The Importance of Diurnal Corticosterone Rhythms in Regulating

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THE IMPORTANCE OF DIURNAL CORTICOSTERONE RHYTHMS IN REGULATING

MOOD

A thesis submitted

To Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Arts

DEVANSHI MITESH MEHTA

August 2019

Except for previously published materials

Thesis written by

Devanshi Mitesh Mehta

B.S., Kent State University, 2017

M.S., Kent State University, 2019

Approved by

John D. Johnson______, Advisor

Ernest J. Freeman______, Chair, Department of Biological Sciences

James L. Blank______, Dean, College of Arts and Sciences TABLE OF CONTENTS

TABLE OF CONTENTS ...... iii

LIST OF FIGURES ...... v

ACKNOWLEDGEMENTS ...... vi

CHAPTERS

I. INTRODUCTION...... 1

Depression...... 1

Stress……...... 2

HPA axis...... 3

Glucocorticoids...... 4

Diurnal Rhythms of Corticosterone...... 5

Circadian rhythms and Circadian genes...... 7

Corticosterone and Period genes...... 9

Hypothesis...... 10

II. METHODS AND MATERIALS...... 12

Study design...... 12

Animals……...... 14

Adrenalectomy...... 15

Corticosterone Replacement...... 16

Blood and Tissue collection...... 16

Quantitative PCR...... 18

Forced Swim test...... 18

Open Field test...... 19

iii Saccharin Preference test...... 19

Statistics…...... 20

III. RESULTS…...... 22

IV. DISCUSSION…...... 41

V. REFERENCES ……………………...... 51

iv LIST OF FIGURES

Figure 1. The hypothalamus-pituitary-adrenal axis...... 4

Figure 2. Diurnal levels of Corticosterone...... 7

Figure 3. The hypothesis model……………..………………………...... 10

Figure 4. Circadian Rhythm across 24 hours in nocturnal animals...... 11

Figure 5. Timeline for study 1...... 13

Figure 6. Timeline for study 2...... 14

Figure 7. The rhythm of CORT after bilateral adrenalectomy (Study 1) ...... 25

Figure 8. Sucrose-preference test...... 26

Figure 9. Total immobility time in 5-min forced swim test...... 27

Figure 10. The rhythm of CORT after bilateral adrenalectomy (Study 2) ...... 32

Figure 11. Blood glucose test...... 33

Figure 12. PER2 mRNA expression in the PVN...... 34

Figure 13. PER2 mRNA expression in the BNST...... 35

Figure 14. PER2 mRNA expression in the cAMY...... 36

Figure 15. Saccharin-preference test...... 37

Figure 16. Total immobility time in 5-min forced swim test...... 38

Figure 17. Total distance moved in 10 mins open field test...... 39

Figure 18. Total duration in center zone in 10 mins open field test...... 40

v ACKNOWLEDGEMENTS

I would like to thank my parents, Mitesh V Mehta and Parul Mehta, for their constant support and encouragement. I would like to thank my brother, Parth Mehta, for giving me strength and stability.

I would like to thank Dr. John D Johnson for his continuous guidance throughout my academic career at Kent State University. Lastly, I would like to extend my gratitude to my senior lab members Adam Kulp, David Barnard and Amy Ionadi for contributing their time and efforts in my project.

CHAPTER 1

INTRODUCTION

DEPRESSION

Major depressive disorder (MDD) is one of the most common disabling mental health disorders

(1, 2), affecting up to 20% of the world population (3). Despite its widespread effect, it currently has limited therapeutic treatments. The core symptoms of depression include anhedonia (i.e. loss of interest in pleasurable activities), low mood, weight changes, sleep disturbances, retardation, loss of energy and concentration, and/or suicidal thoughts (4-6). This neuropsychiatric disorder can result in major alterations in emotional, motivational, neurovegetative and cognitive processes (7). MDD also shows strong associations with high mortality rates and other medical conditions such as heart diseases, diabetes, and stroke (4, 8). MDD often co-occurs with other psychiatric disorders, most common being anxiety, where 51% of those who have depression also suffer from anxiety disorders (9). Dysregulation of the hypothalamus-pituitary-axis (HPA) axis, which regulates cortisol levels, is often associated with MDD (10-14). Severely depressed patients that have high concentrations of cortisol (12) or are treated with synthetic glucocorticoid have greater risk of suffering from depressive episodes (15). Supporting this view, in depressed

1 patients, MDD is strongly correlated to elevated circulating cortisol levels (12), circadian dysregulation of cortisol (16), and/or impaired glucocorticoid receptor negative feedback of the

HPA axis (17). Repeated stress exposure can result in hyperactivity and dysregulation of the

HPA axis, which may be why stress is one of the major predisposing factors in the development of major depression.

STRESS

Stress can be defined as the behavioral and physiological response to a stimulus, crucial in adaptation to external demands and survival (18). This response requires proper engagement of the central and peripheral system, to produce an adequate response to the threat and then enable the body to return to biological equilibrium once the stressor is terminated (19). This response is primarily mediated by the HPA axis. The association between stress and depression was initially made from clinical observations of abnormalities of stress reactivity in depressed patients (10).

These clinical cases primarily included dysfunction of the HPA axis caused by chronic or acute stress. Repeated activation of the HPA axis increases allostatic load on the body due to consequent activation of the different systems related to fight-flight responses (20, 21). As the

HPA axis plays a major role in short-term and long-term effects of the stress on the body, it is expected that repeated activation of this system affects the neural adaptation of the stress-related circuits, which could then increase the risk of developing these mental health disorders (22, 23).

Under a stressful situation, activation of brainstem catecholaminergic neurons and neurons in the hippocampus and amygdala contribute to the activation of the HPA axis.

2 HPA AXIS

The hypothalamic-pituitary-adrenal axis is a biological system that serves as the major neuroendocrine mediator of stress responses (20). As shown in Figure 1, the activation of this system starts with the neurons in the paraventricular nucleus (PVN) of the hypothalamus. These neurons release corticotropin releasing hormone (CRH), which in turn act on the anterior pituitary to release adrenocorticotropic hormone (ACTH). ACTH is secreted into the circulation where it activates the production and release of glucocorticoids (GCs) by the adrenal gland (24).

To reinstate homeostasis, negative feedback mechanisms are initiated; the GCs act on the hippocampus, PVN and the anterior pituitary gland to inhibit further release of CRH and ACTH

(25). Glucocorticoids, specifically, cortisol in humans or corticosterone (CORT) in rodents, is the end product of the HPA axis (26) along with being the principal biomarker for HPA axis activity

(20).

Figure 1: The hypothalamus-pituitary-adrenal axis. This figure shows the hypothalamus-pituitary-adrenal axis and corticosterone being the end product of this axis. CRH=corticotropin releasing hormone; ACTH=adrenocorticotropic hormone; CORT=corticosterone.

GLUCOCORTICOIDS

Corticosterone mediates the body’s stress response and prepares the body for fleeing or fighting by mobilizing energy sources (i.e. glucose) and modulating the immune system (27). This steroid hormone released by the adrenal gland, specifically the adrenal cortex, functions in majority of the tissues by affecting the physiological homeostasis in a highly gene-specific manner (8). It

4 also affects several outputs of the brain either through rapid activation of the cellular systems or by controlling the expression of various genes through slower acting effects of cellular adaptations (28, 29). Glucocorticoids, specifically corticosterone, exerts these effects via two major receptors, the low-affinity glucocorticoid receptors (GR) and the high-affinity mineralocorticoid receptors (MR) (30). The observed metabolic changes observed during a stressful situation is majorly mediated by the GRs due to the presence of high CORT. In case of repeated and/or constant activation of this system, as seen under chronic stress conditions, we observe elevated levels of CRH (31), ACTH and CORT (32), reduced glucocorticoid sensitivity

(33), and delayed return to basal CORT levels compared to control animals (34). Chronic exposure to GCs leads to structural and functional changes in the brain regions such as decreases in neurogenesis in the hippocampus, decreases in synaptogenesis in prefrontal cortex and/ or increases in synaptogenesis in the amygdala (35, 36). These changes can further be responsible for modulating the stress response, contributing to the pathophysiology of various mood disorders (36). Many propose that the structural changes characterized by this reduced glucocorticoid receptor sensitivity, plays a causal role in the onset of depressive-like behaviors.

At the very least, it can be hypothesized that glucocorticoids represent the common link between dysregulation of the HPA axis caused by stress and its effects observed on mood and behavior.

However, more recent literature points to the significance of the intrinsic diurnal rhythm of

CORT in addition to its mere presence, for optimal functioning of the body processes (37).

DIURNAL RHYTHMS OF CORTICOSTERONE

The master pacemaker, suprachiasmatic nucleus (SCN), located in the hypothalamus, is responsible for regulating the daily rhythms in behavior and physiology including the circadian

5 variation in plasma ACTH and CORT levels (38-40). Signals from the SCN help in generating an intrinsic circadian rhythm of CORT, which is established due to the body’s increased need for glucose during an animal’s active phase than its rest phase. This results in higher levels of CORT during an organism’s active phase and lower levels during its resting phase (41). Thus, in nocturnal animals, like rats used in the research presented here, glucocorticoid release is normally high during nighttime and low during the daytime. During the time of stress, activation of the HPA axis results in transient rise in CORT to meet the metabolic demands during the time of stress but returns to its diurnal rhythm thereafter. The reliable diurnal rhythm of CORT allows it to function as a circadian signal (or zeitgeber) for peripheral tissues so that the metabolic response of cells is in rhythm to the time of the day the animal is active. It does this by entraining the rhythms of various circadian clock genes, which are present in the subordinate clocks downstream of the SCN (42, 43). Interestingly, this is important as CORT rhythms are shown to affect the expression of several circadian genes in many cells throughout the body including in limbic brain regions, and changes in these regions can affect the mood and behavior of the animal.

Figure 2: Diurnal levels of corticosterone. Neurons in the SCN mediate the diurnal rhythm of glucocorticoids via regulating the activation of PVN neurons. Neurons in the PVN release CRH into the portal blood that acts on the pituitary to release ACTH. ACTH acts on the adrenal cortex to release glucocorticoids. Blood collected from the tail vein of male Fischer rats at various time points throughout a 24h period demonstrate the diurnal rhythm of CORT with low CORT levels during the “light” (inactive) phase 0-12h and elevated CORT levels during the “dark” (active) phase 12-24h (data collected by Amy Ionadi, unpublished).

CIRCADIAN RHYTHMS AND CIRCADIAN GENES

In mammals, circadian rhythms are regulated both globally by the master clock in the SCN, and locally via the core circadian genes in brain and periphery that control tissue-specific rhythmic outputs (44). The SCN, which receives input from the retina and allows light to serve as a zeitgeber, keeps the cycle set to 24h. However, since the SCN cannot have direct connections to every cell in the body, the circadian rhythm of a large number of cells throughout the body are entrained by the HPA axis and the rhythm of CORT (45). This entrainment of the molecular

7 oscillators allows the circadian clock to optimally drive daily cyclic rhythms in behavior and physiology, essential for adaptation to our cyclic environment (46). Disruption in the normal cellular expression of clock genes results in numerous health impairments including insulin resistance, obesity, type 2 diabetes, and cardiovascular disease (47). Psychiatric diseases like seasonal affective disorder, bipolar disorder, anxiety, and depression as well as some neurodegenerative disorders such as Parkinson’s disease are often associated with a host of deficiencies that can possibly be attributed to circadian dysregulation and abnormal rhythms (48-

51). Spencer et al. (2013) demonstrated that knockdown of a particular circadian gene called,

PERIOD1/PERIOD2, in the nucleus accumbens results in increased anxiety-like behavior in a dark/light box and on an elevated plus maze (52). This supports the premise that disruption of circadian clock genes, particularly PERIOD genes, can lead to impaired emotional behavioral responses.

Circadian genes are widely expressed in the brain and other peripheral tissues (53). There are several circadian genes that interact in negative/positive feedback loops to create synchronous and self-sustaining rhythms over a period of approximately 24 hours (38, 53-55). The circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like protein 1 (BMAL1) are two central components of this system. These two genes encode helix-loop-helix PAS transcription factor and induces the expression of the Period (PER1, PER2, and PER3) and

Cryptochrome (CRY1 and CRY2) genes (56-58). The CRY and PER, in turn, inhibit the activity of CLOCK and BMAL1, thus creating a negative feedback loop that has a 24h cycle (52, 54).

Previous work has shown that daily rhythms of PER2 are uniquely dependent on the diurnal rhythm of CORT released by the adrenal glands (46, 54).

CORTICOSTERONE AND PERIOD GENES

Glucocorticoids are strong zeitgebers for many cells outside the SCN. Glucocorticoids bind to nuclear hormone receptors and interact with glucocorticoid response elements (GRE) in the genome to regulate target gene expression (59). So et al. (2009) demonstrates the influence glucocorticoids have on circadian clock components in both mouse and human mesenchymal stem cells. They identified ten clock components that show shifts in transcriptional oscillations following glucocorticoid receptor activation; three of these (PER1, PER2, and E4bp4) had highly conserved glucocorticoid receptor binding sequences located near their transcription start site and demonstrate rapid changes in transcript levels within 4h of glucocorticoid receptor stimulation (8). Of these, PER2 is particularly interesting because in nocturnal rodents it is rhythmically expressed in multiple limbic areas, including the central nucleus of the amygdala

(cAMY) and bed nucleus of the stria terminalis (BNST) (44, 60-62).

BNST and cAMY are commonly referred to as the central extended amygdala and both these nuclei are important for the output of negative emotional responses and stress-related behaviors

(63, 64). Dr. Shimon Amir’s laboratory demonstrated that peak PER2 expression in these brain areas is approximately 1h after lights-off and the nadir is 1h after lights-on in rats (62), which corresponds to the diurnal rhythm of CORT. Bilateral adrenalectomy abolishes the PER2 rhythms in the BNST and cAMY but had no effect in the SCN or in other limbic brain regions such as basolateral amygdala or hippocampus (65). Providing animals with CORT in drinking water, at a dose that results in normal diurnal levels of circulating CORT, rescues the PER2 rhythm in the BNST and cAMY (66). This suggests that CORT plays a selective role in the

9 regulation of the PER2 rhythms in BNST and cAMY. Thus, these brain regions became of interest as their optimum functioning may be dependent on the diurnal rhythm of CORT, and they have important roles in the control of emotional and motivational states.

HYPOTHESIS

Our hypothesis states that removal of the circadian rhythm of CORT will flatten the circadian gene expression of PER2 within the CEA and BNST, thereby resulting in disruption of emotional behaviors.

Figure 3: The hypothesis model. The hypothesis model for this study states that continuous disruption of normal diurnal rhythm of CORT (caused by constant basal levels of CORT supplied by the osmotic minipump) leads to a disruption in the normal rhythm of circadian genes (PER2). This would affect the normal functions of the limbic area, resulting in depression.

Figure 4: Circadian rhythm across 24 hours in nocturnal animals. We hypothesized that replacement of diurnal rhythm of CORT with constant basal levels of CORT will lead to flattening of PER2 rhythms (yellow line) in cAMY and BNST. The blue line represents the normal rhythm of PER2 gene observed in limbic brain regions like BNST and cAMY.

11 CHAPTER 2

METHODS AND MATERIALS

STUDY DESIGN

STUDY 1: The purpose of Study 1 was to determine if implantation of an osmotic minipump that delivered a constant dose of CORT at 20 ug/hour would be sufficient to eliminate the diurnal rhythm of CORT and maintain basal physiological levels of CORT in adrenalectomized rats.

Behavioral testing was also done to check for any depressive-like behaviors. All animals were given a baseline assessment of their sucrose preference prior to either bilateral adrenalectomy or sham surgery. To eliminate the rhythm of CORT, adrenalectomized rats were implanted with an osmotic minipump subcutaneously that provided a constant dose of CORT (20 ug/hour). After 4 days recovery period, sucrose-preference test was performed on Day 5 post-surgery and another one was performed on Day 12 post-surgery. Animals were exposed to forced-swim pre-exposure

1h after lights-off on Day 11 and a 5-min forced-swim test was conducted on Day 12.

Additionally, blood samples were collected to measure the amount of corticosterone in the blood.

On Day 8 post-surgery, blood samples were collected at 08:00hrs and 20:00hrs. On Day 13 post- surgery, blood samples were again collected at 08:00hrs and 20:00hrs and the animals were then euthanized.

Figure 5: Timeline for Study 1. A flowchart depicting the timeline of Study 1.

STUDY 2: The purpose of study 2 was to test if elimination of the diurnal rhythm of CORT was sufficient to reduce peak PER2 levels in the cAMY and BNST, and whether this was associated with the onset of depressive-like and anxiety-like behaviors. The design was similar to Study 1, except a sham group and two groups of adrenalectomized animals were included: one implanted with an osmotic minipump that delivered 20 ug/h CORT and a second group implanted with an osmotic minipump that delivered 2 ug/h CORT. On Day 4 post-surgery, blood samples were collected at 08:00hrs and 20:00hrs for measurement of plasma CORT and glucose. Saccharin- preference tests were performed from Day 6 to Day 11 post-surgery. All animals were exposed to a forced-swim tests on Day 9 one hour after lights off and a 5 mins forced-swim test was

13 conducted on Day 10 post-surgery at 20:00hrs. Additionally, on Day 9 post-surgery, all animals were exposed to a 10 mins open field test one hour after lights off. On Day 11 post-surgery, blood samples were again collected at 08:00hrs and 20:00hrs and the animals were then euthanized, and brains collected to measure the PER2 mRNA levels in the selected brain regions.

Figure 6: Timeline for Study 2. A flowchart depicting the timeline of Study 2.

ANIMALS

Care and use of the animals were in accordance with protocols approved by the Kent State

University Institutional Care and Use Committee. Adult male Fischer rats (250-300g) were used for all studies in this investigation. Animals were single-housed in Plexiglas cages and provided environmental enriched (e.g. plastic whiffle balls, PVC tubes). Food and water were available

14 ad libitum. Adrenalectomized rats were given saline solution (0.9% NaCl) to drink post-surgery due to the loss in aldosterone. Animals were maintained on a 12 h light-dark cycle beginning at

07:00 h at constant temperature (22 0C). Animals were given two weeks to adjust to being single- housed, which was done to be consistent with past studies from our laboratory. Animals were handled daily to habituate animals to manipulations.

ADRENALECTOMY

The entire bilateral adrenalectomized (ADX) or sham-operated surgery was performed under isoflurane and this was done using a dorsal approach. After the dorsal and lateral sides of the animal was shaved, the area was cleaned with a betadine swab sticks and ethanol three times. All the instruments were cleaned with ethanol and sterilized using a bead-sterilizer between each surgery. Bilateral horizontal incisions were made at the bottom of the rib cage using a #10 scalpel blade. The connective tissue was then broken using a pair of scissors. After identifying the adrenals in the abdominal cavity, adrenal glands were removed using forceps and surgical scissors. Next, the abdominal musculature was sutured using an absorbable plain gut suture. The skin wound was then closed with stainless steel wound clips. This procedure was performed on both sides. Adrenalectomized animals were then implanted with a pump by making another incision on the dorsal side of the animal and between the shoulder blades. An osmotic minipump, with the appropriate dose of CORT, was then implanted subcutaneously between the muscle and skin. The incision was closed with stainless steel wound clips. An intraperitoneal (I.P.) injection,

Ketoprofen (2mg/kg), was given to the rats immediately after the surgery and again 24h later to reduce the pain. The completeness of adrenalectomy was verified by visual inspection during surgery and later by the assessment of the plasma CORT after the animals recovered from

15 surgery. Adrenal-intact controls underwent sham surgery. Sham surgeries were identical to adrenalectomy surgeries except adrenals were touched by the surgical instruments but not removed. Sham animals were maintained on regular tap water.

CORTICOSTERONE REPLACEMENT

Adrenalectomized animals were implanted with Alzet 2ML2 osmotic minipumps to provide constant exogenous corticosterone post-adrenalectomy. Pumps were loaded with CORT to provide 20 ug/hour or 2 ug/hour of CORT. CORT was purchased from Sigma-Aldrich and mixed with poly ethylene glycol (PEG). This mixture was heated until the CORT was dissolved. PEG was diluted in saline in the ratio of 1:4. This solution was then injected in the pumps using 1.4” sterile needle connected to a 5cc syringe. The pumps were then immersed in a beaker containing

0.9% saline solution at least 4 hours before its implantation. Saline was also provided to this group in their drinking water to compensate for the loss of regulation of salts that is normally maintained by the adrenal glands.

BLOOD AND TISSUE COLLECTION

Two blood samples were collected in each study following adrenalectomy to verify loss of the diurnal rhythm. The blood was collected from the tail vein one hour after light on (08:00hrs) and one hour after lights off (20:00hrs) on two separate days. For this, rats were placed in towels to restrain them and then their tail vein was nicked with a #15 scalpel blade for blood collection

(20-80 ul). The samples were then centrifuged at 4 0C and 13,000 r.p.m. for 10 mins. Plasma was extracted and stored at -20 0C. Plasma corticosterone levels were then assessed using enzyme- linked immunosorbent assay (EIA) by ENZO Life Sciences. Before starting the EIA, the plasma

16 was diluted 1:50 in water and heated at 70 °C in a water bath for 1 hour. The manufacturer’s protocol was followed thereafter except the dissociation buffer was not used as the CORT was already dissociated from the corticosterone binding globulin due to heating. For each animal, average CORT values at 08:00h and 20:00h were calculated by averaging the two plasma CORT values taken at the designated time on two separate days.

For blood glucose measurements, OneTouch UltraMini glucometer was used. We used the collected blood samples from tail nicks, to measure the blood glucose levels, however, this was done only at 20:00h on both days. Blood glucose levels were not measured during the day time

(08:00h). The manufacturer's protocol was followed to perform a control routine on the meter.

To measure the blood glucose levels, a new strip was inserted in the meter for every blood sample. A drop of blood was placed on the strip and readings shown on the meter were recorded.

For brain tissue collection, all animals were euthanized by rapid decapitation one hour after lights off, which corresponds to peak PER2 expression. Brains were dissected and stored at -80

0C until tissue was processed. Three brain regions, the paraventricular nucleus (PVN), central amygdala (cAMY), and bed nucleus of stria terminalis (BNST), were extracted by brain punches.

A cryostat machine was used to slice the brain until the desired brain region was observed. Then a 14-gauge punch tool was used to punch out the brain region from both sides. The BNST was punched approximately at Bregma -0.72 mm; Interaural 8.28 mm coordinates. The PVN was punched approximately at Bregma -1.32 mm; Interaural 7.68 mm coordinates. The cAMY was punched approximately at Bregma -1.56 mm; Interaural 7.44 mm coordinates. These punched brain areas were then stored in 2.0 ml graduated tubes until processed for qPCR.

17 QUANTITATIVE PCR

RNA isolation and quantitative real-time PCR (RT PCR) was used to process these brain tissues.

Tubes were spun briefly to pellet the brain tissues at the bottom of the tubes for better sonication.

Dissected brain tissues were sonicated for 10 seconds in 90 ul of extraction buffer from Qiagen

RNAeasy Mini kit. All samples were kept on ice. The lysate was transferred to new purification columns and 250 ul of conditioning buffer was added. Next, the manufacturer's protocol was followed, and total RNA was extracted by using the Qiagen RNAeasy Mini Kit, which included a DNAse treatment. Quality and concentration of RNA was measured using a Nanodrop.

Approximately 12.3 ng/ul of RNA for BNST, 9.8 ng/ul of RNA for PVN and 11.2 ng/ul of RNA for cAMY was reverse transcribed using the High-Capacity cDNA Reverse Transcription kit

(Thermofischer). Using polymerase chain reaction (PCR), the target cDNA was amplified. All qPCR assays were carried out in triplicate using the Brilliant III Ultra-fast qPCR Master Mix and

TaqMan Gene Expression Assays probes (m1 Per2 gene; ID: Rn01427704). GAPDH, a housekeeping gene (g1 Gapdh FAM; ID: Rn01775763), was used as a reference to normalize the cDNA quantities and the 2-DDct method was used to analyze relative gene expression.

FORCED SWIM TEST

The forced-swim test is used to measure depressive-like behavior in the rat. For the forced swim test (FST), rats were introduced into a plastic cylinder (40 cm deep, 20 cm in diameter) filled with water at 23-25 0C up to a height of 25 cm from the base and forced to swim for 10 mins in the pre-exposure session. Twenty-four hour after the first exposure, animals were reintroduced into the same cylinder, and then their 5-min swim session was recorded on a video recorder.

After each swim session, the rat was removed from the cylinder, dried with towels, and returned

18 to its home cage. Water in the cylinder was renewed between subjects and the cylinder was cleaned with 70% EtOH solution. The videos were recorded by a video recorder that was placed above the tube. EthoVision XT software was used to measure total time spent immobile for each animal. An increase in time spent immobile is considered a sign of behavioral despair and a symptom of depression. All testing was done 1-2h after lights off and under red light conditions

(approximately 4 lux).

OPEN FIELD TEST

The open field test is used for measuring anxiety-like and exploratory behavior. The open field apparatus used was 61cm x 61cm x 43cm and made of transparent plexiglas. Each trial was 10 mins and a video recorder were placed above the maze. EthoVision XT software was used to divide the floor into 4 x 4 squares (16 squares), and center of the open field was defined as the middle 2 x 2 square (4 squares). The maze was in a test room, which was lit by a red lamp for background lighting. To maintain sterility, 70% ethyl alcohol was used on the maze between each rat. After the test was conducted, EthoVision XT software was used to analyze and track the total distance travelled by the rat and the amount of time spent by the animal in the center zone.

Animals with increased anxiety spend less time in the center zone. All testing was done 1-2h after lights off under red light conditions.

SUCROSE/SACCHARIN PREFERENCE TEST

Preference for either sucrose or saccharin (compared to water) is often used to measure anhedonia in rats. A switch from sucrose to saccharin was made after Study 1. This was done as sucrose is known to have an additional caloric value to it. Thus, it is possible that animals

19 consume more sucrose to maintain their body weights, and this is concerning as it can increase the sucrose-preference of the animals, resulting in a false indication of absence of anhedonia. As saccharin contains only a sweet value to it and no such caloric value, saccharin was used in

Study 2 for proper results. For sucrose/saccharin preference test, rats were habituated to 0.1% saccharin or 1% sucrose solution for 4 days by exposing two identical bottles either filled with saccharin solution or water. Intake on the last two days was used to calculate baseline levels of preference. After surgery, the rats were allowed to recover for several days prior to sucrose/saccharin preference testing. For adrenalectomized animals, sucrose/saccharin was delivered in saline water with CORT. Sucrose/saccharin preference was defined as the ratio of the volume of sucrose/saccharin vs total volume of total liquid intake (sucrose/saccharin plus water consumed) * 100.

STATISTICS

To compare the amount of CORT present in the blood at 08:00hrs and at 20:00hrs between the sham animals and animals with pumps, a repeated measures ANOVA was used. Further post-hoc analysis was done when significant interactions between time and group were observed. For sucrose- or saccharin-preference, repeated measures ANOVA was used and post-hoc analyses was performed if any significant differences were observed. To compare the immobility time in the forced-swim test, behavioral in the open field, and glucose levels between groups, an independent samples t-test was performed in Study 1 and a one-way ANOVA was performed in

Study 2. A one-way ANOVA was also used to compare the fold changes in PER2 mRNA levels between control rats and animals that received a constant dose of CORT via osmotic minipumps.

Post-hoc analyses, LSD and Tukey HSD, were used to look for any significant difference

20 between the groups when a significant effect was observed in the one-way ANOVA. Statistical significance threshold was set at 0.05 for all the analyses and interactions were followed by post- hoc analysis.

21 CHAPTER 3

RESULTS

Study 1: For this pilot experiment, we used 7 male rats to determine if the 20 ug/hour dose of

CORT in the osmotic mini-pump would provide constant baseline levels of CORT. These rats were divided randomly into two groups, sham (n=3) and pump (n=4). The sham animals underwent sham surgery whereas animals with pump underwent bilateral adrenalectomy. The pump group received exogenous CORT via an osmotic mini pump with a dose of 20 ug/hour of

CORT. To check if the adrenalectomies were successful and to examine CORT levels in these animals, blood samples were taken twice via tail vein nicks (Day 8 and Day 13) both 1 hour after lights off and lights on. Two behavior testing, sucrose-preference and forced swim tests, were done on the animals to check for any depressive-like behavior in the rats after the elimination of diurnal rhythm of CORT.

The effects of adrenalectomy and replacement with an osmotic pump on the rhythm of corticosterone.

Sham animals showed a normal rhythm of CORT, i.e. lower during the morning (1.3 ug/dl) and higher during the night (37.3 ug/dl). Whereas the adrenalectomized animals with CORT replacement in osmotic minipumps had constant circulating CORT levels throughout the day of

21.7 ug/dl at 08:00hrs and a mean of 21.6 ug/dl at 20:00hrs (Figure 7). A repeated measures

ANOVA revealed a significant interaction between time and group [F (1,5) = 8.074; p = 0.036].

22 In sham controls, a repeated measure ANOVA revealed CORT levels at 20:00hrs were significantly higher than that present at 08:00hrs [F (1,2) = 28.923); p=0.033], confirming the normal diurnal rhythm of CORT in these animals. Whereas in adrenalectomized animals with a pump, the levels of CORT present at 08:00hrs and at 20:00hrs were not significantly different from each other [F (1,3) = 0.00; p=0.991]. This demonstrates that we successfully eliminated the rhythm of CORT in the “pump” group and the osmotic mini pump successfully delivered a constant dose of CORT to these animals that was within the natural diurnal rhythm of CORT.

However, since CORT levels were higher than nadir levels in sham animals, we elected to add a second, lower (2ug/hr) dose of CORT in Study 2.

The effects of adrenalectomy and CORT replacements with an osmotic pump on emotional behaviors of the animal.

To check for any depressive-like behaviors after the elimination of the rhythm of corticosterone, we conducted a sucrose preference test (1% sucrose solution) on Days 5 and 10 and a 5-mins forced swim test on Day12 post-surgery, where the total immobility time was measured. A baseline percent of sucrose preference was the average sucrose preference taken over two days before surgery, and as expected, baseline sucrose preference was not significantly different

(Figure 8). Two sucrose-preference tests were taken, one 5-days post-surgery and one 10-days post-surgery. However, a repeated measures ANOVA showed that there were no significant differences between the sucrose preference of the two groups post-elimination of the CORT rhythms in the group of animals with pumps. Although, a trend of decreased sucrose preference was seen in the pump group as compared to the sham rats [F (1,6) = 5.586; p = 0.064], indicating an inclination of showing anhedonic-like behaviors in these animals. Total immobility time

23 (secs) in a 5-mins forced-swim test was similar between the sham animals and animals with a pump (Figure 9). An independent samples t-test showed that there were no significant differences in the total immobility time between the two groups.

Figure 7: The rhythm of CORT after bilateral adrenalectomy. This figure shows the levels of CORT (ug/dl) present at 08:00hrs and 20:00hrs. In sham animals, the mean levels of CORT present at 08:00hrs was 1.32 ug/dl with a SEM of 0.1684 and the mean levels of CORT present at 20:00hrs was 37.29 ug/dl with a SEM of 6.77. In the group of animals with pump, the mean levels of CORT present at 08:00hrs was 21.74 ug/dl with a SEM of 8.41 and the mean levels of CORT present at 20:00hrs was 21.63 ug/dl with a SEM of 8.77. * represents a significant difference between the levels of CORT in the sham animals at 08:00hrs and 20:00hrs and a significant difference in the levels of CORT between the sham animals and pump animals at 20:00hrs.

Figure 8: Sucrose-preference test. This figure shows a change in the percent of sucrose preference between groups from their baseline preference to their preference on Day 10 post-surgery. The baseline preference of sham animals was a mean of 81.66% with a SEM of 11.4 and that of pump animals was a mean of 85.20% with a SEM of 8.073. The sucrose preference of sham animals on Day 5 post-surgery was a mean of 68.79% with a SEM of 7.00 and that of pump animals was 78.37% with a SEM of 6.69. The sucrose preference of sham animals on Day 10 post-surgery was a mean of 91.72% with a SEM of 3.64 and that of pump animals was 72.04% with a SEM of 6.55. A repeated measures ANOVA revealed that there were no significant differences between the sucrose-preferences of the two groups post-adrenalectomy.

Figure 9: Total immobility time in 5-min forced swim test. The figure shows the total immobility time (secs) of the two groups in a 5-mins forced swim test on Day 12 post-surgery at 20:00hrs. The mean immobility time of sham animals was 76 secs with a SEM of 52.7 and that of the animals with a pump was 89 secs with a SEM of 37.48. An independent samples t-test revealed that there were no significant differences between the total immobility time in these two groups of animals.

27 Study 2: In this experiment, we used 24 male rats. These rats were randomly divided into three groups (n=8/group): Sham, 20 ug/hr CORT “High Dose” and 2 ug/hr CORT “Low Dose”. Three animals died during or shortly after surgery, which left n=8 Sham, n=6 High Dose, and n=7 Low

Dose. Blood samples were collected twice, Day 4 post-surgery and Day 11 post-surgery at

08:00hrs and 20:00hrs to examine the rhythm of CORT and test circulating glucose levels.

Behavior testing was performed on Days 6-11 to check for any depressive-like behaviors. Brains from these animals were collected on Day 11 post-adrenalectomy, to examine the effects of adrenalectomy and constant CORT treatment on PER2 mRNA levels in the three selected limbic brain regions (PVN, BNST and cAMY).

The effects of adrenalectomy on elimination of the intrinsic circadian rhythm of CORT.

Similar to study 1, sham animals showed a normal rhythm of CORT with lower levels during the morning (2.25 ug/dl) and higher levels during the night (42.5 ug/dl). Whereas in the group of animals with high dose of CORT, the blood samples revealed CORT levels to be a mean of 1.75 ug/dl at 08:00hrs and a mean of 3.15 ug/dl at 20:00hrs (Figure 10). In the group of animals with low dose of CORT, we measured the CORT levels to be a mean of 0.44 ug/dl at 08:00hrs and a mean of 0.41 ug/dl at 20:00hrs. As expected, repeated measures ANOVA revealed a significant interaction between time and group [F (1,19) = 14.310; p < 0.001]. In the sham animals, a repeated measures ANOVA revealed levels of CORT present at 20:00hrs were significantly higher than that present at 08:00hrs [F (1,8) = 21.096; p = 0.002], confirming the normal diurnal rhythm of CORT in these animals. In contrast, neither the high dose nor the low dose groups showed significant differences in the levels of CORT at 08:00hrs and at 20:00hrs [F (1,5) =

3.709; p = 0.112] and [F (1, 6) = 0.27; p = 0.874], respectively. This demonstrates that we

28 successfully eliminated the rhythm of CORT in both the pump groups and the osmotic mini pump successfully delivered a constant dose of CORT to these animals. A one-way ANOVA showed that the amount of CORT present at 08:00hrs was not significantly different in all three groups of animals [F (2,21) = 2.622; p = 0.099]. Whereas, the level of CORT present at 20:00hrs was significantly different [F (2,21) = 15.405; p < 0.001]. Post-hoc tests (Tukey HSD) showed that levels of CORT in sham animals were significantly greater than both high dose (p=0.001) and low dose (p < 0.001) groups. A repeated measures ANOVA comparing the groups of animals with high dose and low dose of CORT revealed a significant between-subjects effect [F

(1,11) = 12.178; p = 0.005] demonstrating the high dose of CORT resulted in significantly greater circulating CORT levels compared to the low dose of CORT.

The effects of elimination of the intrinsic circadian rhythm of CORT on plasma glucose.

Glucose levels were tested from the blood samples collected on Days 4 and 11 post-surgery to validate the levels of CORT replacement were sufficient to maintain normal glucose levels.

Glucose tests were performed only on the blood collected one hour after lights OFF (20:00hrs).

Levels of glucose (mg/dl) present in the blood at 20:00hrs were similar between groups (Figure

11). A one-way ANOVA test showed no significant differences in the glucose levels between the three groups [F (1,21) = 0.188; p = 0.831]. Hence, no further post-analysis was performed.

The effects of elimination of the intrinsic circadian rhythm of CORT on expression of PER2.

To test our hypothesis, that constant basal levels of CORT would reduce peak PER2 gene expression in selected brain areas, PER2 mRNA levels were measured from the PVN (control),

BNST, and cAMY 1hr after lights-off on Day 11. As expected, no differences in PER2 mRNA

29 expression were observed between groups in the PVN (Figure 12). A one-way ANOVA showed there were no significant differences observed in the fold change of levels of PER2 mRNA between the sham, high dose and low dose group [F (2,18) = 1.162; p = 0.338]. This replicates previous findings that PER2 expression within the PVN is not altered by diurnal rhythms of

CORT (24). Within the BNST (Figure 13), a one-way ANOVA also revealed no significant changes between the levels of PER2 mRNA between the sham, high dose and low dose groups

[F (2,21) = 2.764; p = 0.088]. However, there was a trend in the fold change in PER2 mRNA between sham animals and animals given the low dose of CORT, such that the low dose group was trending to have an increase in PER2 expression. As expected, changes in PER2 mRNA expression where observed in the cAMY (Figure 14). A one-way ANOVA showed that in cAMY there was a significant difference in the fold change of PER2 mRNA between the three groups [F (2,21) = 4.170; p = 0.032]. Post-hoc analysis (Tukey HSD) revealed that there was a significant decrease in the PER2 mRNA levels in the low dose group when compared to sham animals (p=0.031). However, there were no significant changes in the PER2 mRNA levels between the high dose animals and sham animals (p=0.705).

The effects of elimination of the intrinsic circadian rhythm of CORT on emotional behaviors.

To check for depressive-like and anxiety-like behaviors after the disruption of the rhythm of

PER2 gene, we conducted a saccharin preference test (0.1% saccharin solution) from Days 6-11 post-surgery, an open field test on Day 9 post-surgery and, a 5 mins forced swim test on Day 10 post-surgery. A baseline percent of saccharin preference was taken two days before surgery, and as expected, baseline saccharin preference was not significantly different (Figure 15). Multiple saccharin-preference tests were taken consequently from Day 6 to Day 11 post-surgery. Post-

30 surgery, high levels of saccharin preference were maintained on Days 6-9. However, from Days

9-11 post-surgery, a decline in the saccharin preference of all three groups was observed. A repeated measures ANOVA showed that there was a significant effect of time in the saccharin preference of the three groups [F (6,90) = 5.422; p < 0.001]; however, there was no significant effect of time and group interactions [F (12,90) = 0.910; p = 0.540]. No significant differences were seen in the saccharin-preference between the three groups.

Total immobility time (secs) in a 5 mins forced swim test was similar between the sham animals, animals with high dose, and animals with low dose of CORT (Figure 16). A one-way ANOVA showed that there were no significant differences in the total immobility time between the three groups [F (2,18) = 1.021; p = 0.383]. Similarly, total distance moved (cm) in a 10 mins open field test was similar between the sham animals, animals with high dose, and animals with low dose of CORT (Figure 17). A one-way ANOVA showed that there were no significant differences in the total distance moved between the three groups [F (2,21) = 0.265; p = 0.770].

Additionally, the total duration of time animals spent in the center zone (secs) of the open field were the same between groups (Figure 18). A one-way ANOVA showed that there were no significant differences in the time these animals spent in the center zone between the three groups [F (2,21) = 0.997; p = 0.387].

Figure 10: The rhythm of CORT after bilateral adrenalectomy. This figure shows the levels of CORT (ug/dl) present in the groups at 08:00hrs and 20:00hrs. In sham animals, the mean levels of CORT present at 08:00hrs was 2.25 ug/dl with a SEM of 0.765 and the mean levels of CORT present at 20:00hrs was 42.54 ug/dl with a SEM of 8.71. In the group of animals with high dose of CORT, the mean levels of CORT present at 08:00hrs was 1.75 ug/dl with a SEM of 0.39 and the mean levels of CORT present at 20:00hrs was 3.15 ug/dl with a SEM of 0.94. In the group of animals with low dose of CORT, the mean levels of CORT present at 08:00hrs was 0.44 ug/dl with a SEM of 0.13 and the mean levels of CORT present at 20:00hrs was 0.414 ug/dl with a SEM of 0.13. * represents the significant difference between the levels of CORT in the sham animals at 08:00hrs and 20:00hrs and the significant difference in the levels of CORT between the sham animals and pump animals at 20:00hrs.

Figure 11: Blood glucose test. This graph shows the levels of glucose (mg/dl) present in the three groups one hours after lights OFF (20:00hrs). The sham animals, the mean glucose levels were 132.38 mg/dl with a SEM of 6.25. The animals with a high dose of CORT had a mean of 127.91 md/dl with a SEM of 6.23, and the animals with a low dose of CORT had a mean of 133.97 mg/dl with a SEM of 5.2. There were no significant differences in glucose levels in the three groups.

Figure 12: PER2 mRNA expression in the PVN. This graph shows the fold changes in the PER2 mRNA expression present in PVN in the three groups one hours after lights OFF (20:00hrs). The sham animals, the mean fold change was 1.00 with a SEM of 0.036. The animals with a high dose of CORT had a mean of 1.3 with a SEM of 0.41, and the animals with a low dose of CORT had a mean of 1.6 with a SEM of 0.41. There were no significant differences between the PER2 mRNA levels in the three groups.

Figure 13: PER2 mRNA expression in the BNST. This graph shows the fold changes in the PER2 mRNA expression present in BNST in the three groups one hours after lights OFF (20:00hrs). The sham animals, the mean fold change was 1.00 with a SEM of 0.058. The animals with a high dose of CORT had a mean of 1.3 with a SEM of 0.33, and the animals with a low dose of CORT had a mean of 1.79 with a SEM of 0.34. There were no significant differences between the PER2 mRNA levels in the three groups.

Figure 14: PER2 mRNA expression in the cAMY. This graph shows the fold changes in the PER2 mRNA expression present in cAMY in the three groups one hours after lights OFF (20:00hrs). The sham animals, the mean fold change was 0.998 with a SEM of 0.028. The animals with a high dose of CORT had a mean of 0.925 with a SEM of 0.257, and the animals with a low dose of CORT had a mean of 0.49 with a SEM of 0.18. * shows that there was a significant decrease in the PER2 mRNA levels in low dose group when compared to sham animals.

Figure 15: Saccharin-preference test. This figure shows the change in percent saccharin preference between the three groups from their baseline preference to their preference on Day 11 post-surgery. The baseline preference, taken two days before adrenalectomy, of sham animals was a mean of 93.47% with a SEM of 1.16 and that of pump animals with high dose was a mean of 94.3% with a SEM of 0.818, pump animals with low dose was a mean of 93.88% with a SEM of 0.472. The saccharin preference of sham animals on Day 6 post-surgery was a mean of 92.6% with a SEM of 0.514, that of pump animals with high dose was 93.4% with a SEM of 1.625 and pump animals with low dose was a mean of 92.9% and a SEM of 1.00. The saccharin preference of sham animals on Day 11 post-surgery was a mean of 86.5% with a SEM of 0.767, that of pump animals with high dose was 91.3% with a SEM of 1.23 and pump animals with low dose was 88.22% and a SEM of 1.706. There were no significant differences in the saccharin-preferences of the three groups post-adrenalectomy. However, we did observe a significant decrease in saccharin preference across time.

Figure 16: Total immobility time in 5-min forced swim test. The figure shows the total immobility time (secs) of the three groups in a 5-mins forced swim test on Day 10 post-surgery at 20:00hrs. The mean immobility time of sham animals was 39.7 secs with a SEM of 10.5, that of the animals with a high dose was 20.06 secs with a SEM of 8.2, and animals with low dose had a mean of 33.6 secs with SEM of 5.07. There were no significant differences between the total immobility time in these three groups of animals.

Figure 17: Total distance moved in 10 mins open field test. The figure shows the total distance moved by the animals of the three groups in a 10 min open field test on Day 9 post-surgery at 20:00hrs. The mean distance moved by sham animals was 3701.98 cms with a SEM of 536.25, that of the animals with a high dose was 3924.86 cms with a SEM of 437.15, and animals with low dose had a mean of 3354.19 cms with SEM of 549.18. There were no significant differences in the total distance moved between these three groups of animals.

Figure 18: Total duration in center zone in 10 mins open field test. The figure shows the duration the three groups of animals spend in the center zone (secs) during a 10 mins open field test, conducted on Day 9 post-surgery at 20:00hrs. The mean duration of sham animals in the center zone was 98.25 secs with a SEM of 19.96, that of the animals with a high dose was 84.08 secs with a SEM of 24.73, and animals with low dose had a mean of 60.06 secs with SEM of 13.6. There were no significant differences between the amount of time these three groups of animals spent in the center zone during the test.

40 CHAPTER 4

DISCUSSION

The studies presented here tested the hypothesis that elimination of the intrinsic diurnal rhythm of CORT would disrupt the rhythm of PER2 expression in targeted limbic regions, resulting in alterations in the mood and behavior of the animal. It is known that the rhythmic secretion of glucocorticoid hormones is governed by the SCN clock in mammals (67). In turn, this rhythm of glucocorticoids plays a major role in the modulation of several clock gene expressions, mainly the PERIOD genes (68, 69). It is believed that one of the key roles of these circadian oscillators in the brain is to maintain the optimal functioning of neural cells by regulating basic processes at the cell and tissue levels. Therefore, changes in the expression of these genes, may affect the functioning of these circuits, and ultimately, alter the behavior of the animal.

To test the hypothesis, we performed bilateral adrenalectomy that removed the intrinsic source of corticosterone in the animals, thus causing an elimination of the circadian rhythm of CORT. An osmotic minipump was implanted subcutaneously that provided the animals with constant levels of CORT. As the rhythm of PER2 in some cells is entrained and modulated by CORT, we predicted that replacing diurnal rhythms of CORT with constant low basal levels would lead to disruption of PER2 rhythms and constant low PER2 mRNA levels. Furthermore, this decrease in the PER2 levels, specifically in limbic brain regions like the BNST and cAMY, would impair

41 their optimum functioning and lead to mood changes in the animals resulting in altered emotional behaviors.

The blood samples collected from tail veins one hour after lights off and lights on, confirmed the expected differential levels of CORT between the sham animals and groups of animals with a pump. The sham animals showed a regular circadian rhythm of CORT whereas animals implanted with an osmotic minipump showed constant levels of CORT throughout the period of

24 hours. The animals that were given a higher dose of CORT (20 ug/hour) showed basal circulating CORT levels that were comparable to levels of CORT observed in sham animals during their circadian nadir. Animals that received the lower dose of CORT (2 ug/hour) had circulating CORT levels that were below normal endogenous levels.

Because adrenal hormones, specifically glucocorticoids, are known to regulate glucose levels in the body, bilateral adrenalectomy could affect the animal’s need for glucose. Therefore, to make sure that any observed differences in behavior were not due to low glucose availability in adrenalectomized animals, a blood glucose test was performed. As expected, the glucose levels between the three groups did not change due to surgery and implantation of an osmotic minipump, indicating that the animal’s glucose levels were not affected by adrenalectomy. The glucose levels were essential to validate the adrenalectomies and insertion of pumps, and to make sure if we observed changes in saccharin preference, it would not due to hypoglycemia caused by the removal of adrenal hormones. As no differences in blood glucose levels were observed, it indicated that animals implanted with an osmotic pump had sufficient access to blood glucose even after the adrenal hormones were removed.

Next, the PER2 mRNA levels in the PVN, BNST and cAMY were analyzed. This was the core of our hypothesis as we predicted a decrease in the PER2 mRNA in these selected limbic brain regions, after the normal circadian rhythm of CORT was replaced by constant low basal levels of the hormone. The first brain region analyzed was the PVN, a nucleus that receives afferent inputs from many important integrative centers like the hypothalamus, SCN, pons and medulla (70), allowing it to play essential roles in neuroendocrine and autonomic regulation (71, 72). We used

PVN as a control brain region and predicted no changes in the PER2 levels between sham rats and animals with pump, as Amir’s work had previously showed that PER2 expression remained unchanged in the PVN after bilateral adrenalectomy (46, 58). Statistical analysis backed this up and revealed no significant differences in the PER2 levels between the three groups. Next, we analyzed BNST. This brain region plays a crucial role in mood regulation and in anxiety related behaviors, and PER2 expression in this region was previously shown to be influenced by circulating CORT and reduced after ADX (65, 66). Therefore, we expected to see decreased levels of PER2 mRNA in animals implanted with a pump compared to sham animals however, statistical analysis revealed no significant differences in the fold change of PER2 expression between the three groups. If anything, we observed a trend of higher PER2 mRNA levels in the low dose group (Dose 2 ug/hour) when compared to sham animals. The last limbic brain region analyzed was the cAMY. This brain region also plays a major role in stress and fear related behaviors including depression and anxiety (73), and previous research shows PER2 influenced by CORT in this region (58, 66). Thus, we had expected to see a change in Per2 mRNA levels caused by constant basal levels of CORT. Post-hoc analysis revealed that animals supplied with

43 a lower dose of CORT (Dose 2 ug/hour) had significantly lower PER2 mRNA expression when compared to sham animals.

Sucrose or saccharin preference tests were conducted on the animals to check for the presence of anhedonic behavior after the removal of diurnal rhythm of CORT. The sucrose preference tests conducted on Day 5 and Day 10 post-surgery in Study 1, showed a trend where animals with pump had lower preferences for sucrose when compared to sham animals, indicating depressive- like behavior in these pump animals. However, the reduction in sucrose preference was not significant. This lack of significance could very well be attributed to the smaller sample size

(sham=3; pump=4) used in this pilot study. To correct this limitation, sample size was increased in Study 2 to 8 animals per group. In addition to this, the preference of the animals was measured for 6 consecutive days, to obtain a more thorough assessment of their saccharin preference. After observing this trend of lower sucrose preference in the animals with constant levels of CORT in

Study 1, we hypothesized to observe a significant decrease in the saccharin preference in the animals with pump in Study 2. Yet, the saccharin preference tests of sham, high dose and low dose animals conducted on Days 6-11 post-surgery in Study 2, showed no significant changes before and after the surgery between the three groups. Moreover, a general decrease was observed in the preferences of all three groups after Day 8, and this decrease was observed until the last day of preference test. We predict that this peculiar drop in the saccharin preference of all the animals in Study 2 might be because of the overlap between the preference tests and other behavior tests conducted during the night. Starting from Day 9, behavior tests, namely open field and forced swim tests, were conducted on the animals during their active period. On Day 11 post-surgery, blood samples were collected from these animals one hour after lights on and off.

44 This overlap with other behavior tests conducted on those days lead us to think that this drop in preference of all three groups might be due to stress the animals underwent while these behavior tests were being conducted or blood samples were being taken, masking the predicted decrease in preference of the animals with a pump. Another plausible reason for not observing anhedonic behaviors in the groups of animals with pumps in Study 2, can be because of the duration of the experiment. When we compared the drop in the preferences observed in Figure 8 to the general decrease observed in Figure 15, it did not correlate. In addition to this, Figure 8 from Study 1 showed a trend of lower sucrose preference of animals with pump when compared to sham animals, but this was not seen until Day 10 post-surgery. As the osmotic minipumps used for this study can supply constant doses of CORT only up to 14 days, we designed our studies in a way that the experiments were completed in 2 weeks. Because of this short duration of the experiment, it is possible that the elimination of the diurnal rhythm of CORT and its replacement with constant levels of the hormone, was not sufficient to change the PER2 expression and cause a reduction in saccharin preference of the animals. Thus, to get a clearer picture of the animal’s preference for saccharin in the future, one could separate the days when saccharin tests are conducted from when other behavioral tests are done and increase the time of our study to provide sufficient time for the constant levels of CORT to disrupt the rhythm of PER2 gene.

To check for other depressive-like behaviors in animals with a CORT pump, a forced swim test was conducted on Day 12 in Study 1 and Day 10 in Study 2 at 20:00h. In this forced swim test, total immobility or total floating time was measured. Greater immobility or floating time points to the presence of behavioral despair in the animal (74). In both Study 1 and Study 2, we saw no significant difference in the total immobility time between the sham animals and animals with

45 pump, indicating that there was no depressive-like behavior in the animals given a constant dose of CORT. Another behavior test, namely open field test, was added in Study 2, to analyze the onset of anxiety-like behavior in addition to depressive-like behavior. The addition of an anxiety test was important as research shows that 51% of those with depression also suffer from anxiety disorders (9). A 10 mins open field test was conducted on Day 9 post-surgery, at 20:00h in Study

2. Two measurements were taken during this test- total distance moved and the amount of time spent in the center zone, to check for anxiety-like behaviors. The open field area was divided into two zones, a middle center zone and a peripheral outer zone. If a rat spends less time in the center zone, it would indicate presence of anxiety-like behaviors (75). The total distance moved was also measured and was predicted to have no significant changes between the three groups. This was done to make sure that if we observed any differences in the duration spent in the center zone, it would primarily be due to anxiety and not the general reduction in distance moved by the animal. As expected, the total distance moved by animals was not significantly different between the sham animals, animals with high dose and animals with low dose.

However, no significant differences between the three groups were observed in the duration spent by the animals in the center zone, indicating absence of any anxiety-like behavior after the reduction of PER2 mRNA levels in cAMY.

All of the above observed results partially support the first part of our hypothesis by showing that constant low basal levels of CORT cause a decrease in the PER2 mRNA levels in cAMY for animals with low doses of CORT. The second part of our hypothesis, however, was not supported as we did not observe any significant behavior changes in the animals with constant

46 levels of CORT, checked via saccharin/sucrose preference tests, forced swim tests, and open field tests.

Research indicates that both the abundance and circadian oscillation patterns of PER2 in cAMY and BNST are critically important for expression of normal emotional behavior (76, 77). These regions have been previously shown to exhibit rhythms of PER2 expression that are uniquely dependent on circadian glucocorticoid signaling (46). Amir’s work has shown that in rats, PER2 expression in the BNST and cAMY peaks at ZT13 (one hour into the dark phase) similar to peak levels of circulating CORT and this peak of PER2 expression is blunted by ADX in both of these limbic brain regions (58, 60, 65). However, our findings show that the cAMY appears to be differentially sensitive to removal of the CORT rhythms as this reduction in the PER2 levels were not observed in the BNST. We were not able to replicate Amir’s results of reduction in

PER2 expression in BNST and cAMY in ADX rats due to a few differences between our study designs (65, 66, 69). In Lamont and Amir’s study (2005), the ADX rats were not given any exogenous CORT after the surgery and it was shown that ADX decreased the PER2 expression in cAMY along with completely abolishing the circadian rhythm of the gene (61). This differed from our study, in regards that we gave back constant basal levels of CORT after adrenalectomy.

In Segall et al. (2006), the ADX rats were either given CORT via drinking water or a 100 mg 30- day slow releasing pellets that provided a constant dose of CORT throughout the day (66). For

ADX rats that were given CORT in drinking water, the rhythm of PER2 was reestablished in both BNST and cAMY. However, they showed that CORT replacement via constant-release pellets was not able to restore the circadian rhythms of PER2 in either BNST or cAMY (66).

While ADX and implantation of CORT pellets eliminated the circadian rhythm of PER2, Amir’s

47 studies did not directly compare PER2 levels in this group to PER2 levels in sham controls, thus it is difficult to determine if CORT pellets resulted in constant high or constant low PER2 expression. The slow release pellet used by Segall et al. (2010) resulted in constant CORT levels that were at the peak of the diurnal CORT rhythm, thus one would predict these levels to maintain higher PER2 expression. We decided to use a lower dose of either 20 ug/h or 2 ug/h of

CORT to mimic physiological levels of CORT observed at the nadir of the diurnal rhythm; with the hypothesis this would result in lower PER2 expression in cAMY and BNST. The high dose supplied to the animals with pump (20 ug/h) mimicked the low basal levels of CORT in the sham animals, however, we did not observe any significant effects in the levels of PER2 in animals with high dose of CORT. Whereas, we did see significant changes in PER2 levels in cAMY, of the animals provided with low dose of CORT (2ug/h) even when this dose was lower than basal physiological levels of the sham animals. We think that this low dose of CORT replicates

Lamont et al. (2005), where PER2 levels in the cAMY reduced after ADX with no CORT replacement (61). Lower doses of CORT are of particular interest because impairments in glucocorticoid signaling has been observed in humans with a history of depression, therefore, lower doses of CORT may better model what occurs in depression (78). In addition to this, it could also be that because the brains were collected 1 hour after lights off, we could get PER2 mRNA only at that time point, making it impossible to predict the rhythm of the gene expression in our study. To check our prediction that constant basal levels of CORT would reduce the peak of PER2, resulting in constant levels of PER2 mRNA levels throughout the day in the BNST, it would require us to measure the PER2 expression at several time points, to further help us capture any regulatory effects of changes in glucocorticoids on these local PER2 oscillations.

48 Despite the notion that PER2 rhythms appears to be necessary for the optimal function of the cells and behavior, we did not observe any significant depressive behaviors in animals with pumps (2ug/hr) and lower PER2 expression in the cAMY, as originally predicted. A study conducted at Emory by Michael Davis (2006), focused on the individual and differential role of cAMY and BNST in the regulation of mood. Interestingly, this study demonstrated a heightened role of cAMY for immediate fear expression and the increased role of BNST in long-term anxiety (79). This could suggest that PER2 rhythms in cAMY and BNST might not play a major role in the onset of depressive and anxiety related behaviors in rats but might play an important role in other behaviors such as fear expression or social behavior (80). As we found a significant decrease in the PER2 mRNA levels in the cAMY, we can incorporate other behavior tests that will focus on fear and endocrine changes in the future, to help us tailor our focus on PER2 and its role in cAMY. One of the other reasons for not observing any depressive or anxiety-like behavior in the animals could be the lack of disruption of PER2 rhythms in the BNST. This loss of effect is particularly important as BNST is highly involved in regulating behaviors in response to emotional stimuli (66). As CORT entrains PER2 rhythms in both BNST and cAMY and both these brain regions play an important role in the regulation of mood, we could reason that because PER2 levels did not change significantly in the BNST as well, it was not enough to produce any significant changes in the behavior of the animal. Moreover, these two regions are known to be chemically, anatomically, and functionally homologous regions, making their combined effect on PER2 expression of greater importance (65).

Interestingly, previous literature has shown that PER2 regulates the female reproduction estrous cycle and that sex hormones affect circadian rhythms suggesting a dynamic feedback between

49 the two systems in the brain (81-83). Moreover, research was widely shown that females are more prone to depression (84). Therefore, to completely understand the biological mechanisms underlying gender differences in depressive and anxiety-like behaviors, in our pilot studies we included female rats as well. However, three out of four female rats with pumps died shortly after adrenalectomy. It is unclear why females are more sensitive to adrenalectomy surgery. One of the reasons for this could be a lack of supply of proper dose of CORT in the pumps. Female rats are known to have slightly higher basal CORT and greater CORT responses to stressors, thus may require a higher dose of CORT after adrenalectomy or a bolus injection of CORT at the time of surgery to ensure their survival. As we did not have time to problem solve such a hurdle, only male rats were used in the present investigation. Although it is recognized that these studies will need to be extended to female rats in the future given the fact females are more susceptible to depression and anxiety disorders.

In summary, the results of the present study show that elimination of the intrinsic circadian rhythm of CORT was successfully done by adrenalectomy and pump implantation. The elimination of the rhythm of corticosterone and its replacement by constant low basal levels of

CORT was sufficient to lower the PER2 levels in the central amygdala in animals with low dose of CORT (2ug/hr). However, constant levels of CORT were insufficient to cause a decrease in the PER2 mRNA levels in the BNST. Furthermore, adrenalectomy and low constant levels of

CORT was not sufficient to produce any depressive or anxiety-like behavior in the animals, as checked by saccharin tests, forced swim tests, and open field tests.

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