and

8 parasympathetic nervous system. Interneuron projections are the largest class of SCN efferent and are those that innervate other medial hypothalamic regions (Kalsbeek, Palm et al. 2006).

Investigations with retrograde and anterograde tracers have identified many regions the SCN sends projections or signals to at varying degrees of intensity. These include, but are not limited to, the vasolateral septum (VLS), bed nucleus of the stria terminalus (BST), median pre-optic area (MPO), paraventricular nucleus (PVN), dorsomedial hypothalamus (DMH), arcuate (ARC), supraoptic nucleus (SON), anterior hypothalamic area (AHA), and subparaventricular zone (SPVZ) (Watts and Swanson

1987; Watts, Swanson et al. 1987; Cui, Saeb-Parsy et al. 1997; Leak, Card et al. 1999;

Abrahamson and Moore 2001; Leak and Moore 2001; Kriegsfeld, Leak et al. 2004).

Taken together, these regions work to coordinate circadian information and relay it to other central and peripheral systems though both neuronal and humoral pathways. Many of these regions project to the SCN as well as having connections among themselves, so there are potentially limitless opportunities for indirect effects of SCN output.

The cellular behavior of the areas with SCN projections is beyond the scope of this dissertation. However, hypothalamic regions that have been suggested to be involved in in the regulation of feeding and locomotor activity will be discussed later

(See: Food Anticipatory Activity).

1.4 Molecular Mechanism of the SCN

Circadian rhythms are generated within the SCN by an auto-regulatory transcription/translation feedback loop. There is an extensive literature on the basis of

9 molecular clock function (Reppert and Weaver 2001; Hastings and Herzog 2004;

Okamura 2004). The clock is comprised positive and negative feedback loops (Figure 3).

The positive loop involves the dimerization of the constitutively expressed Clock and

Bmal, and the subsequent binding of CLOCK: BMAL (King, Zhao et al. 1997; Gekakis,

Staknis et al. 1998). The negative arm of the loop consists of two gene families, Period

(Per 1, Per 2, and Per 3) and Cryptochrome (Cry 1, Cry 2, and Cry 3) (Zylka, Shearman et al. 1998). The CLOCK: BMAL complex binds to the E-box element in the promotor region of Per and Cry and initiates their transcription, starting at CT0. As Per and Cry are translated and protein product begins to accumulate in the cytoplasm, they begin to dimerize. These newly formed PER: CRY complexes stabilize the protein and allow it to move back into the nucleus, starting about CT12. Once in the nucleus, the complex blocks CLOCK: BMAL thereby inhibiting the transcription of Per and Cry (Reppert and

Weaver 2002; Hastings and Herzog 2004). There is an additional loop that modulates the positive loop which includes two main genes, Rev Erbα and Rora, whose expression are induced by the CLOCK: BMAL complex. The protein product REV-ERBα inhibits the transcription of Bmal (Preitner, Damiola et al. 2002; Ueda, Chen et al. 2002). Once PER:

CRY blocks CLOCK: BMAL the production of REB-ERBα is inhibited and the suppression of BMAL transcription is alleviated. Therefore, this secondary loop helps to stabilize rhythmic expression of clock genes across 24 hours.

Figure 3: Molecular transcription/translation feedback loop. Adapted from Hardin, P.E. (2004).

1.5 Entrainment to Photic and Non-photic Stimuli

1.5.1 Photic Entrainment

In order for an organism to adapt to an ever changing environment, the organism’s endogenous clock must be capable of responding to environmental stimuli.

In order to investigate rhythms, a general nomenclature has been developed. Organisms with no external stimuli express circadian rhythms as output of the endogenous clock.

The phase of the rhythm is therefore referred to as circadian time (CT). CT12 is the onset of activity in a nocturnal organism. By contrast, the phase organisms in the presence of an external stimuli or “zeitgeber” (German: time-giver) are referred to in zeitgeber time

(ZT) in this case, ZT12 marks the onset of the phase of activity to which an organism is entrained.

Photic cues are the most powerful zeitgebers for the entrainment of the SCN

(Moore 1983). Light is transduced in the retina into a neural signal by intrinsically photoreceptive ganglion cells, and this signal travels along the RHT to innervate the light sensitive ventrolateral core of the SCN where glutamate is released. Glutamate acts on

SCN neurons by activating NMDA receptors, causing an influx of Ca2+ which activates the mitogen-activated protein kinase cascade, leading to the phosphorylation of CAMP- response-element-binding protein (CREB). The activated CREB then binds to the

Ca2+/CAMP response element (CRE) in the promotor regions for Per1 and Per2, initiating their transcription (as reviewed by (Antle and Silver 2005). The initiation of

Per1 is thought to be of critical importance to SCN response to photic stimuli. The core of the SCN communicates with the shell using a variety of neurotransmitters to communicate resetting information with the rhythmic SCN shell neurons.

Light effects the clock in a time dependent manner. Light leads to phase delay in the early subjective night, phase advances in the late subjective night, and has no effect on phase during the subjective day (Daan 1977). Phase response to stimuli varies between photic and non- photic stimuli as seen in a phase response curve (Figure 4).

1.5.2 Non-Photic Entrainment

Photic cues are the strongest zeitgebers for entrainment of the SCN. However, in the absence of photic cues (and, at times, in addition to them), there are several non- photic cues that can affect the circadian clock. Exposure to non-photic stimuli causes phase advances in the subjective day and small phase delays in the subjective night

(Challet, Caldelas et al. 2003). These stimuli are diverse in origin and include temperature (Rensing and Ruoff 2002), food availability (Boulos and Terman 1980), social interactions, pharmacological treatments (Van Reeth, Vanderhaeghen et al. 1988), and induced activity (Reebs, Lavery et al. 1989; Reebs and Mrosovsky 1989; Reebs and

Mrosovsky 1989). Non-photic cues are thought to reach the SCN via several pathways, including serotonergic projections from the RN and NPYergic projections from the IGL.

These cues serve a vital role in maintaining appropriate entrainment.

1.6 Extra-SCN and Peripheral Clocks

The SCN was initially thought to be the one master regulator of all endogenous circadian function, the one ring to rule them all and in entrainment bind them. However, an extensive circadian network of central and peripheral oscillators responsible for the coordination of virtually all organ systems, tissues, and cell types have been shown to

Figure. 4: Phase response curves. The blue line indicates the phase response to non-photic stimuli and the orange line indicates the phase response to photic stimuli.

14 substantially modulate clock function. Central oscillators with varying levels of autonomy have been identified in several brain regions.

The first mammalian autonomous oscillator to be identified was the retina (Tosini and Menaker 1996). Retina cell cultures showed entrainment to LD, temperature compensation, and oscillations in constant conditions (Tosini and Menaker 1996; Tosini and Menaker 1998). The olfactory bulb also shows characteristics of being a self- sustaining autonomous oscillator (Granados-Fuentes 2004). The ability of the retina and the OB to function as autonomous oscillators is significant, suggesting the crucial importance of daily rhythms in sensory function. Later investigations found daily oscillations in gene expression in several hypothalamic and thalamic nuclei, in addition to the amygdala and the cerebellum, as reviewed by (Feillet, Mendoza et al. 2008) (Guilding and Piggins 2007). Interestingly, the neuroendocrine related nuclei, such as the hypothalamic nuclei ARC and PVN, show intrinsic oscillations that require the SCN for synchronization (semi-autonomous oscillators), while other brain regions only maintain oscillations in direct regulation of another oscillator (slave oscillators). Central oscillators are not in phase with one another, with gene expression showing various levels of delay from the SCN, suggesting site specific circadian requirements The SCN’s master role in circadian functioning is further muddied by recent investigations into the methamphetamine sensitive circadian oscillator (MASCO) and the food entrainable oscillator (FEO). Methamphetamines effect circadian functions, such as locomotor activity, onset of activity, and period length, and reinstates rhythmicity in SCN-lesioned rats (Honma, Honma et al. 1986; Honma, Honma et al. 1987). Food restriction in 24 hour

LD cycles also causes changes in locomotor activity and activity onset, and can reinstate

15 rhythms in SCN-lesioned rats (Boulos and Terman 1980; Mistlberger 1994). The

MASCO and FEO are oscillators are beginning to hint toward a broad network of central regulation of circadian rhythms.

Oscillations are present in most all peripheral tissues (Yamazaki, Numano et al.

2000), and these oscillations are the product of the same molecular feedback loops that generate SCN rhythms (Yagita, Tamanini et al. 2001). The SCN synchronizes peripheral tissues through both humoral and neural signals. Interestingly, SCN transplants to SCN- lesioned rats restored SCN communication with kidney tissue and skeletal muscle, but not heart or spleen tissue (Guo, Brewer et al. 2006). Many physiological processes exhibit daily oscillations in function. Some examples include metabolism in the liver, muscle and adipose tissue, urine production, and heart rate (Davidson, Castanon-

Cervantes et al. 2004) (Harfmann, Schroder et al. 2015) (Ptitsyn, Zvonic et al. 2006).

Peripheral gene expression is delayed compared to the SCN by about four hours, suggesting the SCN modulates the rhythms of peripheral tissues (Balsalobre 2002).

Peripheral tissue oscillations are subject to regulation by external stimuli in addition to direction by the SCN. Feeding rhythms entrain many tissues, and body temperature can also reset peripheral rhythms (Stephan 2002).

1.7 Food Anticipatory Activity

Food restriction is used to investigate a variety of behavioral and physiological processes, including memory, weight regulation, and aging (Mistlberger 2009). Food restriction was first noted to lead to an increase in activity almost a century ago(Richter

1922) and scheduled food restriction during the inactive phase has been found to induce

16 food anticipatory activity (FAA) in rats, mice, Syrian hamsters, and rabbits (Mistlberger

1994; Mistlberger and Marchant 1995) as well as fish and other aquatic and marine vertebrates (Azzaydi, Rubio et al. 2007; Sanchez, Lopez-Olmeda et al. 2009; Sanchez and Sanchez-Vazquez 2009). FAA is thought to represent an evolutionary adaptation to take advantage of food resources at abnormal times (Stephan, Laroche et al. 2001), and is defined behaviorally by an increase in activity (demonstrated by wheel running or general cage activity ) 2-3 hours prior to food presentation, (Antle and Silver 2009) and physiologically by increases in blood glucose, corticosterone, and body temperature

(Feillet, Mendoza et al. 2008).

The mechanism by which FAA develops has been studied for close to 40 years.

A review of these potential mechanisms is presented by (Mistlberger 2009). According to Mistlberger, potential mechanisms include 1) a metabolic hour glass timer, 2) interval timing, 3) memory for phase of pacemaker, 4) associative learning, and/or 5) a food entrainable oscillator. A metabolic hourglass timer would increase pre-prandial activity because of the drive for homeostasis. Hunger drives increased activity that ceases when feeding occurs. This is not an example of anticipation, however, as each food presentation would complete another independent cycle based solely on homeostatic need. There is some evidence for interval timing, learning when one event happens in relation to another (when food is present in relation to the LD cycle) in the development of FAA (Stephan 1984). These mechanisms are not sufficient to explain FAA because they both depend on feeding to initiate another cycle. Discrimination of phase

(remembering time of day food is present) and associative learning (pairing the conditioned phase with the unconditioned feeding time to have an appetitive response) do

17 not explain the gradual shift in FAA seen when time of food presentation is shifted.

Because of these reasons, as well as the need for food presentation to occur within circadian intervals the prevailing mechanism is a food entrainable oscillator that has the ability to drive rest- activity cycles.

The food entrainable oscillator is a circadian clock that responding to food availability and affects entrainment (Boulos and Terman 1980; Aschoff and von Goetz

1986; Mistlberger 1994; Stephan 2002). Rats and mice can entrain to restricted feeding in LD and LL, with rats and only certain strains entraining to restricted feeding in DD

(Stephan 1986; Stephan 1986; Castillo, Hochstetler et al. 2004; Abe, Honma et al. 2007).

Further, FAA can develop in LD, DD, and LL (Boulos, Rosenwasser et al. 1980;

Mistlberger, Sinclair et al. 1997). Restricted feeding is usually done in the mid-rest phase to separate anticipatory activity from normal activity of the active phase, but even so,

FAA can develop when food is limited in the active phase (Storch and Weitz 2009). This evidence supports a dual regulation of locomotor activity and rest-wake cycles by a light entrained and food entrained oscillator. Evidence for a role of a FEO in response to scheduled feeding fueled the search for its anatomical location and mechanism. The SCN suppresses activity and promotes rest during the rest phase (Mistlberger 2005). It is theorized that the FEO interacts with the SCN to inhibit the SCN directed suppression of activity (Mistlberger 2006; Moriya, Aida et al. 2009; Landry, Kent et al. 2011). The anatomical origin of the FEO has remained elusive. Studies showing FAA compromised by lesion studies are almost all met with studies that suggest there is no effect of the lesion on FAA; including lesions to the hippocampus, arcuate nucleus of the hypothalamus, area prostema, and olfactory bulbs (reviewed in (Feillet, Mendoza et al.

2008). Interestingly, the DMH has been thought to play a role in the regulation of the levels of activity during the day. The DMH receives information from the periphery regarding feeding activity and energy consumption, and has projections to the ventral pre-optic area to regulate wakefulness. It shares reciprocal projections with the SCN as well as other hypothalamic oscillators, making it an ideal candidate for the FEO. Further, chemical, but not electrolytic, lesions of the DMH impair FAA, suggesting a critical role as part of the FEO. Interestingly, both global and liver specific knock out of Per 2 eliminates FAA, however the presence of Per2 in the liver, while necessary, is not sufficient for FAA (Chavan, Feillet et al. 2016)Thus the FEO is currently thought to be a network of central and potentially peripheral oscillators that work together to direct responses to scheduled feeding.

The SCN drives rhythms in behavior, including the sleep/wake cycle, which in turn drives rhythms in feeding, which entrains many peripheral clocks. Scheduled feeding changes the relationship between peripheral oscillators and the SCN, decoupling the rhythms. Several tissues have been shown to entrain to restricted feeding, many with corresponding shifts in hormonal rhythms critical to food intake and energy balance, including the liver, stomach, intestines, kidneys, heart, lungs, and pancreas. FAA is not effected by rhythms from the stomach, liver, esophagus, or colon, regardless of entrainment to feeding (Davidson, Stokkan et al. 2002). The pancreas and adrenal glands are sites of potential regulation of FAA. The pancreas is reset by restricted feeding

(Damiola, Le Minh et al. 2000), and specifically, rhythms of insulin secreting beta-cells are altered (Marcheva, Ramsey et al. 2010). Insulin expression is increased at expected mealtimes. Glucagon, another pancreatic hormone usually in antiphase with insulin, is

19 decreased at the time of scheduled feeding (Davidson and Stephan 1999; Diaz-Munoz,

Vazquez-Martinez et al. 2000). Adrenal glands entrain to food restriction, and its cortisone secretions develop a bimodal distribution peaking at food presentation and the onset of activity in the active phase (Krieger 1979). This suggests a role in the development of FAA, however, FAA persists in adrenalectomized mice and rats

(Stephan, Swann et al. 1979; Boulos, Rosenwasser et al. 1980). Other potentially mediating hormones are ghrelin and leptin, as reviewed by (Patton and Mistlberger

2013).

1.9 Biological Sex and Circadian Rhythms Biological sex has extensive impacts on both behavior and physiology. Circadian parameters have been well investigated in both male and female mice with minor but significant differences. Addressed herein are the variances among period, photic and non- photic responses, and restricted feeding.

1.9.1 Period

The period of the circadian clock represents the time it takes to complete one cycle under constant environmental conditions, and is usually close to, but not exactly, 24 hours. Sex differences in period are highly species-specific, but even when present, the differences are generally modest. Free-running period in rats and golden hamsters is longer in males than females (Davis, Darrow et al. 1983; Schull, Walker et al. 1989); however, the differences in period are very small, whereas in Octodon degus period is longer in females by approximately half an hour (Labyak and Lee 1995; Lee and Labyak

1997). In mice with a C57BL/6J background, there does not appear to be a sex difference in free-running period (Kuljis, Loh et al. 2013). Despite the limited nature of the sex differences, gonadal hormones have a significant impact on circadian period.

Ovariectomy lengthens circadian period in rats and hamsters, and period is then shortened by replacement of estradiol (Morin, Fitzgerald et al. 1977; Morin, Fitzgerald et al. 1977; Albers 1981). However, no change in period is apparent in mice after ovariectomy (Kuljis, Loh et al. 2013), though estradiol, an estrogen receptor α agonist, or an estrogen receptor β agonist, shorten period in ovariectomized mice (Royston, et al.,

2014). In contrast, reports on the effect of castration on period are varied, one study indicates that it does lengthen period in mice (Daan, Damassa et al. 1975), while another showed no such effect (Schwartz and Zimmerman 1990). It appears that this effect may be dependent on the presence of constant dim red light (as opposed to true constant darkness) (Butler, Karatsoreos et al. 2012). Castration does not result in a change in period in hamsters (Morin and Cummings 1981).

A direct action of gonadal steroids on the SCN would likely be mediated by one or more of the steroid hormone receptors: estrogen receptor α (ERα), estrogen receptor β

(ERβ), androgen receptor (AR), progesterone receptor (PR), or G protein-coupled estrogen receptor 1 (GPER1). ERα, ERβ, and AR are all expressed in the SCN (Ehret and Buckenmaier 1994; Zhou, Blaustein et al. 1994; Shughrue, Lane et al. 1997;

Karatsoreos, Wang et al. 2007; Vida, Hrabovszky et al. 2008), with sexual dimorphisms present in ERβ, and AR (Vida, Hrabovszky et al. 2008). For a full review of the neuroanatomical aspects of sexual dimorphism in the circadian system, see (Bailey and

Silver 2014). In addition, the SCN receives input from other estrogen receptor-positive

21 regions of the brain (De La Iglesia, Blaustein et al. 1999), providing another potential mechanisms for steroid-modulation of SCN function.

1.9.2 Photic responses

There are a number of potential mechanisms by which biological sex, via gonadal steroids, can influence the photic sensitivity of the circadian clock. However, it is not known if the effects that have been found thus far are biologically important, and these effects may vary dramatically by species. In Octodon degus, females adjust to a six-hour advance of the light-dark cycle significantly faster than males (Goel and Lee 1995). In mice, females have larger phase shifts to light (Kuljis, Loh et al. 2013), while gonadectomized male mice have larger phase shifts than intact male mice (Karatsoreos,

Butler et al. 2011). The lengthening of period that occurs when animals are housed in increasing intensities of constant light is also potentiated in gonadectomized animals

(Butler, Karatsoreos et al. 2012). Female mice lacking estrogen receptor alpha, show increased phase shifting responses to light (Blattner and Mahoney 2013). These data are consistent with the idea that both estradiol and testosterone act to reduce the phase shifting effects of light.

1.9.3 Non-photic responses

There has been little work done investigating sex difference in non-photic influences on entrainment. In Syrian hamsters, a couple of studies have been done on the influence of the estrous cycle on circadian responses, but not with direct comparisons to male animals. Females show an estrous-cycle dependent modulation of activity level in response to a non-photic stimulus, such as a cage change or novel wheel exposure, but

22 this led to only modest variability in the size of non-photic phase shifts (Young Janik and

Janik 2003). However, they did note that large shifts during proestrus caused a 1-day delay in the estrous cycle. A similar delay was observed in response to phenobarbital treatment on proestrus, suggesting that large phase shifts caused the circadian clock to

“miss” generating the daily signal needed for the GnRH surge (Legan, Donoghue et al.

2009). However, in order to demonstrate a true sex difference in non-photic responses, it will be necessary to conduct experiments with direct comparisons between males and females, and it is important that this be done in additional species to see if there are common responses. In degus, there are sex differences in the effect of odor on circadian re-entrainment rates to shifts in the light/dark cycle, and these effects are influenced by estrogen, progesterone, and testosterone (Jechura, Walsh et al. 2003; Jechura and Lee

2004).

1.9.4 Food entrainment

When rodents are placed on a restricted feeding schedule, such that food is only available for a limited period of time each day during an animal’s normal sleep period, they show a behavioral response known as food anticipatory activity (FAA). This FAA generally takes the form of increased behavioral activation for a period of about three hours prior to food availability. FAA is particularly notable when animals are provided with a running wheel, as wheel-running during FAA can be more intense than normal nocturnal running. This activity is thought to be stimulated by the action of a circadian clock, as food availability that is timed in non-circadian intervals (e.g., 18 hrs.) does not result in FAA (Petersen, Patton et al. 2014). In addition, FAA persists for several cycles after a return to ad libitum feeding conditions and also does so in the absence of the SCN

(Stephan, Swann et al. 1979; Stephan, Swann et al. 1979). In mice, females have reduced

FAA compared to males (Li, Wang et al. 2015; Michalik, Steele et al. 2015). In a recent study by Li, et al, male mice were shown to have increased FAA, increased food intake and increased weight loss relative to female mice. Additionally, ovariectomy in females led to increases in FAA up to levels seen in intact mice (Li, Wang et al. 2015). A few studies have investigated the role of the reward system on entrainment to feeding using palatable foods. When receiving a high fat food as a snack, male mice exhibit anticipatory activity and females do not (Hsu, Patton et al. 2010). However, females show activity at the time of previous food delivery on subsequent days, suggesting that the females are still timing the arrival of the food but are not showing the anticipatory activity. There is some evidence to suggest that female motivation for sugary/fat foods is modulated by the estrous cycle (Clarke and Ossenkopp 1998). Dopamine is involved in the reward circuitry and is critical for reward seeking behavior, ablation leads to decreases in foraging behavior. Administration of either isoform of Dopamine, D1 or D2, prior to restricted feeding increases FAA (Michalik, Steele et al. 2015). The fact that circadian clock-driven anticipatory activity can occur under both normo-caloric and hypocaloric conditions suggests that there are multiple drivers of food anticipatory activity, a motivational circuit and a homeostatic circuit (Gallardo, Gunapala et al. 2012).

1.10 Overall Aims

The nature of circadian regulation of food entrainment has been the focus of extensive investigation for over four decades. The goal of this dissertation is to investigate the regulation of locomotor output during fasting and restricted feeding

24 conditions utilizing locomotor activity analysis, gene expression, surgery, and food intake assessments. The first aim is to investigate the role of tissue plasminogen activator in the development of locomotor activity during fasting and restricted feeding. We hypothesize that tPA plays a role in regulating the locomotor response to restricted feeding. The second aim is to investigate the role of biological sex in locomotor response to restricted feeding. We hypothesize that males and females will differentially respond to restricted feeding. The third and final aim is to investigate the role of gonadal sex on locomotor response to restricted feeding. We hypothesize that food anticipatory activity is regulated by gonadal hormones resulting in differential FAA between males and females.

CHAPTER II

Regulation of Locomotor Activity in Fed, Fasted, and Food-Restricted Mice Lacking Tissue Plasminogen Activator

2.1 Introduction Circadian rhythms of physiology and behavior are driven by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus (SCN) (Moore and Eichler

1972; Stephan and Zucker 1972). The SCN is directly innervated by retinal ganglion cells which provide the entrainment signals that synchronize SCN rhythms with the environmental light-dark (LD) cycle (Stephan and Nunez 1976; Pickard 1982; Morin and

Allen 2006). The signal transduction pathway that conveys photic information to the

SCN is dependent on the activation of the trkB receptor by brain-derived neurotrophic factor (BDNF) (Liang, Allen et al. 2000).The production of the mature form of BDNF

(Figure 5) in the brain is at least partly dependent on the extracellular activity of tissue- type plasminogen activator (tPA) which converts plasminogen to plasmin, which in turn catalyzes the conversion of proBDNF to mBDNF (Pang, Teng et al. 2004). Both BDNF and trkB are found in the SCN (Liang, Walline et al. 1998; Liang, Allen et al. 2000).

BDNF deficits lead to a decrease in light induced phase shifts (Allen, Qu et al.

2005), and trkB agonists in the SCN block light induced phase shifts of the circadian clock in vivo (Michel, Clark et al. 2006) and glutamate-induced phase shifts in vitro

(Mou, Peterson et al. 2009) . Further, tPA inhibition in vitro decreases phase shifts (Mou,

Peterson et al. 2009). These findings suggest that tPA activity is important for regulating glutamate induced phase shifts. However, nothing is known about whether tPA influences other forms of entrainment, such as the response of circadian clocks to timed restricted feeding regimes. In this study, we test the impact of the loss of tPA on the locomotor activity responses to changes in feeding regime.

When food availability is restricted to a narrow window of time per day, rodents exhibit a behavior known as food anticipatory activity (Bolles and Stokes 1965), which occurs for a 2-3 hour period prior to food availability. This activity appears to be driven by a food-entrainable circadian oscillator, and persists in the absence of a functional SCN

(Stephan, Swann et al. 1979; Marchant and Mistlberger 1997) or critical components of the molecular circadian clock mechanism (Pendergast, Nakamura et al. 2009; Storch and

Weitz 2009). A number of neuroendocrine regulatory factors contribute to appearance of food anticipatory activity (for a review, see (Patton and Mistlberger 2013)), however, the underlying mechanisms are still poorly understood. Because the loss of tPA reduces neuronal plasticity, we hypothesized that mice lacking tPA would have difficulty adapting to timed restricted feeding regimes.

Figure 5: The Fibrinolytic Pathway tPA acts on plasminogen to create the plasmin that converts proBDNF to the biologically active mBDNF form. This then acts on its cognate receptor, trkB, a tyrosine kinase membrane bound receptor.

2.2 Methods Animals. Animals used in this study were age-matched across each experimental group in each study. Two to six-month old male C57BL/6J wildtype mice (WT) and tPA knockout mice (KO) (bred from stock purchased from Jackson Laboratory (Bar Harbor,

ME), backcrossed to C57BL/6J) were used in all experiments. Animals were individually housed in Plexiglas cages equipped with a running wheel. Animals were housed at a temperature of 20°C and had access to water ad libitum. Food was also available ad libitum except as indicated below. In each experiment, WT and KO mice were age-matched. All animal use protocols in this study were approved by the Kent

State Institutional Animal Care and Use Committee and were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Restricted Feeding. Following a two-week baseline activity recording period, mice were deprived of food for 48 hours. Mice were then given four days of free food access before removal at lights off, or zeitgeber time (ZT) 12. Subsequently, food was presented to mice at ZT 6 and removed at ZT 10 (Figure 6). This was continued for eight to ten days at which point experimental protocol varied as detailed below. Body weight was measured during baseline activity, following fast, following free feeding period and after restricted feeding.

Activity Measurement. All cages were equipped with running wheels. Running wheel data was collected as revolutions per minute with either ClockLab (Actimetrics,

Wilmette, IL) or Med Associates (St. Albans, VT) and data was qualified and quantified using ClockLab. Activity profiles were calculated as an average activity per animal and

Figure 6: Food restriction protocol Animals are placed in ad-libitum feeding for two weeks before food removal at ZT 12 and replacement at ZT 12 48 hours later. Animals are given ad-libitum access for 72 hours to recover from fasting affects. Food is removed at ZT 12 prior to food restriction and returned from ZT 6- ZT 10 daily for 10 days.

30 per genotype as follows: baseline, averaged 4 day baseline and restricted feeding averaged over days three through eight. Activity profiles were created using both total revolutions and relative activity, calculated as a percentage of total 24 hour baseline activity. Food anticipatory activity (FAA) was defined as activity measured during the three hours (ZT 3-5) prior to food presentation.

Assessment of baseline activity profiles in a 12:12 LD cycle. Mice were housed in a 12:12 LD cycle and activity profiles were assessed, both in absolute terms and as a percentage of the 24-hour mean for each animal.

Food anticipatory activity in KO mice under light-dark conditions. Male WT and

KO mice were maintained in a standard 12:12 light-dark cycle and underwent the food restriction protocol described above.

Food anticipatory activity in KO mice with reduced light exposure (skeleton photoperiod). Male WT and KO mice were entrained to a 12:12 LD cycle for at least 10 days. Light exposure was then limited to 15 minutes at the onset and offset of the active phase (ZT 12-12.25 and ZT23.75-0). Mice were then given two weeks to acclimate to the skeleton photoperiod, following which food was restricted for eight days. Note that because different running wheels were used for the skeleton photoperiod experiment than the 12:12 LD experiment, running wheel activity levels cannot be directly compared between the two experiments.

Assessment of circadian phase during food restriction. Male WT and KO mice were placed in a skeleton photoperiod after being entrained to a 12:12 light-cycle. Mice were divided into four groups, WT RF and KO RF were food restricted and WT and KO ad/libitum feeding groups (AL) had continuous access to food. After ten days of

31 restricted feeding, mice were released into constant dark conditions with continuous access to food. Mice were allowed to free run for two weeks before phase was measured.

Food intake changes during restricted feeding. Male age matched c57 and KO mice were individually housed in small Plexiglas cages with metal grated cage liners and a PVC pipe for comfort. Weight and food intake was measured daily to generate a baseline. Mice then were food restricted and weight and food intake was measured daily.

Body composition analysis was completed with the use of an EchoMRI (Echo Medical

Systems, Houston, TX) for baseline, after fast, and before and after restricted feeding.

EchoMRI measured fat mass and lean mass, will lean mass being calculated as total body mass minus fat mass.

Statistical Analysis. Comparisons between groups were performed using one-way and two-way ANOVA with repeated measures where appropriate. Planned comparisons between genotypes throughout the 24-hour cycle were assessed using Fisher’s LSD test if the ANOVA showed a statistically significant interaction between genotype and clock time. Significance was ascribed if p < 0.05.

Results Food Restriction in wild-type and knockout mice in a 12:12 light dark photoperiod. During baseline both WT and KO exhibit typical nocturnal locomotor activity.

Nocturnal activity was divided into two discrete bouts of locomotor activity, a high level of activity in early to mid-night ending in a drop of locomotor activity followed by a brief increase in activity ending gradually at ZT 24. However, the level of activity was reduced in KO during the first part of the dark phase in LD from ZT 12-17 (F23, 575 = 2.63, p < 0.001; Figure 7).

KO WT

4500 KO WT 4000 * * * * * 3500

3000 * 2500 2000 1500 1000

500 Average total activity Average total activity (revolutions) 0 12 14 16 18 20 22 24 2 4 6 8 10 ZT

Figure 7 Representative actograms (A) of food restriction protocol in KO and WT mice in LD. Arrows indicate onset and offset of fast and beginning of restricted feeding. Fast includes food removal at ZT 12 and return 48 hours later at ZT 12. B. Average 24 hour activity profile (B), bar indicates dark phase; locomotor activity is higher in WT than KO at ZT 12-17 (p<0.05).

Food availability had an effect on both the pattern and level of locomotor activity in WT and KO. The pattern of locomotor activity was consistent between genotypes across all treatments. Food deprivation leads to increased diurnal activity across genotypes on both days. When food was removed at ZT 12 locomotor activity was suppressed compared to baseline activity during the first portion of the dark phase. KO had decreased activity compared to WT in LD fast day one (F (1, 22) = 4.57, p = 0.044;

Figure 8a). During fast day two locomotor activity increased significantly over WT

(Figure 8b) during both night, ZT 15-18 and day, ZT 5-7, 9, 10 (F(23,529) = 2.23, p

<0.001). There was no difference in weight loss between genotypes (KO: -20.6% ±.008g and WT: -21.7% ±.009g, t29 = 0.937, p = 0.357; Figure 8c). During restricted feeding the baseline differences in raw locomotor activity between genotypes disappeared and there was no difference in nocturnal or food anticipatory activity levels (F (23,547) = 1.18, p=0.253; Figure 9a). Following restricted feeding KO gained less weight than WT (KO:

-.9% ±.01 and WT: 2.91% ±.02, t28 = -1.6509, p = 0.109; Figure 9b).

A. 4000 Fast day 1: total activity 3500 KO WT 3000 2500 2000 1500 1000 500

Average total activity Average total activity (revolutions) 0 12 14 16 18 20 22 24 2 4 6 8 10 ZT

B. Fast day 2: total activity 4000 * 3500 * * 3000 * * * * 2500 * 2000 * 1500 1000 500

0 Average total activity Average total activity (revolutions) 12 14 16 18 20 22 24 2 4 6 8 10 ZT

C. 0 Figure 8: LD Fast induced locomotor activity. A-B show locomotor activity -5 levels across 24 hours, bars indicate -10 night phase. During fast day 1 total locomotor activity (A) is higher in WT -15 than KO (p<0.05) with no effect of time of day. During fast day 2 total locomotor

-20 activity is significantly higher in KO than % Weight % Change -25 WT during both the night (ZT15-18) and the day (5-7, 9,10) (F = 2.23, p KO WT (23,529) <0.001). Weight changes between genotypes are consistent following a 48- hour fast (C).

4500 RF D3-8: total activity 4000 KO WT 3500 3000 Food 2500 Access 2000 1500 1000

500 Average Total Activity Average Total Activity (Revolutions) 0 12 14 16 18 20 22 24 2 4 6 8 10 ZT

B. 6 5 4 3 2 1 0 *

% Weight % Change -1 -2 -3 KO WT

Figure 9: LD Food restriction-induced locomotor activity. Activity profile averages from days 3 through 8 of restricted feeding. Bars indicate night phase and dots indicate food availability from ZT6-10. Total locomotor activity (A) following restricted feeding (B) KO do not increase weight as WT (p<0.05).

Food Restriction in wild-type and knockout mice in a 12:12 skeleton photoperiod

A skeleton photoperiod was used to investigate the effect of light on locomotor activity levels in response to food deprivation and restricted feeding. During baseline both WT and KO exhibit typical nocturnal locomotor activity. Nocturnal activity was divided into two discrete bouts of locomotor activity, a high level of activity in early to mid-night ending in a drop of locomotor activity followed by a brief increase in activity ending gradually at ZT 24. However, the level of nocturnal activity was reduced in KO the in LDsk from ZT12-17 and 21 (F (23,805) = 4.57, p <0.001; Figure 10a, b). As seen in

LD, the pattern of locomotor activity was consistent between genotypes. It is important to note, however, that LDsk activity cannot be compared directly to LD, as different types of wheels were used. On fast day one, KO locomotor activity is less than WT from ZT 13-

15 (F (23,785) = 2.20, p=0.001; Figure 11a). There was no difference in activity levels on fast day two at any time point (F (23,805) = 0.86, p=0.653; Figure 11b). Weight loss was consistent between genotypes following fast day two (KO:-20.7% ± .97 and WT: -18.8%

± 1.3) (t18 = 2.2622, p = 0.267; Figure 11c). During restricted feeding days baseline differences in locomotor activity disappeared (F (23,805) = 0.64, p=0.899; Figure 12a) as a result of increased KO activity. Following restricted feeding KO gained less weight than

WT (KO: .78± 1.12 WT=4.7 ±1.43) (t17 = -2.1281, p = 0.048; Figure 12b).

KO WT

B. 3000 * 2500 * KO WT 2000 * * * 1500 *

1000 *

Average total activity Average 500

0 12 14 16 18 20 22 24 2 4 6 8 10 ZT

Figure 10: LDsk Baseline locomotor activity. Representative actograms (A) of

food restriction protocol in KO and WT mice in LDsk. Arrows indicate onset and offset of fast and beginning of restricted feeding. Fast includes food removal at ZT 12 and return 48 hours later at ZT 12. B. Average 24 hour activity profile (B), bar indicates dark phase; KO locomotor activity is higher in WT than KO at ZT 12-17 and 21 (p < 0.05).

3000 Fast day 1: total activity KO WT 2500 * * * * 2000 * *

1500

1000

500 Average total activity Average total activity (revolutions) 0 12 14 16 18 20 22 24 2 4 6 8 10 ZT B. 3000 Fast day 2: total activity

2500

2000

1500

1000

500 Average total activity Average total activity (revolutions) 0 12 14 16 18 20 22 24 2 4 6 8 10 ZT C. 0

-5 Figure 11: LDsk fast-induced locomotor activity -10 Activity profiles show locomotor activity levels across 24 hours, bars indicate night phase. During fast day 1 total -15 locomotor activity (A) is higher in WT than KO at ZT 13- Weight -20 18 (p<0.05). During day 2 fast total locomotor activity (B) there are no differences between genotypes. Weight -25 changes between genotypes are consistent following a KO WT 48-hour fast (C).

A. 2500 RF D3-8: total activity

KO WT 2000

Food 1500 Access

1000

500 Average total activity Average total activity (revolutions)

0 12 14 16 18 20 22 24 2 4 6 8 10 ZT

B. 7 6 5 4 3

2 % Weight % Change 1 0 -1 KO WT

Figure 12: LDsk food restriction-induced locomotor activity Activity profile averages from days 3 through 8 of restricted feeding. Bars indicate night phase and dots indicate food availability from ZT6-10. Total locomotor activity (A) is not different between genotypes. (A). Following restricted feeding (B) KO do not increase weight as WT (p<0.05).

Assessment of circadian phase during food restriction

The effect of timed restricted feeding on the SCN can be masked by light. When released to constant conditions following restricted or ad libitum feeding there was no genotypic effect on free-running period (F1,23 = 0.60, p = 0.447; Figure 13a,b). However,

RF treatment had an aftereffect on free-running period, shortening the free-running period of RF groups (KO RF: 23.77 ± 0.036, WT RF: 23.75 ± 0.019) compared to AL groups (KO AL: 23.84 ± 0.073, WT AL: 23.91 ± 0.026) (F1, 23 = 8.49, p= 0.008). There was no evidence of an underlying shift in the phase angle of entrainment toward food presentation in LDsk across genotypes (F1, 23 = 2.24, p = 0.148) or treatment (F1, 23 =

0.024, p = 0.631) (KO RF: -.33 ± 0.339, WT RF: .24 ± 0.194, KO AL: -.40 ± 0.188, WT

AL: -.01 ± 0.203; Figure 14a). Additionally, no difference in phase angle of entrainment was seen between genotypes following release from RF in LD to constant conditions (KO

RF= -0.24 ± 0.154, WT RF= 0.15 ± 0.262) (t14 = 2.306, p = 0.1882; Figure 14b).

KO WT

B. * 24.00

23.95

23.90

23.85 ) τ 23.80

23.75 Period Period ( 23.70

23.65

23.60 KO WT KO WT RF AL

Figure 13: LDsk food restriction to constant conditions. Representative Actograms of food restriction protocol in KO and WT (A) in skeleton photoperiod, followed by release into constant dark (indicated by arrow). There is no genotypic difference in period (B), however it there is a significant shortening of free-running period after RF as compared AL (p<0.05).

A. 0.6 RF AL

0.4

0.2

-0.2

Phase Phase Shift (hours) -0.4

-0.6

-0.8 KO WT

0.5 0.4 0.3 0.2 0.1 0 -0.1

Phase Shift (hours) -0.2 -0.3 -0.4 -0.5 KO WT Figure 14: Phase shifts following release to constant dark There were no genotypic or treatment effects seen on phase shifting in either LDsk (A) or LD (B).

Food intake analysis

Since differences in FAA might reflect differences in the motivation for feeding, we compared food intake in KO and WT. There was no difference in food intake (KO:

4.65g ± .09g WT: 4.79g± .07g) during standard LD conditions with food available ad libitum (t18 = 2.262, p = 0.964). There is no effect of genotype on feeding after the 48 hour fast (KO: 5.8g ± .19g WT: 5.7g ± .15g; t18 = 2.262, p < 0.001). During restricted feeding food intake in KO was less than WT (KO: 2.14g ± .06g, WT 2.73g ± .11g; t18 =

2.262, p < 0.001; Figure 15a). There are no genotypic differences in weight at baseline

(KO= 29.21g ±.58g, WT= 28.43g ± .44g; t18 = 2.262, p = 0.297), after 48 hour food deprivation (KO= 23.62 ± .51, WT= 22.22 ± .44; t18 = 2.2622, p = 0.0519), or following

RF (KO=26.27g ± .41g, WT= 26.91g ± .47g, t18 = 2.262, p = 0.316; Figure 15b). Changes in weight following food restriction were due to lean mass. KO lean mass change was less than WT (KO=-3.265g ±.23g, WT= -3.7076g ±.27g; t18 = 2.262, p = 0.001). There was no difference in the loss of fat mass between genotypes (KO = -1.243g ±.11g, WT=-

1.1872g ±.20g; t18 = 2.262, p = 0.657). Following RF there was no genotypic difference in lean mass change (KO=-2.753g± .10g, WT=-2.714g ±.45g; t18 = 2.262, p = 0.823) but fat mass change differed between genotypes (KO=-.78g ± .26g, WT=.3533g ± .10g; t18 =

2.262, p < 0.001; Figure 15c).

7 KO WT A. 6

3 Chow Chow (g) 2 *

0 Baseline Post Fast RF

B. 35 30 25 20 15 Body WeightBody (g) 10 5 0 Baseline Fast RF

Fast RF C. Δ Lean Δ Fat Δ Lean Fat Δ 1 0.5 * 0

(g) -0.5 -1

Mass -1.5 Δ -2 -2.5 -3 -3.5 -4 -4.5 Figure 15: Food intake analysis. There is no difference in food intake (A) at baseline or following food deprivation. During RF KO consume less chow than WT (p<0.05). There are no differences in body weight (B) at any part of the study. Analysis of body composition over the course of the restricted feeding protocol (C) shows no difference in changes in lean or fat mass during the 48-hour fast. During restricted feeding lean mass change is consistent between genotypes but fat mass change is different between genotypes (p< 0.05).

2.4 Discussion

The line of research outlined here was initiated was initiated with the goal examining the role of tPA in regulating circadian rhythms of activity, particularly with regard to circadian clock-driven responses to restricted feeding. The results, however, suggest a more direct role for TPA in modulating locomotor activity output in response to circadian drive. Our initial finding was that tPA knockouts showed reduced nocturnal wheel-running under a standard 12:12 LD cycle. It is likely that this results from the deficiency in mature BDNF in these mice. The positive link between BDNF and locomotor activity has been examined largely in the context of animal models of depression (Siuciak, Lewis et al. 1997; Shirayama, Chen et al. 2002), but not explicitly for the motivated behavior of voluntary wheel-running.

Interestingly, reduced wheel-running in the tPA knockout mice was reversed on day two of a 48-hr fast. During fasting, both WT and KO mice show a second daily peak of activity during the light phase of the light-dark cycle. On day one of the fast, activity is elevated during the light phase in both genotypes, but is still reduced in KO. On day 2, however, nocturnal wheel-running in WT is reduced while it is increased in KO, suggesting an increased activation of the behavior in response to the energetic deficit.

The timing of the behavior also seems to be somewhat altered on day, with the peak in dark-phase locomotor activity delayed slightly in KO mice. Despite the difference in locomotor activity, however, there was no difference in total weight loss during the fast.

The phenomenon of fast-induced increases in activity in rodents is well documented

(Richter 1922), however, our data suggests a role for tPA in the neural processes that

46 regulate this behavior. Furthermore, the increase in wheel-running in KO mice on day 2 of the fast provides evidence that the decrease in wheel-running under baseline conditions is not due to any kind of physical deficiency, but is more likely related to processes relating to the motivation for wheel-running.

Because of the role tPA plays in regulating plasticity in the brain (Salazar,

Caldeira et al. 2016), we had anticipated that KO mice might have some difficulty adapted to a timed restricted feeding schedule. This turned out not to be an issue. Timed restricted feeding totally eliminated the difference in wheel-running activity between KO and WT. A strong bout of activity in the three hours present prior to food presentation was observed in both genotypes, with a compensatory decrease in activity during the latter half of the dark phase. There was a small but statistically significant difference in the change in body weight during the restricted feeding regime, with weight increasing slightly in WT but not in KO. It could be that increases in locomotor activity in KO resulted in increased energy expenditure, however, given the short-term nature (~10 days) of the restricted feeding period we are reluctant to attribute much functional importance to this finding.

Visual inspection of actograms during the course of these experiments led us to hypothesize that KO mice might have a greater increase their activity during the light phase of the LD cycle by virtue of being less sensitive to the masking effect of light on locomotor activity. If so, then repeating the experiment using a skeleton photoperiod would eliminate any masking effects, and WT activity would remain higher than KO during restricted feeding or on day 2 of fasting. The results, however, did not support this hypothesis, at least not entirely. During day 1 of the fast, KO mice still had reduced

47 activity during the subjective night relative to WT. The overall increase in activity in the subjective day was similar in the two genotypes and appeared to be less than that seen in

LD. On fast day two, KO mice increased their activity to match that of the WT mice but did not increase it above WT as was seen in LD. These data cannot be explained by our masking hypothesis. In addition, during restricted feeding in the skeleton photoperiod activity in KO mice matched that of WT, exactly the same result as seen in LD.

Normally, there is an increase in FAA in skeleton photoperiod when compared to a regular LD cycle (Patton, Parfyonov et al. 2013). Unfortunately, due to the use of different running wheels, we cannot quantitatively compare the results in the two photoperiod regimes directly, but we can conclude that the differences in fast and restricted feeding-induced activity between genotypes are not a result of differences in the masking effects of light.

tPA does appear to play a role in the regulation of photic input to the SCN (Mou,

Peterson et al. 2009). After a period of restricted feeding, we released mice into ad/lib feeding and constant darkness to assess the phase of the underlying nocturnal activity rhythm. We found no genotypic differences in free-running period, or phase at the time of the photoperiod transition, suggesting that SCN function was largely unaffected by restricted feeding. However, we did note that restricted feeding had an aftereffect on free-running period in the subsequent constant dark period, irrespective of genotype, in the form of a shortening of the period of the wheel-running rhythm. Since this rhythm is driven by the SCN, it suggests periods of restricted feeding may have a more subtle, long-lasting effect on the SCN.

As a result of seeing small differences in the change in body weight between genotypes during restricted feeding, we conducted a separate study on food intake. The protocol for this experiment differed from the locomotor activity studies in that the mice were housed in cages without running wheels, but which were designed for more accurate assessment of food intake. We found no genotypic differences in food intake during baseline conditions or during refeeding after a 48 hour fast. However, total food intake was significantly reduced in food restricted as compared to ad/lib, and was reduced in

KO compared to WT. This manifested as a small decrease in fat mass in KO that was not present in WT. These data do suggest that from an energetic standpoint the KO mice have a slightly decreased ability to adapt to restricted feeding, but the lack of a difference in the fasted animals suggests that the difference is not metabolic but is more likely behavioral.

Overall, the data presented here suggests that the effects of tPA on locomotor activity are primarily mediated by action in the brain. The most likely route for these effects is through tPA’s regulation of mature BDNF production, and that tPA’s actions are stimulatory for wheel-running locomotor activity. The current literature tPA’s functions in modulating hypothalamic-driven behaviors is sparse; further investigations into the location of tPA’s action in the brain may reveal important pathways that show divergence between locomotor activity driven by circadian rhythms and those driven by energetic demands.

CHAPTER III

Sex differences in food anticipatory activity in response to restricted feeding

3.1 Introduction Circadian rhythms in behavior and physiology are regulated centrally by the suprachiasmatic nucleus (SCN) (see (Dibner, Schibler et al. 2010) for review). The SCN is comprised of bilateral nuclei, consisting of about 20,000 neurons and seated in the ventral portion of the anterior hypothalamus, dorsal to the optic chiasm and bilateral to the third ventricle (Broida and Svare 1983). The SCN responds directly to environmental light (photic cues) via the retino-hypothalamic tract (RHT) that innervates the SCN

(Hendrickson, Wagoner et al. 1972; Moore and Lenn 1972; Pickard and Silverman 1981;

Pickard 1982). This allows the SCN to synchronize internal circadian rhythms with the environment.

The circadian network includes organs, tissues, and cells throughout the body, all of which must maintain stable phase relationships to each other (Yamazaki, Numano et al. 2000; Yagita, Tamanini et al. 2001). In addition, clocks have been detected in numerous regions throughout the brain, with several nuclei having distinct circadian oscillations with varying levels of autonomy including the retina, olfactory bulbs,

50 thalamus, amygdala, and several hypothalamic nuclei (Feillet, Mendoza et al. 2008).

Analysis of rhythmic gene expression in these regions suggests that they maintain stable, but independent phases relative to one another, suggesting site specific requirements for circadian phase (Guilding and Piggins 2007). Circadian oscillations have been found in all peripheral tissues to date, following the same molecular feedback loop that generates

SCN rhythmicity (Yagita, Tamanini et al. 2001). Many of these peripheral oscillations are more sensitive to effects of non-photic stimuli including feeding, temperature, and pharmacological interventions, than the central oscillations in the SCN (Stephan 2002).

Food restriction was first noted to lead to an increase in activity almost a century ago (Richter 1922). Scheduled food restriction during the inactive phase induces food anticipatory activity (FAA) in rats, mice, Syrian hamsters, and rabbits, in addition to some non-mammalian species (Mistlberger 1994; Mistlberger and Marchant 1995). This activity is thought to be stimulated by the action of a circadian clock, as food availability that is timed in non-circadian intervals (e.g., 18 hrs) does not result in FAA (Petersen,

Patton et al. 2014). FAA is defined behaviorally by an increase in activity (demonstrated by wheel running or general cage activity ) 2-3 hours prior to food presentation (Antle and Silver 2009), and physiologically by increases in blood glucose, corticosterone and body temperature (Feillet, Mendoza et al. 2008). During ad-libitum feeding, circadian rhythms of the SCN and peripheral tissues are coupled, with physiology and behavior regulated primarily by the output of the SCN. In mice, scheduled feeding causes SCN rhythmicity to become decoupled from peripheral oscillators, with the SCN not responding to feeding changes (Damiola, Le Minh et al. 2000). Several tissues entrain to restricted feeding, many with corresponding shifts in hormonal rhythms critical to food

51 intake and energy balance, including the liver, stomach, intestines, kidneys, heart, lungs, and pancreas. A recent study showed that a liver- specific deletion in Per2 abolished

FAA while its overexpression rescued FAA, suggesting a peripheral origin for FAA

(Chavan, Feillet et al. 2016).

At the time the experiments described in this chapter were initiated, there had been little published literature on potential sex differences in food anticipatory in mice.

However, there have now been two labs to address the differences seen in female food anticipatory activity. In mice, females have reduced FAA compared to males (Li, Wang et al. 2015; Michalik, Steele et al. 2015). Male mice were also shown to have increased food intake and increased weight loss relative to female mice (Li, Wang et al. 2015).

When receiving a high fat food as a snack, male mice exhibit anticipatory activity and females do not (Hsu, Patton et al. 2010). However, females show activity at the time of previous food delivery on subsequent days, suggesting that the females are still timing the arrival of the food but are not showing the anticipatory activity. Dopamine is involved in the reward circuitry and is critical for reward seeking behavior, ablation leads to decreases in foraging behavior. Administration of Dopamine D1 or D2 agonists prior to restricted feeding increases FAA (Michalik, Steele et al. 2015). The fact that circadian clock-driven anticipatory activity can occur under both normo-caloric and hypocaloric conditions suggests that there are multiple drivers of food anticipatory activity, a motivational circuit and a homeostatic circuit (Gallardo, Gunapala et al. 2012).

Our goal in this investigation was to explore the female response to fasting and food restriction and examine potential mechanisms for this difference.

3.2 Materials and Methods Animals

Animals used in this study were age-matched across each experimental group in each study. Adult male and female C57BL/6J (WT) mice, 2-5 months old, were housed on a

12:12 light-dark (LD) cycle prior to the experiments. Animals were individually housed in Plexiglas cages equipped with a running wheel, were housed at a temperature of 20°C, and had access to water ad libitum. Food was also available ad libitum except where indicated. All animal use protocols in this study were approved by the Kent State

Institutional Animal Care and Use Committee and were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the

National Institutes of Health.

Food Restriction Protocol

Following a two-week baseline activity recording period, food was removed and mice were fasted for 48 hours. Mice were then given four days of free food access before removal at ZT 12. Subsequently, food was presented to mice at ZT 6 and removed at ZT

10. Restricted feeding procedure was continued for eight to ten days at which point experimental protocol varied as detailed below. Body weight was measured during baseline activity, following fast, following free feeding period and after restricted feeding.

Behavioral Monitoring and Assessment

All cages unless otherwise noted were equipped with running wheels. Two types were used, a wired traditional stainless steel wheel and a plastic wireless dish-wheel. There is

53 no difference in the acclimation of novice mice between types. Running wheel activity was monitored with Med Associates’ Wheel Manager or Actimetric’s Clocklab.

Locomotor activity was exported to Microsoft Excel and running wheel activity calculated as total revolutions as well as activity relative to average 24 hour baseline.

Peripheral Gene Expression

Adult male and female C57BL/6J (WT) were placed either under the food restriction protocol or continued ad libitum feeding. Animals were sacrificed via cervical dislocation and liver samples were collected at the height of FAA (ZT5) on the sixth day and flash frozen and stored at -80 C. Liver tissue was homogenized and total RNA was extracted and purified using Qiagen Rneasy kits with Dnase treatment. Total RNA was then converted into cDNA for use in real-time quantitative PCR (qPCR) using a reverse transcription kit from Qiagen. All samples analyzed using qPCR for the expression of two genes using stock primer/probe sets from IDT (Per1 and Per2). Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) expression was used as the control gene. Relative gene expression was determined by converting all samples to a relative expression value through the use of a 2-

ΔCT, with ΔCT being the average expression of the triplicates from the output from the qPCR minus the average for GAPDH for the triplicates. Significant differences in gene expression were evaluated using two-way ANOVAs, with time of sex and treatment as factors. Interactions between factors were analyzed using a Tukey-Kramer Multiple

Comparison Test. Significance was ascribed for all statistical tests if p < 0.05.

3.3 Results Food restriction in male and female wild-type and knockout mice in a 12:12 light dark photoperiod WT Mice During baseline activity recording both male and female mice exhibited a similar distribution of nocturnal locomotor activity across time. Females showed increased activity compared to males at ZT 12 and midway through the active phase (ZT 19-21) (F

(23,368) = 1.57, p= 0.005; Figure 16a, b). Males weighed significantly more at baseline

(Males: 26.88g ± 0.33g; Females: 22.23g ± 0.54g; t18 = 7.14, p < 0.001; Figure 1c). On fast day one, male activity increased, compared to baseline, at ZT 1-15, 20-21, and 24 (F

(23, 276) = 0.20, p<.005) and female activity increased, compared to baseline, at ZT 4-12,

22-24 (F (23, 46) = 0.10, p<.005). There were no sex differences seen in fast day one activity (F (23,368) = 1.36, p= 0.12; Figure 17a). On day two of the fast, nocturnal locomotor activity was decreased compared to fast day one in males from ZT 1,6-11, 16-

24 (F(23, 276)= 3.80, p<0.005) and females from ZT 4-11 and 13-24 (F(23, 46)= 0.20, p<.005). There was no sex difference in activity during fast day two (F (23,368) = 0.40, p=

0.99; Figure 17b). Weight loss following the fast was not significantly different between sexes (Male: -21.7% ± 1.2% Female -25.6% ± 1.5%, t18 = 1.18, p = 0.086; Figure 17c).

During restricted feeding, locomotor activity distribution is similar between sexes and males show a small but not statistically significant elevation in food anticipatory activity

(F (23,368) = 1.27, p= 0.19; Figure 18a) compared to females. There are no sex differences in weight change following restricted feeding (Male: -2.9% ± 2.6% Female 2.2% ± 6.3%, t16 = 1.69, p = 0.11; Figure 18b).

B. WT Baseline 5000 * WT M WT F

4000 * 3000 * * 2000

1000

Average hourly Average hourly activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

C. Wild-type Baseline Weight

30 25 * 20 15 10 Weight(g) 5

0 Male Female Figure 16: Wild-type male vs female baseline locomotor activity. Representative actograms (A) for wild-type male and female mice under a 12:12 LD cycle over the duration of study, Activity profile for baseline ad- libitum feeding (B) indicates increased nocturnal activity at onset (ZT 12) and midway through the active phase (ZT 19-21), Data is represented as mean total activity in each one hour bin throughout the 24 hour light-dark cycle. Dark bar represents lights off. Male mice have increased weight compared to females at baseline (C). Asterisks denote significance (P < 0.05).

A. WT Fast Day 1

7000 WT M WT F 6000 5000 4000 3000 2000 1000 Average hourly Average hourly activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

B. WT Fast Day 2 4000 WT M WT F 3500 3000 2500 2000 1500 1000 500 Average hourly Average hourly activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT Figure 17: Wild-type male vs female fast-induced activity. Activity profiles of WT male and C. Fast Induced weight change, % female mice during (A) the first 0 day of 48 hour fast (B) the -5 second day of 48 hour fast. There was no difference in -10 activity. Data is represented as -15 total activity in each one hour

% Change % bin throughout the 24 hour light- -20 dark cycle. Dark bars indicate -25 lights off. Weight loss was -30 consistent between males and WT Male WT Female females (C). Asterisks denote significance (P < 0.05).

A. WT RF D3-8 5000 WT M WT F 4000

3000

2000

1000 Average hourly hourly Average activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

B. Restricted Feeding Weight Change, % 0.1

0 Weight change, %

-0.1 WT Male WTFemale

Figure 18: Wild-type male vs female food restriction- induced activity. Activity profiles of WT male and female mice during days 3-8 of food restriction (A), There is no difference in activity between male and female. Data is represented as mean total activity in each one hour bin throughout the 24 hour light-dark cycle. Dark bars indicate lights out. Weight changes are consistent between sexes (C).

KO Mice During baseline both male and female mice exhibited a similar distribution of nocturnal locomotor activity across time. There was no difference in baseline locomotor activity between male and female KO mice (F (23,334) = 0.39, p= 0.88; Figure 19a, b).

Male KO mice are significantly heavier than females (Male: 31.3g ± 0.6g; Female 27.2g

± 1.4g; t18 = 2.26, p = 0.04; Figure 19c). On fast day one, male activity increased compared to baseline at ZT 5-15 and 20 (F (23, 252) = 0.20, p<.005) and female activity was no different from baseline (F (23, 46) = 0.29, p = 0.99). There were no sex differences seen in fast day one activity (F (23,298) = 1.50 p= 0.07; Figure 20a). On day two of the fast locomotor activity was decreased compared to fast day one in males from ZT 5-6,11-16, and 20 (F(23, 227)= 3.58, p<.005) and female activity was no different from baseline (F(23,

46)= 0.84, p = 0.66). There was no sex difference in activity during fast day two (F (23,368)

= 0.40, p= 0.99; Figure 20b). Weight change was similar between sexes (Male: -20.5%

± .8%; Female -21.7% ± 2.6%; t18 = 0.571, p = 0.57; Figure 20c). During restricted feeding locomotor activity was similar between sexes with no time interaction (F (23,334) =

0.75, p= 0.79; Figure 21a). Weight changes were consistent between sexes (Male: -0.9%

± 1.1 %; Female 2.5% ± 3.5%; t18 = 1.23, p = 0.24; Figure 21b).

Clock gene expression in restricted fed male and female mice during food anticipatory activity

For Per1, there was a significant effect of restricted feeding, with elevated expression during restricted feeding (F (1, 28) = 10.38, p= 0.003). Expression in females was higher than in males, but this difference was not statistically significant (F (1, 28) =

2.67, p= 0.11). There was no interaction between sex and feeding condition (F (1, 28) =

0.04, p= 0.84). For Per2, there was a significant effect of sex, with expression in females being significantly greater than in males (F (1, 28) = 4.81, p= 0.037). There was no significant effect of restricted feeding (F (1, 28) = 0.81, p= 0.37) and no significant interaction between restricted feeding and sex (F (1, 28) = 0.23, p= 0.64; Figure 22).

B. KO Baseline 5000 KO M KO F

4000

3000

2000

1000 Average hourly hourly Average activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

Figure 19: KO male vs female C. baseline locomotor activity. Representative actograms (A) for tPAKO Baseline Weight tPAKO male and female mice 40 under a 12:12 LD cycle over the duration of study, Activity profile 30 * for baseline ad-libitum feeding (B) 20 show no differences between Grams male and female. Data is 10 represented as mean total activity in each one hour bin throughout 0 KO M KO F the 24 hour light-dark cycle. Dark bar represents lights off. Male mice have increased weight compared to females at baseline (C). Asterisks denote significance (P < 0.05).

A. KO Fast Day 1 7000 KO Male KO Female 6000 5000 4000 3000 2000 1000 Average hourly hourly Average activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT B. KO Fast Day 2 4000 KO Male KO Female 3500 3000 2500 2000 1500 1000 500 Average hourly Average hourly activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

C. Fast Induced weight change, % 0 -5 -10 -15 -20 -25

-30 Weight change, % KO Male KO Female

Figure 20: Wild-type male vs female fast-induced activity. Activity profiles of KO male and female mice during (A) the first day of 48 hour fast and , (B) the second day of 48 hour fast, Data is represented as mean percentage of total activity in each one hour bin throughout the 24 hour light-dark cycle.(C) Average baseline weights change for male and female mice. Vertical bars indicate lights off. Asterisks denote significance (P < 0.05).

KO RF D3-8 5000 KO Male KO Female 4000

3000

2000

1000 Average hourly Average hourly activity 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

RF Weight Change % 7 6 5 4 3 2 1 0 Weight change, % -1 -2 -3 KO Male KO Female

Figure 21: KO male vs female food restriction-induced activity. Activity profiles of KO male and female mice during days 3-8 of food restriction (A), Data is represented as mean percentage of total activity in each one hour bin throughout the 24 hour light-dark cycle.(B) Average post food restriction weight change for male and female mice.

600% *

500%

400%

300% * *

Relative Relative Expression 200%

100%

0% Per 1 Per 2

Female AL Male AL Female RF Male RF

Figure 22: Liver Clock gene expression. For Per1, there was a significant effect of restricted feeding, with elevated expression during restricted feeding. Expression in females was higher than in males, but this difference was not statistically significant. There was no significant interaction term. For Per2, there was a significant effect of sex, with expression in females being significantly greater than in males. There was no significant effect of genotype and no significant interaction.

3.4 Discussion This line of research developed from our study on the role of tissue plasminogen activator (tPA) in locomotor response to food restriction (see Chapter 2 and (Moreland

2010)). In both wild-type (WT) and tPA knock out (KO) mice we noticed that females showed decreased levels of food anticipatory activity compared to males (effect occurred within genotypes). Additionally, based on our initial analysis of Chapter 2 we thought

(incorrectly) that KO mice showed increased levels of FAA. Therefore, as an aside we sought to determine if the mechanisms were connected while addressing the potential reduction of FAA in females.

In this study we investigated the relationship between biological sex, tPA and locomotor activity in response to food challenges. To do so, we quantified wheel running activity during ad-libitum, fasting, and food restriction conditions. Consistent with published data, we saw that WT females have increased wheel running activity compared to males during ad-libitum conditions (Bartling, Al-Robaiy et al. 2016). Interestingly, we found that in the KOs there was no difference in wheel running activity between male and female animals. tPA is used to treat ischemia in humans, and appears to have a reduced efficacy in women (Buijs, Uyttenboogaart et al. 2016) suggesting that at least some aspects of tPA function may differ between sexes.

Fast-induced increases in diurnal locomotor activity and metabolic impacts are well documented (see (Jensen, Kiersgaard et al. 2013) for review). This increase in activity is thought to represent an increase in food seeking behavior. There were no differences in fast-induced activity between males and females in either WT or KO. This was different from what we expected to see. Based on our preliminary findings we

65 expected females would have reduced levels of locomotor activity during fasting compared to males. These data indicate that females do not respond differently to fasting conditions.

Food restriction induces a change in activity distribution, with mice exhibiting a period of robust locomotor activity 2-3 hours prior to food presentation. During food restriction we found no difference in either total wheel running or distribution between males and females of either genotype. Females show reduced FAA in other research from our lab (see Chapter 4 and (Moreland 2010)) and that is consistent with published research (Li, Wang et al. 2015; Michalik, Steele et al. 2015). The lack of a sex difference in this study could be a false negative result, arising from random variation and a relatively small sample size.

In the second part of this study, we asked how peripheral expression of period gene expression might differ between males and females in response to food restriction.

Food restriction causes the advance of period gene expression in peripheral tissues involved in feeding toward the time of food presentation without changing the phase of the SCN in mice (Damiola, Le Minh et al. 2000; Hara, Wan et al. 2001). We hypothesized that period gene expression in females may not respond to food restriction in a manner consistent with males, reflecting differences in FAA. To test this, we compared the expression of clock genes Per1 and Per2 in the liver in food restricted and ad libitum fed males and females at ZT 5, one hour prior to food presentation. We selected the liver because it is involved in feeding and the circadian rhythms of liver tissues are known to be shifted during food restriction. We found that Per1 was elevated in food restricted compared to ad libitum fed mice, with no significant effect of sex which

66 is consistent with published data ((Li, Wang et al. 2015). Interestingly, Per2 was increased in females compared to males with no effect of food restriction. This appears to be a novel finding in female mice. In mice fed exclusively during the day (with 12 hour food access) the phase of both Per1 and Per2 are inverted (Damiola, Le Minh et al. 2000;

Mukherji, Kobiita et al. 2015). A recent paper showed that a liver-specific deletion of

Per2 was enough to abolish FAA (Chavan, Feillet et al. 2016). However, in this study we see no change in Per2 during food restriction, suggesting that Per2 alone cannot be responsible for the origin of FAA in the liver. It is possible that a difference in Per2 expression in females causes a change in the response of the liver to food restriction and subsequent behavioral and locomotor activity changes.

In conclusion, this study demonstrated that female mice do exhibit both fast- induced and food anticipatory activity with no difference in activity levels. Based on our other data we suspect this is a false negative due to a small sample size. This issue is addressed in Chapter 4. We also found no link between tPA and biological sex, as there was no difference in effect between WT and KO. Additionally, we find that Per1 expression responds to food restriction in a similar fashion in males and females, and present a novel finding that Per2 expression in females is elevated compared to males at

ZT 5.

CHAPTER IV

Role of gonadal hormones in the locomotor response to food challenge

4.1 Introduction

The mammalian circadian network is regulated by the suprachiasmatic nucleus

(SCN), which coordinates daily central and peripheral cycles of molecular, physiological and behavioral activity (Mohawk, Green et al. 2012). To do this, the SCN must integrate a broad and complex set of information ranging from external sensory input to internal central and peripheral physiological conditions. The response of the SCN to these conditions can depend on biological sex. Gonadal hormones estrogen and testosterone are present in both the central and peripheral systems of adult mammals and can serve to modulate circadian function (see Chapter 3 Introduction).

Gonadal hormones affect the SCN through interaction with gonadal hormone receptors. Androgen receptors (AR) have been identified in the SCN of mice

(Karatsoreos, Wang et al. 2007) and rats (Zhou, Blaustein et al. 1994) and in females AR expression is less than half that of males (Iwahana, Karatsoreos et al. 2008). There are two classical estrogen receptors, estrogen receptor α (ER α) and estrogen receptor β (ER

β). ER β has been localized in the SCN (Vida, Hrabovszky et al. 2008) and has a five-

68 fold increase in expression compared to ER α expression (Vida, Hrabovszky et al. 2008;

Bailey and Silver 2014). Progesterone (PR) mRNA expression in mice is co-localized with estrogen receptor mRNA (Lauber, Romano et al. 1991) while human SCN neurons contain progesterone receptors (Kruijver and Swaab 2002).

Estrogen and circadian rhythms

Activity onset in intact females is dependent upon circulating estrogen concentration which varies with the estrous cycle (Morin, Fitzgerald et al. 1977). The earliest onsets correspond with peak estradiol concentrations (pro-estrous and estrous in hamsters) (Baranczuk and Greenwald 1973), estrous only in rats (Albers, Gerall et al.

1981). Consistent with reports that estrogen advances the onset of activity rhythms is its effects on period in mice, rats and hamsters. OVX rats and hamsters exhibit a lengthened period of wheel running that is shortened by the delivery of exogenous estrogen (Morin,

Fitzgerald et al. 1977; Morin, Fitzgerald et al. 1977; Albers, Gerall et al. 1981) .

Removal of endogenous estrogen via ovariectomy reduces wheel running activity in mice (Ogawa, Chan et al. 2003), rats (Albers 1981), and degus (Labyak and Lee 1995).

Replacement with 17-β estradiol rescues intact levels of activity (Rodier 1971). These studies support a role of estrogen as enhancing locomotor activity in rodents. Estrogen implants into the preoptic area increase wheel running, demonstrating a direct effect of estrogen on locomotor activity (Fahrbach, Meisel et al. 1985) and since ER α is expressed more than ER β, it was suggested that the estrogen mediated effect on locomotor activity is through ER α. In an elegant follow up study, Ogawa, et al. investigated the role of ER

α and ER β by utilizing ER α and ER β knock out mice. WT and ER β KO mice were

69 ovariectomized and treated with exogenous estrogen and had an increase in activity. ER

α KO with same treatment mice did not increase activity levels, supporting the previous finding that ER α is a component in estrogen mediation of locomotor activity (Ogawa,

Chan et al. 2003). This is further supported by Royston, et al. with the demonstration that stimulation of ER α leads to an increase in locomotor activity whereas ER β does not affect locomotor activity but does affect other circadian parameters (Royston, Yasui et al.

2014).

Estrogen regulates energy balance (Wade and Schneider 1992; Alonso-

Magdalena, Ropero et al. 2008; Foryst-Ludwig and Kintscher 2010) and has a basal

(tonic) and cyclic (phasic) effect in the mediation of food intake and feeding in females of several species including mice, rats, hamsters, and humans (Eckel 2004). In rats, decreased food intake occurs at the peak of circulating estrogen concentrations (Butcher,

Collins et al. 1974; Blaustein and Wade 1976) at pro-estrus. In rats, ovariectomy leads to an increase in food intake (dependent upon meal size) and weight gain (Wade 1975;

Blaustein and Wade 1976; Asarian and Geary 2002) which is restored to intact levels with physiological levels of estrogen (Tarttelin and Gorski 1973; Czaja, Butera et al.

1983; Asarian and Geary 2002). The decrease in food intake does not appear to be dependent upon peripheral effects of estrogen on metabolism (Palmer and Gray 1986).

Stimulation of the VMH with 17-β estradiol decreases food intake in rats (Wade and

Zucker 1970). The PVN has been suggested to mediate estrogen’s effects on feeding in some studies (Berthoud 2002; Leibowitz and Wortley 2004) but not in others (Dagnault and Richard 1994; Hrupka, Smith et al. 2002). By blocking ER β with an ICV infusion of anti-sense ER the effects of estrogen on food intake and body weight are blocked in

70 rats (Liang, Akishita et al. 2002) suggesting that ER β is responsible for the mediation of food intake by estrogen. However, ER α mutant OVX mice treated with 17-β estradiol do not have the expected decrease in food intake, suggesting that in mice ER α is responsible for estrogen’s effects (Garey, Morgan et al. 2001).

Progesterone and circadian rhythms

Progesterone has not been investigated as extensively as estrogen or testosterone, yet given its rhythmic cycling with estrogen over the estrous cycle, it merits consideration. When given in conjunction with estrogen in rats it has no effect on activity levels. However, when given in low doses to intact mice there is a decrease in activity. Further, OVX mice treated with progesterone have no further decrease in activity but OXV mice treated with estrogen have rescued activity levels until being given progesterone (Rodier 1971). Additionally, when OVX mice are treated with progesterone there is no effect however when given in conjunction with estradiol the effects of estradiol treatment alone are blocked (Takahashi and Menaker 1980). This supports the suggestion that progesterone acts in opposition to estrogen and that the effects of progesterone on locomotor activity are through the estrogen mediated pathway.

In some cases the effect is so profound as to remove the effects of estrogen within one activity cycle (Wollnik and Turek 1988). The effects of estrogen and progesterone on feeding vary with nutrient composition and food availability (Yu, Geary et al. 2008). In normal feeding conditions, the administration of estrogen alone to OVX Sprague-Dawley rats reduced food intake levels compared to both co-administration of estrogen or progesterone or progesterone alone. However, in a binge food paradigm (food access

71 given for one hour, there was no difference in food intake effect between estrogen, progesterone, or co-administration (Yu, Geary et al. 2011). In OVX Fischer rats, co- administration of estrogen and progesterone increases food intake to a lesser extent than estrogen alone (Varma, Chai et al. 1999). Taken together, these reports suggest progesterone acts in opposition to estrogen to regulate food intake.

Testosterone and circadian rhythms

Testosterone most likely mediates circadian rhythms at the SCN by binding to androgen receptors (AR) which are highly localized to the ventro-lateral area of the SCN

(Karatsoreos, Wang et al. 2007). Castration leads to a reduction of AR expression in the

SCN which is rescued by testosterone implants. However, the increased expression of

AR in castrated testosterone treated mice does not neatly correlate with behavioral changes seen following castration (Model, Butler et al. 2015). Castration in mice leads to a lengthened free running period and a shift in activity distribution, with mice losing the onset bout of activity and the peak of activity being at the end of the active phase (Daan,

Damassa et al. 1975). Male mice in DD show decreased activity and shifts in its distribution. When treated with testosterone propionate (TP) mice demonstrate restored locomotor activity distribution as well as free running period (Daan, Damassa et al. 1975;

Butler, Karatsoreos et al. 2012). In hamsters, castration or short photoperiod leads to a decrease in precision of locomotor activity onset as well as cohesion (Morin and

Cummings 1981).

Testosterone treatment has no effect on locomotor activity levels in intact males

(Eleftheriou, Elias et al. 1976) or females (Li and Huang 2006). Castration leads to a

72 decrease in locomotor activity that is rescued with exogenous testosterone (Broida and

Svare 1983). However, given the possibility of aromatization of exogenous testosterone to estrogen, Roy and Wade gave castrated rats 17-β estradiol, testosterone, dihydrotestosterone (DHT), or oil and found that 17-β estradiol has the greatest impact on activity levels, followed by testosterone. Neither DHT nor oil effect the locomotor activity levels in castrated rats (Roy and Wade 1975; Karatsoreos, Wang et al.

2007).These data suggest that the effect of testosterone on locomotor activity is through an estrogen mediated pathway. To address this issue Merc-25, an estrogen antagonist, was given to castrated rats treated with either 17-β estradiol or testosterone and decreased activity in both groups (Roy and Wade 1975).

According to Chai, ET. al., testosterone has been implicated in feeding behavior in rats (Kakolewski, Cox et al. 1968; Gentry and Wade 1976), mice (Petersen 1978;

Blank, Korytko et al. 1994), and hamsters (Slusser and Wade 1981) (Chai, Blaha et al.

1999). Castrated males consume less food and decrease weight gain relative to intact males, an effect that is reversed by systemic physiological TP. The loss of testosterone causes a decrease in meal number (Petersen 1978). The regulation of feeding behavior via testosterone is thought to be a slower time course than estrogen, given that in mice it takes close to a month for these effects to become apparent (Gentry and Wade 1976).

Exact effect of circulating testosterone via castration on overall food intake is uncertain, with studies reporting a decrease in overall feeding and others reporting a decrease in meal numbers that is compensated for by increased feeding per meal (Petersen 1978;

Nunez 1982).

Rationale

Female mice exhibit less FAA than males, and we sought to investigate the mechanism for this difference. Females show no daily changes in FAA that might correspond to the estrous cycle, suggesting that variation in estrogen levels are not responsible for the difference between males and females. Therefore we hypothesized that testosterone might be responsible for sex differences in FAA. To test this hypothesis, male and female mice were randomly selected for gonadectomy (GDX) and sham procedures and placed on 4-hr daily restricted feeding. We then added groups in which testosterone or estradiol were replaced in GDX mice prior to being placed on 4-hr daily restricted feeding. To further test this hypothesis, in a second cohort of animals we delivered exogenous hormones to GDX males and females to validate the role of gonadal steroids in FAA. Our goal in this study was to investigate the role of circulating hormones in the differential locomotor response to restricted feeding. A recent study suggested that gonadal hormones are involved in sex differences seen in FAA, possibly through the mediation of ghrelin (Li, Wang et al. 2015). It is within the context of these and other finding that this current work will be addressed.

4.2 Materials and Methods

Animals Animals used in this study were age-matched across each experimental group in each study. Adult male and female C57BL/6J (WT) mice, 2-5 months old, were housed on a 12:12 light-dark (LD) cycle prior to the experiments. Animals were individually

74 housed in Plexiglas cages equipped with a running wheel, were housed at a temperature of 20°C, and had access to water ad libitum. Food was also available ad libitum except where indicated. All animal use protocols in this study were approved by the Kent State

Institutional Animal Care and Use Committee and were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the

National Institutes of Health.

Silastic capsules

Silastic capsules were made using Dow Corning silastic tubing (0.062ID,

0.125OD) cut to 20mm sections. Tubes were closed on one end by filling with silicon and allowed to dry for 24 hours. 17β-Estradiol (36 μg/mL) and Testosterone (500 μg/mL) were independently dissolved in sesame oil. 10mm of Estradiol in sesame oil,

Testosterone in sesame oil, or sesame oil was injected into the capsule and the ends were closed with silicon. Capsules were allowed to dry 24 hours before being cut down to

14mm. Capsules were stored in covered weight boats for up to one week before implantation.

Surgical Procedures

Animals were randomly assigned to either GDX, sham, or intact surgical groups

(Figure 23).

Pre-operative procedure

Both male and female mice were bilaterally gonadectomized or underwent identical sham surgeries to remove endogenous gonadal steroids. Mice were weighed and placed into an induction chamber into which an isoflurane/oxygen mixture was routed for

75 initial anesthetization. Once anesthetized, a nose cone with a slow flow of the mixture was placed over the nose and mouth. Hair was shaved and skin disinfected with 70% ethanol followed by chlorhexidine.

Operative Procedure

Males. A small incision was made in the skin of the scrotal region followed by a small incision in the muscle of the abdominal wall. Testes and vas deferens were dissected bilaterally using forceps heated in a bead sterilizer to cauterize the wound.

Muscle wall was sutured and skin was closed using surgical clips.

Females: A small incision was made in the dorsal posterior area directly proximal to the leg followed by a small incision in the abdominal wall. Ovary and fallopian tube were dissected using forceps heated in a bead sterilizer to cauterize the wound. Muscle wall was sutured and skin was closed using surgical clips. Access to second ovary was created with a bilateral incision and dissection.

Silastic capsule implantation

When silastic capsules were used they were implanted immediately following

GDX during the same procedure. Silastic capsules were stored in sterile saline for 24 hours and soaked in sterile alcohol for two hours prior to implantation. Skin was shaved between the shoulder blades and cleaned with 70% Ethanol and Chlorhexidine. A small vertical incision was made in the skin only. Forceps were used to loosen the skin and allow for the implant to rest vertically along the back and not create any uncomfortable turning of the implant. Skin was closed with surgical clips.

Post-operative procedure

Following operation all GDX and sham animals were placed in clean small heated

Plexiglas cages for no less than ½ hour. Animals were then returned to animal facility to heal for at least 7 days. Following 7 days surgical clips that had yet to fall out were removed.

Food Restriction Protocol

Figure 23: Experimental Design. For experiment one, males and females were randomly selected for intact, sham, or GDX groups. These groups were then randomly divided into fast/food restricted (RF) and ad libitum groups (A). For experiment two, males and females were gonadectomized and randomly chosen for control or testosterone/estrogen (respectively) implants. All mice were fasted and food restricted (B).

4.3 Results

Effect of gonadectomy on locomotor response to food challenge in male and female mice Baseline conditions Activity Baseline activity and weight levels were measured two weeks after surgical procedures. For purposes of statistical analysis, baseline RF/ad lib groups were collapsed because at this point they remained identical groups. Representative actograms of male

(Figure 24) and female (Figure 25) experimental groups show baseline, fast, and food restriction activity. Overall, females had greater activity than males across treatments (F

(1, 98) =7.54, p = 0.007; Figure 26). In both male and female, intact and sham mice exhibited similar levels and distribution of activity, while GDX mice had reduced activity

(F (2, 98) = 28.48, p < 0.001; Figure 27a, b). Females had higher activity than male from

ZT 13-22 (F (23, 46) = 5.41, p < 0.001; Fisher’s LSD p < 0.05; Figure 28). There was no interaction between sex and treatment (F (2, 98) = 0.71, p = 0.49), however, we analyzed male and female data separately for illustration purposes. Intact differs from sham from

ZT 15-18 while both intact and sham are higher than GDX from ZT 12-22 (F (46, 2253)

=18.98, p < 0.001; Fisher’s LSD p < 0.05; Figure 29a, b).

Weight

Baseline weights in males are not different between intact, sham, and GDX groups (F (2, 49) = 1.33, p = 0.27). Baseline weight in GDX females was increased compared to intact and sham (F (2, 45) = 13.9, p < 0.001). Males weighed more at baseline than females (Figure 30a, b).

nset nset

lib lib

Castrated

12:12 LD actograms for fast/food restricted and ad and restricted fast/food for 12:12actograms LD

Sham Sham

Representative Representative

Intact

hour fast and the onset of restricted feeding. Bars indicate lights off. off. lights indicate Bars feeding. restricted of onset the and hour fast -

Ad libitum libitum Ad Restricted Fast/Food

Figure 24: Male representative actograms. actograms. representative 24: Male Figure the o indicate Arrows protocol. feeding restricted of the course over animals male GDX and sham, intact, control of 48

lib lib

Ovariectomized

course of restricted feeding protocol. Arrows indicate the onset the onset indicate Arrows protocol. feeding restricted of course

Representative 12:12 LD actograms for fast/food restricted and ad restricted fast/food for 12:12 actograms LD Representative

Sham Sham

Intact

hour fast and the onset of restricted feeding. Bars indicate lights off. off. lights indicate Bars feeding. restricted of onset the and hour fast

Figure 25: Female representative actograms. actograms. representative Female 25: Figure the over animals female GDX and sham, intact, control of 48

Ad libitum libitum Ad Restricted Fast/Food

Baseline

14 * * 12

8 *

4 Average activity Average activity (rev/min)

0 Male Intact Female Male Sham Female Male GDX Female GDX Intact Sham Figure 26: Baseline total activity sex comparison. Comparison of total activity levels between males and females at baseline. Females have increased activity (p <0.05) compared to males regardless of treatment.

A. Baseline: Male 14

4 * Average activity Average activity (rev/min) 2

0 Male Intact Male Sham Male GDX

Baseline: Female B. 14

8 6 * 4

Average activity Average activity (rev/min) 2

0 Female Intact Female Sham Female GDX

Figure 27: Male and female total baseline activity. Overall activity levels of male (A) and female (B) at baseline. Within each sex, GDX animals have decreased locomotor activity compared to intact and sham animals (p < 0.05).

Baseline 40 Male Intact Male Sham Male GDX Female Intact Female Sham Female GDX 35

15 Average Average activity(rev/min)

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

Figure 28: Baseline 24 hour activity profile sex comparison. Overall activity levels of male (A) and female (B) at baseline. Within each sex, GDX animals have decreased locomotor activity compared to intact and sham animals. Bars indicate dark phase. Significance omitted due to figure complexity. See text for details.

A. Baseline: Male 40 Male Intact Male Sham Male GDX

35 # 30 #

(rev/min) 25 # 20

15 * * * 10 * Average activity Average activity * * * 5 * * * * 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11

B. Baseline: Female 40 Female Intact Female Sham Female GDX 35 # # # 30

15 *

10 *

* * * Average activity Average activity (rev/min) 5 * *

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

Figure 29: Baseline 24 hour male and female activity profiles. 24 hour activity profile shows baseline activity in male animals (A) and female (B) animals. Regardless of sex, GDX animals had decreased activity compared to sham and intact from ZT 12-21. Sham differed from intact from ZT 16-18. Bars indicate dark phase. Significance is noted at p < 0.05.

Baseline Weight

30 * 25

Weight(g) 15

0 Male Intact Male Sham Male GDX Female Female Female Intact Sham GDX

Figure 30: Baseline weights in males and females. Overall males have increased weight over females. There is no effect of surgical treatment on weight in males. GDX increased weight in females compared to both sham and intact.

Fasting conditions Activity During fast day one GDX fasted mice have less activity than both intact and sham fasted in both male and female (F (2, 46) = 3.48, p < 0.001). Fasted groups have increased activity compared to ad-lib from ZT 12-16 and ZT 21-22 and this increase is comparable between males and females (F (23, 46) = 2.60, p < 0.001) with no difference in overall activity levels (F (1, 46) = 2.58, p = 0.11; Figure 31a, b). There is no difference between sexes (F (1, 46) = 0.37, p = 0.55; Figure 32). During fast day two GDX fasted mice continue to have decreased overall activity (F (2, 46) = 22.8, p <0.001) compared to sham and intact fasted mice. Fasted intact mice were the only group to show an increase in locomotor activity compared to ad lib (F (1, 46) = 5.95, p = 0.017; Figure 33a, b). Females have increased activity compared to males from ZT 18-20 (F (23, 46) = 1.88, p = 0.007;

Fisher’s LSD p < 0.05) with no overall difference in activity (F (1, 46) = 2.90, p = 0.095;

Figure 34).

Weight Change

There was no difference in weight loss following 48 hour fast between surgical groups (F (2, 46) = 0.69, p = 0.51). There was also no difference in weight loss between treatments in female mice ((F (2, 32) = 0.05, p = 0.95). Fasted groups had a greater weight loss than ad lib groups in both male ((F (1, 46) = 23.51, p < 0.001) and female ((F (1, 34) =

11.71, p < 0.001) with no significant interaction between treatment and condition (Figure

35a, b).

A. 60 Male Fast Day 1 Activity Profile

50 Male Intact Ad Lib Male Intact RF Male Sham Ad Lib Male Sham RF Male Cast Ad Lib Male Cast RF 40

20 Average Activity Average Activity (rev/min) 10

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 Fast 1 ZT

B. 60 Female Fast Day 1 Activity Profile

50 Female Intact Ad lib Female Intact RF Female Sham Ad Lib Female Sham RF Female OVX Ad Lib Female OVX RF 40

20 Average Activity Average Activity (rev/min) 10

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 Fast 1 ZT

Figure 31: Fast day 1 male and female activity profiles. 24 hour activity profiles show activity in male animals (A) and female (B) animals. Bars indicate dark phase. Significance omitted due to figure complexity. See text for details.

Fast Day 1 Activity Profile 40 Male Intact RF Male Sham RF Male Cast RF Female Intact RF 35 Female Sham RF Female OVX RF

15 Average Activity Average Activity (rev/min) 10

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 Fast 1

Figure 32: Fast day 1 male vs female fasted groups 24 hour activity profiles. Fast day 1 24 hour activity profile compares male and female 24 hour activity profiles. Bars indicate dark phase. Significance omitted due to figure complexity. See text for details.

A. Male Fast Day 2 Activity Profile 60 Male Intact Ad Lib Male Intact RF Male Sham Ad Lib Male Sham RF 50 Male Cast Ad Lib Male Cast RF

10 Average Activity Average Activity (rev/min)

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 Fast 2 ZT

Female Fast Day 2 Activity Profile B. 60 Female Intact Ad lib Female Intact RF Female Sham Ad Lib Female Sham RF 50 Female OVX Ad Lib Female OVX RF

10 Average Activity Average Activity (rev/min)

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 Fast 2 ZT

Figure 33: Fast day 2 male and female 24 hour activity profiles. Shown here in

male animals (A) and female (B) animals fasted and ad libitum animals. Bars indicate dark phase. Significance omitted due to figure complexity. See text for details

Fast Day 2 Activity Profile 45 Male Intact RF Male Sham RF Male Cast RF Female Intact RF Female Sham RF Female OVX RF 40

15 Average Activity Average Activity (rev/min)

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 Fast 2

Figure 34: Fast day 2 male vs female fasted groups 24 hour activity profiles. Bars indicate dark phase. Significance omitted due to figure complexity. See text for details.

A. Fast Weight Change 4 2 0 -2 -4 -6 -8 -10 -12 Average WeightAverage Loss,% -14 -16 -18 AL RF AL RF AL RF Intact Sham GDX Male

Fast Weight Change B. 4 2 0 -2 -4 -6 -8 -10

-12 Average WeightAverage Loss,% -14 -16 AL RF AL RF AL RF Intact Sham GDX Female

Figure 35: Fast- induced weight changes in male and female mice. There was no difference between surgical groups in weight loss in males (A) or females (B) and all fasted groups lost weight compared to ad lib controls.

Food Restriction

Activity

During restricted feeding, males have higher overall activity levels than females

(F (1, 49) = 6.81, p = 0.012). In both male and female animals GDX resulted in reduced activity (F (2, 49) = 10.57, p < 0.001) with no effect of sex (F (2, 49) = 0.32, p = 0.73; Figure

36) indicating the effects of GDX did not differ between males and females. Male activity was higher from ZT 3-6 (prior to food presentation) and ZT 13-15 (early active phase) (F (46, 1127) = 6.62, p < 0.001; Fisher’s LSD p < 0.05; Figure 37). There was no interaction between sex and treatment, however, we analyzed male and female data separately for illustration purposes. Intact mice have increased activity compared to sham only at ZT 5, and both sham and intact mice differ from GDX at ZT 12-18 (F (46,

1127) = 6.62, p <0.001; Fisher’s LSD p < 0.05; Figure 38a, b). There were no differences in overall activity levels between ad lib and food restricted groups with the exception of sham groups (Figure 39a, b). Food anticipatory activity was higher in males than females

(F (46, 1127) = 6.62, p < 0.001). There is no effect of sham or GDX on food anticipatory activity in males. Female sham and GDX mice show increased food anticipatory activity compared to intact mice (stats; Figure 40).

Weight

In males, sham animals during restricted feeding had lost more weight than any other group (treatment: (F (1, 37) = 13.72, p < 0.001), interaction: (F (2, 37) = 11.71, p <

0.02). There were no other differences in weight change in male animals. In females,

93 intact animals gained more (F (2, 34) = 4.86, p = 0.01), regardless of RF (F (1, 34) = 0.42, p =

0.52), with no interaction (F (2, 34) = 0.36, p = 0.70; Figure 41a, b).

Restricted Feeding Activity levels

12 *

6 *

4 * Average Activity Average Activity (rev/min)

0 Male Intact RF Male Sham RF Male Cast RF Female Intact Female Sham Female OVX RF RF RF

Figure 36: Total food restriction activity in food restricted males and females. GDX mice had reduced activity compared to both intact and sham regardless of sex. Females had reduced activity levels compared to males. Significance is noted at p < 0.05.

Food Restriction Activity Profile 50 Male Intact RF Male Sham RF 45 Male Cast RF Female Intact RF 40

Average Activity Average Activity (rev/min) 10

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 RF D3-8

Figure 37: Food restriction male vs female food restricted 24 hour activity profiles. Average hourly activity food restriction days 3-8. Bars indicate dark phase. Significance omitted due to figure complexity. See text for details

Male Restricted Feeding Activity Profile A. 50 Male Intact Ad Lib Male Intact RF 45 Male Sham Ad Lib Male Sham RF Male Cast Ad Lib Male Cast RF 40 35 30 25 20 15

10 Average Activity Average Activity (rev/min) 5 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 RF D3-8 ZT

B. Female Restricted Feeding Activity Profile 50 Female Intact Ad lib Female Intact RF 45 Female Sham Ad Lib Female Sham RF Female OVX Ad Lib Female OVX RF 40 35 30 25 20 15 10

5 Average Activity Average Activity (rev/min) 0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 RF D3-8 ZT

Figure 38: Food restriction male and female 24 hour activity profiles. Male (A) and female (B) mice in food restricted and ad lib groups. Significance omitted due to figure complexity. See text for details

A. Food Restriction 18 16 14 12 10

8 * 6 4

2 Average activity Average activity (rev/min) 0 Intact Ad Lib Intact RF Sham Ad Lib Sham RF GDX Ad Lib GDX RF Male

Food Restriction 18 16 14 12 10 * 8 6 4

2 Average activity Average activity (rev/min) 0 Intact Ad lib Intact RF Sham Ad Lib Sham RF GDX Ad Lib GDX RF Female

Figure 39: Food restriction total activity in males and females. Comparable activity levels between ad lib and restrict fed groups during restricted feeding. Male and female sham mice show differences between food restriction and ad lib groups. Significance is noted at p < 0.05.

90 * 80

Average Actiivty Average Actiivty (rev/min) 20 * 10

0 Male Intact Male Sham Male Cast RFFemale Intact Female Female OVX RF RF RF Sham RF RF

Figure 40: Food anticipatory activity sex comparison. In males, there is no difference in FAA between surgical groups. Females show an increase in FAA in sham and GDX groups. Overall male FAA is higher than female. Significance is noted at p < 0.05.

RF Weight Change 8 A. 6

4 * 2 0 -2 -4 -6

-8 Average WeightAverage Loss,% -10 -12 AL RF AL RF AL RF Intact Sham GDX Male

RF Weight Change 5 B. 4 3 2 1 0 -1 -2 -3

-4 Average WeightAverage Loss,% -5 AL RF AL RF AL RF Intact Sham GDX Female

Figure 41: Food restriction-induced weight changes in males and females. Post food restriction weight change. Male sham RF lost weight with no other differences. Female intact mice gained more weight regardless of treatment. Significance is noted at p < 0.05.

100

Gonadal Steroid Replacement

Testosterone Replacement in Males

Testosterone replacement increased baseline nocturnal activity levels from ZT 12-

20 (F (23,368) = 7.91, p < 0.001; Fisher’s LSD p < 0.05; Figure 42a, b) and overall baseline locomotor activity levels (t (16) = 3.85, p = 0.001; Figure 43a). Testosterone treated mice weighed more at baseline (t (18) = 3.01 p= 0.008; Figure 43b). Testosterone treated mice had a slight though not significant increase in activity levels on fast day 1 (t (16) = 2.112 p= 0.051; Figure 44a). The increase in activity during fast day one appears to be largely restricted to nocturnal activity with no significant difference between groups (F (23,368) =

1.9, p = 0.248; Figure 44b). On fast day two testosterone treated mice had more activity than controls (t (16) = -3.25, p = 0.005; Figure 45b). This increase in activity is distributed over both the light and the dark phases with significant increases from ZT 12-19 and ZT

4-6 (F (23,368) = 3.39, p < 0.001; Fisher’s LSD p < 0.05; Figure 45a). There was no difference in weight loss following fast (t (18) = 0.35, p = 0.073; Figure 45c). During food restriction there is no effect of testosterone treatment (t17=0.898, p = 0.38; Figure 46a, b) and no interaction with time, meaning levels of food anticipatory activity are consistent between groups (F (23, 391) = 0.43, p = 0.99) (Figure 46c).

101

Baseline: Male Activity Profile 25 B. * Male Oil Male Testosterone

20 * 15 * * 10 *

Average activity Average activity (rev/min) * * 5 *

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

Figure 42: Testosterone baseline activity profile. Representative actograms of locomotor activity over duration of food restriction protocol in both GDX oil-treated and testosterone treated males (A). 24 hour activity profile (B) shows an increase in baseline nocturnal locomotor activity from ZT 12-20. Bars indicate dark phase. Significance is noted at p < 0.05.

102

Baseline Activity: Male A. 6

Average activty Average activty (rev/min) 1

0 Baseline

Male Oil Male Testosterone

B. Baseline Weight: Male 28.5 28 27.5 27 26.5 26

Weight Weight (g) 25.5 25 24.5 24 23.5 Pre Fast

Male Oil Male Testosterone Figure 43: Testosterone replacement overall baseline and weight. Total baseline locomotor activity is increased in testosterone treated mice compared to control (A). B. Baseline weight is lower in testosterone treated mice (B). Significance is noted at p < 0.05.

103

A. Fast 1 Male Oil Male Testosterone 16

Average Average activity(rev/min) 4

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

B. Fast Day 1 Activity 6

1 Average activty Average activty (rev/min) 0 Fast 1

Male Oil Male Testosterone

Figure 44: Testosterone replacement fast day 1. (A). There is a slight though non- significant increase in nocturnal activity in testosterone treated mice (B). Fast Day 1 overall activity is not significantly different. Bars indicate dark phase. Significance is noted at p < 0.05. .

104

A. Fast 2 40 Male Oil Male Testosterone

Average activity Average activity (rev/min) 10

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

B. Fast Day 2 Activity C. Fast Weight Loss 18 Male Oil Male Testosterone 16 %Change 0 14 -2 12 -4 10 -6 8 -8 6 -10 4

% Weight change Weight % -12 Average activty Average activty (rev/min) 2 -14 0 Fast 2 -16 Male Oil Male Testosterone

Figure 45: Testosterone replacement fast day 2. Testosterone treatment increases both nocturnal and diurnal locomotor activity (A) leading to a greater overall activity (B). There was no difference in weight loss with testosterone treatment (C). Significance is noted at p < 0.05.

105

A. Male Food Restriction Activity Profile 35 RF D3-8 Male Oil RF D3-8 Male Testosterone

10 Average activity Average activity (rev/min) 5

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

B. C. Food Restriction Activity Food Anticipatory Activity 8 3

7 2.5 6 2 5

4 1.5

3 1

2 Average activty Average activty (rev/min) Average activity Average activity (rev/min) 0.5 1

0 0 RF D3-8 Male ORC + Oil Male ORC + T

Figure 46: Testosterone replacement food restriction There is no difference locomotor activity at any point during food restriction (A,B) and no difference in food anticipatory activity (C).

106

Estrogen Replacement in Females

Estrogen treatment in females had no effect on activity at any time (F (23,322) =

38.67, p = 0.11; Figure 47a, b) and baseline activity levels were consistent between treatments (t (14) = 2.31, p = 0.22; Figure 48a). Females treated with estrogen weighed significantly less at baseline (t (17) = 2.70, p = 0.015; Figure 48b). Estrogen did not alter levels at any particular time (F (23,322) = 1.39, p = 0.11; Figure 49a) or have a significant effect on overall activity levels compared to vehicle (t (12) = 1.424 p=0.17; Figure 49b).

On fast day two, there were no differences between estrogen treatment and vehicle across time (F (23,322) = 0.69, p = 0.11; Figure 50a) or in overall activity (t (13) = 0.825, p = 0.424;

Figure 50b). There are no differences in weight change following fasting between estrogen and vehicle (t (17) = 1.46 p = 0.16; Figure 50c). There was no effect of estrogen treatment on overall activity levels during food restriction (t (15) = 1.074, p = 0.299;

Figure 51b). Estrogen treated mice appeared to have decreased nocturnal activity and show no differences in food anticipatory activity levels (F (23, 345) = 1.44 p = 0.088; Figure

51a, c).

107

Baseline: Female Activity Profile B. 40 Female Estrogen Female Oil

Average activity Average activity (rev/min) 10

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

Figure 47: Estrogen replacement baseline activity profile. Representative actograms of locomotor activity over duration of food restriction protocol in both GDX oil-treated and estrogen treated females (A). 24 hour activity profile shows no difference in nocturnal activity. Bars indicate dark phase. Significance is noted at p < 0.05.

108

Baseline Activity A. 12 Female Oil Female Estrogen

Average activty Average activty (rev/min) 2

0 Baseline

Baseline Weight: Female

B. 24.5

23.5

22.5 *

22 Weight Weight (g) 21.5

20.5

20 Pre Fast

Female Oil Female Estrogen

Figure 48: Estrogen replacement overall baseline and weight. Estrogen treatment does not change overall locomotor activity at baseline (A). Baseline weight is lower in estrogen treated mice (B). Significance is noted at p < 0.05.

109

Fast 1 A. Female Estrogen Female Oil 35

10 Average Average activity(rev/min) 5

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

Fast Day 1 Activity 14

Average activty Average activty (rev/min) 2

0 Fast 1

Female Oil Female Estrogen

Figure 49: Estrogen replacement fast day 1. Fast Day 1 24 hour activity profile shows no difference in hourly activity (A). Fast Day 1 overall activity is similar between estrogen treated and control groups (B). Bars indicate dark phase. Significance is noted at p < 0.05.

110

A. Fast 2 Female Estrogen Female Oil 40

10 Average activity Average activity (rev/min) 5

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

B. C. Fast Day 2 Activity Fast Weight Loss 25 %Change 20 0

15 -5 10 -10

% Weight change Weight % Average activty Average activty (rev/min) -15 0 Fast 2

Female Oil Female Estrogen -20 Female Oil Female Estrogen

Figure 50: Estrogen replacement fast day 2. Estrogen treatment does not affect locomotor activity at any point during fast day 2 (A,B). Bars indicate dark phase. Significance is noted at p < 0.05. Fast induced weight loss in control and estrogen treated GDX females are comparable (C).

111

A. Female Food Restriction Acitivity Profile

40 RF D3-8 Female Estrogen RF D3-8 Female Oil

Average activity Average activity (rev/min) 5

0 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 ZT

Female Food Restriction Activity B. C. Food Anticipatory Activity 2.5 12 Female Oil Female Estrogen

10 2

8 1.5

6 1 4

0.5 Average Average activity(rev/min)

Average activty Average activty (rev/min) 2

0 0 RF D3-8 Female: OVX + E Female OVX + Oil

Figure 51: Estrogen replacement food restriction. There is no difference locomotor activity at any point during food restriction (A,B) and no difference in food anticipatory activity (C).

112

4.4 Discussion

Daily temporal food restriction leads to food anticipatory activity, which is lower in females compared to males (Moreland 2010; Li, Wang et al. 2015; Michalik, Steele et al. 2015). Our goal in this investigation was to evaluate the role of circulating gonadal hormones in the locomotor response to both fasting and food restricted conditions by removing them via gonadectomy and then replacing the gonadal hormones with exogenous hormones. Our overall finding is that sex differences in locomotor response to food restriction are present and persist in the absence of circulating gonadal steroids.

This suggests that an activational effect of gonadal hormones is not responsible for sex differences in the response of wheel-running activity to timed restricted feeding.

Baseline activity during ad-libitum feeding

During ad-libitum feeding female mice have a 20-50% higher level of wheel running activity compared to males (Lightfoot 2008), which is comparable to the data presented in this chapter. Onset of activity in females cycles with estrogen levels, with the earliest onset of activity corresponding to the highest estrogen concentrations (Albers

1981; Wollnik and Turek 1988; Kuljis, Loh et al. 2013). We do not see this in our data.

The magnitude of variability in onset of activity is species dependent and mice c57BL/6J mice do not show estrous driven changes in activity onset as compared to hamsters

(Kuljis, Loh et al. 2013). Gonadal hormones play an important role in maintaining normal activity levels. Gonadectomy of both female (Albers 1981; Labyak and Lee 1995;

Ogawa, Chan et al. 2003) and male (Broida and Svare 1983) mice lead to large decreases in activity levels. Our data shows that both male and female gonadectomized mice show

113 reduction in locomotor activity. We see no difference in the effect of gonadectomy on locomotor activity between sexes, suggesting that the sex differences seen are mediated by other mechanisms. Gonadal hormones have organizational effects that create sexual dimorphisms in neuron characteristics and influence the electrical properties of the SCN and other regions (Bailey and Silver 2014). This could potentially modify the central response to food challenges. In addition to organizational effects of gonadal hormones chromosomes themselves have a small but potentially significant role in the sex differences seen in activity duration (Kuljis, Loh et al. 2013).

Additionally, the replacement of 17β-estradiol in females (Rodier 1971) and testosterone proprionate in males (Broida and Svare 1983) rescue intact activity levels.

In our study, testosterone treatment increased baseline activity compared to control castrated mice. We didn’t observe any effect of 17β-estradiol treatment on baseline locomotor activity levels compared to control ovariectomized mice.

Total daily energy intake is higher in males than females when standardized for body size (Woodward and Emery 1989; Whitaker, Totoki et al. 2012) with feeding behavior different across species (Strohmayer and Smith 1987). Food intake is defined by both meal size and duration. Interestingly, females have an earlier onset of feeding, consuming more in the light phase than males and less than males in the dark phase

(Chen, Wang et al. 2015). Gonadal hormones play a significant role in metabolism and feeding behavior. Estrogen is important for energy balance (Wade and Schneider 1992;

Foryst-Ludwig and Kintscher 2010) and regulates feeding behavior in response to energy depletion and repletion (Ibrahim and Briski 2014). Food intake is closely coupled with the estrous cycle, with minimum food intake during proestrus (peak estrogen) in rats and

114 mice (Blaustein and Wade 1976). In females, ovariectomy increases food intake and body weight by increasing meal size (Wade 1975; Blaustein and Wade 1976) and exogenous replacement of 17β-estradiol restores intact feeding patterns and food intake

(Tarttelin and Gorski 1973; Czaja, Butera et al. 1983). Progesterone serves as an agonist to estradiol effects with little known independent effects (Wade 1975; Czaja 1978) .

Testosterone has not been investigated to the extent of estrogen. Orchiectomy decreases food intake and body weight by decreasing meal size (Gentry and Wade 1976; Petersen

1978). Testosterone treatment increases all three factors (Chai, Blaha et al. 1999). In our study, baseline weight was measured two weeks following gonadectomy and shows an increase in weight in females and no difference with castration in males. This was consistent with the literature for females but not for males.

Fast-induced activity

Fasting conditions typically induce a change in the pattern of locomotor activity with nocturnal activity initially dampened followed by a burst of activity during the light phase. In previous studies (see Chapters 2 and 3) this activity is robust and persists for both days of fasting. In this study, only intact male fasted mice showed a significant increase in activity over ad libitum controls. Males and females respond differently to energy repletion following a 24 hour fast, with males losing more weight than females followed by overeating and females eat less (food intake compared to pre-fast values)

(Valle, Catala-Niell et al. 2005). Interestingly, fasted GDX mice showed significantly less activity compared to intact mice yet there was no difference in weight loss following the fast. Testosterone replacement in males increased locomotor activity across the 48

115 hour fast compared to castrated controls with no difference in weight loss between testosterone treated and control groups. In females we saw no effect of 17β-estradiol replacement on locomotor activity or weight loss.

Food Restriction

Food restriction leads to FAA in mice and other rodents (see (Mistlberger 1994) for review). In the present study, during food restriction males have higher total activity than females and higher levels of FAA. This is not consistent with chapter 3 but is consistent with other work from our lab and benefits from a much larger sample size.

This data is also consistent with published reports (Li, Wang et al. 2015; Michalik, Steele et al. 2015). Gonadectomized males and females have reduced overall activity levels during food restriction. However, food restricted GDX animals show no difference in overall activity compared to ad lib GDX suggesting GDX mice do not respond to food restriction differently than intact animals. In males, castration has no effect on levels of

FAA or body weight during food restriction. In females, both ovariectomy and sham groups are increased compared to intact, so we cannot conclude that there is a significant role for estrogen from our data. Ovariectomized mice lost weight compared to intact mice, but there was no difference in restricted fed or control groups suggesting this effect was not based on feeding status. It is unknown what after effects the 48 hour fast has on the metabolic demands during subsequent restricted feeding. In contrast to published reports, we see no effect of testosterone proprionate in males or 17β-estradiol in females on FAA levels. Li, et al (2015) suggests that androgens increase and estrogens decrease levels of FAA. Our data do not support that claim. Testosterone treatment increased

116 overall activity levels but had no effect on FAA. Since administration of 17β-estradiol in females had no effect, it is possible that the delivery of hormone was insufficient. This could be lack of physiologically relevant concentrations and/or the time of release was less than was expected. A dose response study might reveal more about potential roles for gonadal steroids in FAA. The data presented herein do not provide evidence in support of gonadal hormone mediation of sex differences in food anticipatory activity.

Conclusions This study provides evidence that the food anticipatory activity is present in both male and female mice with a significant reduction in females and that the sex differences in locomotor activity, regardless of condition (ad-libitum feeding/fast/food restriction), are not dependent upon gonadal hormones. Food restriction initiates a complex response that involves changes in locomotor activity, peripheral circadian gene expression, motivation and reward circuitry, activity distribution, metabolic changes, and is also a major environmental stressor. Any of these factors, in combination with organizational effects or central activational effects could be responsible for mediating this difference in response to food restriction. There is not much published on FAA in females, but a new report suggests that motivational differences may attenuate the levels of FAA seen in females compared to males (Michalik, Steele et al. 2015).

117

Chapter V

Global Discussion

The studies conducted and described in this dissertation focus on behavioral output during fasting and food restriction. We utilized a tissue-plasminogen knock-out mouse to investigate the role of the SCN in modulating the effects of light on locomotor activity. During these investigations, we noted a sex difference in the response to food restriction. Female mice showed reduced food anticipatory activity (FAA) compared to males. We sought to investigate the reason for this difference.

Behavioral modification during food restriction includes FAA and truncated late active phase activity. In our studies, we utilized wheel running as a measure of FAA.

The physiological impacts of timed feeding are varied and include changes to metabolism and increased stress. In rodents, the SCN and peripheral tissues become uncoupled when food is restricted to a specific time outside the active phase, causing an advance of peripheral rhythms (including stomach, liver, pancreas, intestines, etc) toward food presentation. Based on our data, there is no difference in the advance of Per1 or Per2 in

118 the liver suggesting that differences in activity are not based on changes in gene expression in the liver.

The patterns and amplitude of locomotor activity in both intact males and females is dependent upon circulating hormones. At baseline, females have higher locomotor activity than males. The exact reason for this difference is unknown. Ovariectomy leads to a dramatic decrease in wheel running activity in female mice and orchiectomy also leads to a dramatic decrease in locomotor activity in males. This appears to be the main effect of gonadectomy and exogenous hormone replacement during fasting and food restriction as pertains to locomotor activity. In fasting conditions there is no difference between male and female activity. During food restriction males have greater overall activity and greater levels of FAA than females. Differences between male and female activity levels persist in GDX groups, which suggests that the sex difference in locomotor activity levels is not driven solely by activational effects of gonadal hormones.

Future Directions

Activational effects of peripheral gonadal hormones are not sufficient to explain the sex differences in FAA. It is likely that this difference is rooted in the brain. A first step would be to identify regions of the brain that are involved in or respond to food restriction and determine what regions respond differently in females. There are several hypothalamic nuclei that would be excellent locations to begin including the dorsomedial hypothalamus, arcuate nucleus, and the paraventricular nucleus. Identifying brain regions that are differently affected by food restriction would allow for the isolation of potential

119 mechanisms for this difference. One consideration that has not been taken into account is the relationship between stress and food restriction as it pertains to the differences seen in males and females. Female rats show higher plasma corticosterone concentrations following food restriction (Lenglos, Mitra et al. 2013). It is possible that this difference in stress response is related to the locomotor activity seen in response to restricted feeding.

Concluding Statement

The work in this dissertation focused on understanding the physiological factors that differentially induce locomotor activity. Food restriction provides an excellent model through which we can perturb normal functioning to shed light on internal variation in physiology. This work demonstrated a potential role of tPA in the levels of locomotor output as well as providing evidence for the low levels of food anticipatory activity in females. Further, it suggests that these differences are not based on activational effects of gonadal hormones. Taken together these data demonstrate the complexity inherent the behavioral responses seen in restricted feeding.

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