Sleep Disturbances After Chronic Alcohol Consumption

Home , Alcohol and cortisol, Melatonin

SLEEP DISTURBANCES AFTER CHRONIC ALCOHOL CONSUMPTION:

HOMEOSTATIC DYSREGULATION OR

CIRCADIAN DESYNCHRONY?

RONG GUO

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Program in Neuroscience

JULY 2016

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of RONG GUO find it satisfactory and recommend that it be accepted.

______Steven M. Simasko, Ph.D., Co-Chair

______Heiko T. Jansen, Ph.D., Co-Chair

______Barbara A. Sorg, Ph.D.

______Ilia Karatsoreos, Ph.D.

ACKNOWLEDGEMENT

First, I would like to thank my mentors, Drs. Steven Simasko and Heiko Jansen. They have not only helped me grow my knowledge in the field but also deepened my understanding of science and research. They are supportive, encouraging, and inspiring. I’m glad that we were able to work together and turn my initial research interest in sleep and alcohol into such a nice project. I appreciate their help and guidance both in my studies and in my career. It is my pleasure to be part of their team. I would also like to thank my committee members, Drs. Barbara

Sorg and Ilia Karatsoreos, for their help during numerous occasions in my research. They have provided insightful thoughts that helped shape my project and this dissertation. They also have given me great advice regarding my future career. I have learned a lot from them and it was an honor to be able to work with them.

I would like to thank the past and present members of the Simasko lab and the Jansen lab, especially Jamie Gaber, Christi Pedrow, and Brandon Hutzenbiler, and many others who were willing to help me along the path of my graduate work. I’d like to give my special thanks to

Derrick Phillips, who has been a great classmate, lab-mate, office ‘roommate’, and dear friend. It would not have been possible to accomplish much without all their enthusiastic help. I would also like to thank all members of the department of Integrative Physiology and Neuroscience at

WSU, especially the office and vivarium staff. I appreciate their kindness and patience with me.

In addition, I would like to thank the financial support from the Alcohol & Drug Abuse

Program at WSU, which supported the initiation of my projects.

Finally, I would like to express my gratitude to my dear families and friends for their unconditioned love, support, and faith. It means the world to me to have them in my life.

iii

SLEEP DISTURBANCES AFTER CHRONIC ALCOHOL CONSUMPTION:

HOMEOSTATIC DYSREGULATION OR

CIRCADIAN DESYNCHRONY?

Abstract

by Rong Guo, Ph.D. Washington State University July 2016

Co-Chairs: Steven M. Simasko Heiko T. Jansen

Chronic alcohol use often leads to sleep disturbances such as insomnia and sleep fragmentation, but the underlying mechanisms are not fully understood. Sleep is regulated by two main processes: a homeostatic process representing the build-up of sleep pressure during wake and a circadian process integrating sleep timing with environmental cues. It is possible that chronic alcohol exposure impairs sleep through either process or both. This dissertation presents investigations to address these possibilities.

We first determined if chronic alcohol exposure impaired the homeostatic regulation of sleep by using a sleep deprivation challenge. We found that alcohol-treated rats showed robust and relatively normal compensatory increase in sleep time and sleep intensity (as measured by slow wave amplitude) within 24 hours after the challenge, but the recovery sleep of these rats was delayed and fragmented. These results suggest that chronic alcohol exposure may weaken the stability of sleep states, leading to sleep fragmentation, but the homeostatic regulatory mechanisms were most likely intact. We next determined if chronic alcohol treatment altered the integrity of circadian systems by examining diurnal rhythms of body temperature, locomotor,

iv plasma corticosterone, and Per1 expression in the master pacemaker (suprachiasmatic nucleus,

SCN) and in the HPA axis (pituitary and adrenal glands). We found that after chronic alcohol exposure these processes still exhibited a 24-hour cycle, but the cycles were less robust and more variable. Most importantly, the phase relationships among diurnal physiological rhythms, and between the central and peripheral molecular clocks, were significantly altered leading to desynchronized circadian processes. Lastly, we placed rats under constant darkness to remove the external light Zeitgeber and found that constant darkness stabilized the dampened rhythms and normalized the blunted distribution of sleep/wake time caused by chronic alcohol exposure.

Taken together, our studies suggest that chronic alcohol exposure: 1) weakens the stability of sleep states leading to unstable and fragmented sleep, 2) compromises the ability of SCN to synchronize internal clocks and to integrate sleep timing with the external light Zeitgeber, and 3) does not prevent the animal from responding to the build-up of sleep pressure.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT ...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

DEDICATION...... xii

CHAPTER ONE

GENERAL INTRODUCTION ...... 1

Alcohol and Alcoholism ················································································· 3

A. Alcoholic Beverages ··············································································· 3

B. Alcoholism and Alcohol Use Disorder (AUD) ················································ 4

C. Alcohol-related Pathologies ······································································ 6

D. Alcohol and the Central Nervous System ······················································ 8

E. Summary ··························································································· 10

Alcohol-related Sleep Disturbances ··································································· 11

A. Acute Alcohol and Sleep ········································································· 12

B. Chronic Alcohol and Sleep ······································································ 15

C. Sleep Disturbances and Relapse ································································ 16

D. Treatment Options for Sleep Disturbances in AUD ·········································· 17

E. Possible Mechanisms ············································································· 18

Sleep and Sleep Regulation ············································································· 20

A. Sleep Measurements ·············································································· 20

B. Sleep Stages ························································································ 21

C. Rodent Sleep vs. Human Sleep ·································································· 22

D. Sleep Regulation ·················································································· 23

E. Two-Process Model of Sleep Regulation ······················································ 24

Circadian Rhythms ······················································································· 27

A. Molecular Clocks and Circadian Genes ························································ 27

B. The Suprachiasmatic Nucleus (SCN) ·························································· 28

C. Interaction between Alcohol, Sleep, and Circadian Rhythms ······························ 28

References ································································································· 32

CHAPTER TWO

CHRONIC ALCOHOL CONSUMPTION DELAYS AND FRAGMENTS RECOVERY

SLEEP AFTER SLEEP DEPRIVATION IN RATS ...... 46

Abstract ···································································································· 47

Introduction ······························································································· 48

Materials and Methods ·················································································· 52

Results ····································································································· 57

Discussion ································································································· 62

References ································································································· 77

CHAPTER THREE

CHRONIC ALCOHOL CONSUMPTION IN RATS LEADS TO DESYNCHRONY IN

DIURNAL RHYTHMS AND MOLECULAR CLOCKS ...... 82

Abstract ···································································································· 83

Introduction ······························································································· 84

vii

Materials and Methods ·················································································· 87

Results ····································································································· 92

Discussion ································································································· 96

References ······························································································· 110

CHAPTER FOUR

CONSTANT DARKNESS ALLEVIATES SLEEP AND CIRCADIAN DISRUPTIONS

AFTER CHRONIC ALCOHOL EXPOSURE IN RATS ...... 115

Abstract ·································································································· 116

Introduction ····························································································· 118

Materials and Methods ················································································ 122

Results ··································································································· 126

Discussion ······························································································· 129

Reference ································································································ 140

CHAPTER FIVE

GENERAL DISCUSSION ...... 143

Summary of Finding ··················································································· 143

Possible Candidate Mechanisms ····································································· 145

A. Body Temperature ·············································································· 145

B. The Corticotrophin-Releasing Factor (CRF) System, HPA Axis, and Glucocorticoids

(GCs) ··································································································· 147

Implications ····························································································· 157

Reference ································································································ 159

viii

LIST OF TABLES

CHAPTER TWO

Table 2. Sleep Parameters after Sleep Deprivation ...... 75

CHAPTER THREE

Table 3-1. Light vs. Dark Characteristics of Body Temperature and Locomotor Activity . 107

Table 3-2. Rhythm Parameters for Body Temperature and Locomotor Activity ...... 108

Table 3-3. Results of Discrete Wavelet Transform (DWT) Analysis of Body Temperature

and Locomotor Activity ...... 109

CHAPTER FOUR

Table 4. Rhythm Parameters for Body Temperature ...... 139

LIST OF FIGURES

CHAPTER TWO

Figure 2-1. State Time in Wake, NREMS, and REMS during Sleep Deprivation and druing

the 24 Hours after...... 68

Figure 2-2. Latency to NREMS and REMS after Sleep Deprivation ...... 70

Figure 2-3. Slow Wave Amplitude (SWA) after Sleep Deprivation ...... 71

Figure 2-4. Spectral Power Distribution after Sleep Deprivation...... 72

Figure 2-5. Body Temperature and Locomotor Activity before and after Sleep Deprivation .

...... 74

CHAPTER THREE

Figure 3-1. Rate of Body Weight Change ...... 101

Figure 3-2. Representative Actograms of Body Temperature and Locomotor Activity from a

Control and an Alcohol-treated Rat...... 102

Figure 3-3. Diurnal Rhythms of Body Temperature and Locomotor Activity during the

Baseline Period and during the 6-week Alcohol Treatment ...... 103

Figure 3-4. Effects of Chronic Alcohol Exposure on Plasma Corticosterone (CORT)...... 104

Figure 3-5. Effects of Chronic Alcohol Exposure on Per1 Expression in the SCN and in the

Pituitary and Adrenal Glands ...... 105

CHAPTER FOUR

Figure 4-1. Representative Actograms of Body Temperature from a Control and an Alcohol-

treated Rat ...... 131

Figure 4-2. Body Temperature in L:D during Chronic Alcohol Exposure...... 132

Figure 4-3. Sleep in L:D during Chronic Alcohol Exposure ...... 133

Figure 4-4. Body Temperature in D:D during Chronic Alcohol Exposure ...... 134

Figure 4-5. Sleep in D:D during Chronic Alcohol Exposure...... 135

Figure 4-6. Body Temperature in D:D during Withdrawal of Alcohol ...... 136

Figure 4-7. Sleep in D:D during Acute Withdrawal from Alcohol ...... 137

Figure 4-8. Temperature in L:D after Prolonged Abstinence ...... 138

DEDICATION

This dissertation is dedicated to my loving family and dear friends.

xii

CHAPTER ONE

GENERAL INTRODUCTION

Sleep disturbances such as insomnia and sleep fragmentation are common among active and abstinent alcoholic patients. Chronic insomnia not only can impair their health but also can increase their risk of relapse during abstinence. The mechanisms by which chronic alcohol use disrupts sleep and the connection between sleep disturbances and relapse are still not fully understood. The goal of this dissertation is to present new findings from my studies that contribute to the understanding of the mechanisms of sleep disturbances after chronic alcohol consumption.

This opening chapter presents relevant literature in the field of alcoholism, alcohol-related sleep disturbances, sleep, circadian rhythms, and the interactions among alcohol, sleep, and circadian rhythms. The two main processes in sleep regulation include a homeostatic process and a circadian process. Whether chronic alcohol exposure impairs sleep through the homeostatic process, the circadian process, or both remains to be elucidated. This chapter validates the need to address this fundamental gap in our knowledge of sleep disturbances associated with alcohol use disorder.

The second chapter determines whether chronic alcohol treatment impairs the homeostatic regulation of sleep by examining the rebound sleep of control and ethanol- treated rats after 6 hours of acute sleep deprivation. We focus on the amount, intensity, and quality of the compensatory rebound sleep in the 24-hour recovery period after sleep deprivation.

The third chapter addresses whether chronic alcohol exposure affects the integrity of circadian systems by examining its effects on diurnal rhythms of body temperature, locomotor activity, plasma corticosterone concentrations, and ex vivo Per1 expression in the master pacemaker (suprachiasmatic nucleus, SCN) and in peripheral clocks within the hypothalamic–pituitary–adrenal (HPA) axis (pituitary and adrenal glands). We measure multiple parameters on multiple levels to emphasize the significance of internal synchrony. Proper phase relationships among various peripheral rhythms and between the central and the peripheral clocks are key to synchronized and optimized physiological functions of the human body.

In the fourth chapter, we test the hypothesis that if the primary effect of alcohol is to desynchronize the circadian control system, then removing the external Zeitgeber should lessen circadian desynchrony and correct some of the disturbed sleep patterns. We found that placing the animals in constant darkness reversed the redistribution of sleep that occurred in the normal 12 hour light:dark situation.

The final chapter summarizes the findings of my studies and concludes that the primary effect of chronic alcohol exposure on sleep is to compromise the ability of the

SCN to maintain a tight synchronization among internal clocks and to integrate sleep timing with the external light Zeitgeber, rather than a direct effect on mechanisms underlying recovery sleep to a homeostatic challenge. This chapter also points out future research directions to explore the mechanisms underlying alcohol-induced circadian desynchrony and the potential applications of this knowledge to the treatment of other alcohol-related pathologies and other mental disorders.

Alcohol and Alcoholism

A. Alcoholic Beverages

Alcoholic beverages have existed for thousands of years and are now widely produced and consumed throughout the world on a daily basis. Alcoholic beverages were first fermented from rice, honey, and fruits, and could be dated back to the Stone Age.

Ancient wine was first produced in the Near East between 8,500 and 4,000 B.C.; beer has been brewed in China by 7,000 B.C.; spirits were developed after the distillation of alcohol became possible in the twelfth century (Forbes, 1970; McGovern, 2003;

McGovern et al., 2004). Drinks in ancient times were served not only as beverages but also as medicines and offerings to gods (Blum, 1969; French, 1890; Roueche, 1963).

Later, alcoholic beverages became luxury goods restricted to the upper social classes.

Nowadays, alcohol from various alcoholic beverages is one of the most accessible and commonly used licit drugs in modern society (SAMHSA, 2015).

In the United States, alcoholic beverages are very popular and the alcohol consumption levels have been relatively high since the 1990s. Based on reports from the

National Institute on Alcohol Abuse and Alcoholism (NIAAA) in 2014, more than 87% of people 18 years or older in the US have consumed alcohol in their life (NIAAA, 2014).

According to the global status report on alcohol and health by the World Health

Organization (WHO), the adult per capita consumption of pure alcohol in the form of alcoholic beverages in 2005 worldwide equaled 6.13 liters, and the consumption in the

United States equaled 9.44 liters (WHO, 2014).

B. Alcoholism and Alcohol Use Disorder (AUD)

Excessive consumption of alcohol for prolonged periods of time often leads to the development of alcoholism, characterized by compulsive alcohol use, tolerance to alcohol, and alcohol withdrawal (Brower, 2003b). Alcoholism is currently diagnosed as alcohol use disorder (AUD) according to the Diagnostic and Statistical Manual of Mental

Disorders (DSM)-V issued by the American Psychiatric Association. Patients who meet any two or more of the eleven criteria listed under the DSM-V within a year would be diagnosed with AUD. The more criteria a patient would meet, the more severe the AUD would be. Common symptoms of AUD include compulsive alcohol craving, excessive drinking, loss of control of drinking, continued drinking despite aversive consequences, alcohol tolerance, physical dependence, and withdrawal.

AUD is a serious social problem in the United States, negatively affecting more than 17 million people each year (SAMHSA, 2015). According to a survey in 1993, there are more than 700,000 patients who receive alcoholism treatment each day, 13.5 percent of which receive hospital or residential treatment while 86.5 percent of which receive outpatient treatment (NDATUS, 1993). In 2012, about 1.4 million adults are receiving

AUD treatment each year (NIAAA 2014). Estimated economic costs of alcohol abuse in the US in 1998 are around $184.6 billion, including $26.3 billion of direct costs for health care, treatment, and medical consequences, $134.2 billion of indirect costs for loss of productivity, and $24.1 billion of other costs such as crime and vehicle crashes (NIAAA,

2000). Recent surveys show that AUD creates an annual economic burden of $223.5 billion in the US (CDC, 2006).

Many biological, psychological and sociological factors contribute to the prognosis of AUD. Genetic factors, which may account for 40 to 60 percent of the risk, interact with many environmental factors leading to the development of addiction in general (Goldman, 2005). A family history of heavy drinking can significantly increase the risk of AUD (Dinwiddie and Reich, 1993). Relevant genes can affect personality characteristics such as impulsivity, contributing to the increased risk of general substance abuse. In addition, certain variations of genes can also specifically increase the risk of alcohol-related disorders. For example, isozymes of key alcohol metabolism enzymes, e.g. alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which influence the duration of the presence of alcohol and its metabolites in the body, may contribute to the genetic vulnerability to alcoholism (Garver, 2001). Low sensitivity to the effect of alcohol may also predispose individuals to heavy drinking later in life

(Schuckit, 1985).

Addiction is a neurocircuitry disorder driven by the brain reward/motivation system associated with the euphoric or pleasant experience during drug use and by the brain anti-reward /stress system associated with the dysphoria and negative affect during withdrawal (Koob, 2013). Over repeated exposure to alcohol, the positive reinforcing effects of alcohol would diminish due to long-term neuroadaptations within the reward system, leading to reward deficits. At the same time, the brain anti-reward system would be activated, leading to the transition from impulsive drug taking to compulsive drug seeking for the alleviation of negative consequences such as withdrawal. Plenty of studies have demonstrated that alcohol can affect critical brain areas involved in the brain reward system, such as dopaminergic neurons in the ventral tegmental area (VTA), the nucleus

5 accumbens (NA), the central nucleus of the amygdala (CeA), and the prefrontal cortex

(PFC) (Koob, 2014). Alcohol promotes dopamine releases in the NA, and during withdrawal, extracellular dopamine drops (Boileau, 2003; Rossetti, 1992). The negative emotional affect of alcohol withdrawal is proposed to be driven by the brain stress system through excessive hypothalamic-pituitary-adrenal (HPA) axis activity and the up- regulation of the corticotropin-releasing hormone (CRH) system in the amygdala (Hellig,

2007; Koob, 2008).

C. Alcohol-related Pathologies

Misuse of alcohol can contribute to the development of many diseases and health problems. Because alcohol can easily diffuse through tissues and organs, alcohol consumption can affect most of the major physiological systems in the body. It often takes years of excessive alcohol use before individuals realize that they have a real problem, and by that time severe damages have often been done to their health. In fact,

AUD is the most significant contributor to diseases and injuries associated with alcohol use. Moreover, alcohol consumption is responsible for fetal alcohol syndrome (FAS).

Gestational drinking can lead to babies with various FAS mental and/or physical symptoms and disabilities.

Gastrointestinal and liver diseases are commonly comorbid with AUD. Humans consume alcohol mostly via drinking. After ingestion alcohol is readily absorbed into the bloodstream mainly through the stomach and later metabolized by the liver. Alcohol use contributes to the risk of gastroesophageal reflux disease (GERD) by increasing the secretion of gastrin and HCl and by reducing the muscle tone of the esophageal sphincter

(Bujanda, 2000; Chen, 2010; Matsuo, 1972). Alcohol use also increases the risk of gastritis and pancreatitis (Bujanda, 2000; Migliori, 2004). Individuals with heavy alcohol use often have high risks of colon and rectal cancer (Klatsky et al., 1988). Alcohol use can also cause various liver diseases such as fatty liver, liver cirrhosis, acute alcoholic hepatitis, and even liver cancer (Grewal and Viswanathen, 2012). The liver damage may be partially mediated through the leaky gut caused by chronic alcohol use (Keshavarzian et al., 1999). In addition, alcohol damages the liver by affecting lipid metabolisms. After alcohol is transported to hepatocytes, it is oxidized into acetaldehyde with a massive production of NADPH, which speeds up lipid biosynthesis and causes accumulation of fatty acids in hepatocytes (Zhou et al., 1998). The accumulation of fatty acids damages liver after the initial buffering actions of releasing fatty acids into the plasma and/or changing them into triglycerides have exhausted (Baraona, 1998). Recent studies suggest that chronic alcohol exposure may cause liver and gut pathologies via disruptions of clock gene expression (Summa et al., 2013). Maldigestion and malnutrition are also indirect consequences of chronic alcohol consumption in parallel with damaged gut and liver functions.

Alcohol use can contribute to the development of many cardiovascular diseases.

Holiday heart syndrome (or paroxysmal atrial arrhythmias), can occur after acute alcohol ingestion due to the fast potassium depletion (Ettinger, 1978; Fauchier, 2003). Chronic alcohol use may cause hypertension, and contribute to dilated cardiomyopathy by affecting the apoptosis of myocytes (Piano, 2002; Richardson, 1986). Heavy drinking also increases the risk of stroke by affecting cerebral vascular smooth muscles (Li, 2004;

Reynolds, 2003).

Chronic alcohol consumption also weakens the immune system (Redwine, 2003;

Sander, 2002). The cellular immunity of alcoholics is lower than that of non-alcoholics probably due to sleep disruption (Redwine, 2003). The nature killer (NK) cell activity, interleukin (IL)-6 formation, and IL-6/IL-10 ratio are also decreased in alcoholic patients

(Adinoff et al., 2003).

D. Alcohol and the Central Nervous System

Alcohol can pass through the blood-brain barrier and reach the central nervous system (CNS). Effects of acute alcohol exposure on the CNS are proposed to be biphasic: stimulatory during the ascending phase of the plasma alcohol concentration and sedative during the descending phase (Pohorecky, 1978). Alcohol has been shown to affect most of the major neurotransmitter systems, such as acetylcholine, γ-Aminobutyric acid

(GABA), glutamate, norepinephrine, dopamine, serotonin, and adenosine (Chastain,

2006). The two main targets of alcohol are GABA and glutamate receptors. Alcohol can inhibit glutamate activity, increase GABA activity, and inhibit the release of acetylcholine, causing a temporary sedative effect (Littleton, 1998). Acute alcohol in high doses can suppress respiratory function and may lead to alcohol intoxication or even death. The norepinephrine system is proposed to oppose the sedative effects of alcohol.

Low doses of alcohol increase norepinephrine levels and high doses decrease norepinephrine levels in rats (Rossetti, 1992). Norepinephrine-mutant mice are hypersensitive to alcohol administration (Weinshenker and Schroeder, 2007).

Chronic alcohol consumption can cause many long-lasting neural changes in the

CNS. Common neuroadaptations include decreased inhibitory responses of GABA

8 receptors and increased glutamate activities (Becker, 1999). During abstinence, changed basal activities may be responsible for mediating some withdrawal symptoms. For example, norepinephrine levels are elevated during abstinence (Hawley, 1994). Drugs such as benzodiazepine, clonidine, and propranolol were proposed to be used during alcohol withdrawal because they could reduce the activity of the norepinephrine system.

In addition, the effects of chronic alcohol on the CNS may be direct and/or indirect. For example, Korsakoff’s syndrome is not unusual among alcoholics. Common symptoms of

Korsakoff’s syndrome include anterograde and/or retrograde amnesia, confabulation, lack of insight, apathy and loss of content in conversation. Actually, chronic alcohol use in human alcoholics is often associated with loss of learning and memory, and similar effects on learning and memory are also found in rats (Fadda and Rossetti, 1998; Walker,

1978). Korsakoff’s syndrome is caused by vitamin B1 deficiency as a result of the malnutrition following chronic alcohol consumption. Alcoholics tend to use alcohol beverages as the major caloric source instead of regular meals because alcoholic beverages themselves contain high amounts of calories and because alcoholics usually cannot maintain a healthy and regular diet habit. Moreover, chronic alcohol consumption is comorbid with many neurological and/or psychological disorders, such as neurodegeneration, major depression, anxiety disorders, insomnia, seizure and epilepsy, and psychosis. The mechanisms by which chronic alcohol consumption contributes to these problems are still not fully understood.

E. Summary

Alcohol is one of the most accessible and widely consumed drugs in modern society. It is legal; it is broadly advertised; it is also rather cheap. Chronic alcohol use can be problematic and can lead to the development of alcohol use disorder. Like other drugs of abuse, alcohol use disorder (alcoholism) is a real disease that involves long-term neuroadaptations in the brain reward system and the brain stress system. Due to the nature of alcohol to be highly soluble in water, alcohol could affect both the central nervous system and massive peripheral tissues and organs in human bodies. Chronic alcohol use can contribute to the development of many diseases and health problems.

Alcohol-related Sleep Disturbances

The main interest of this dissertation lays on sleep disturbances after chronic alcohol exposure, especially in the context of alcohol use disorder (AUD). Sleep problems such as difficulty falling asleep, loss of total sleep time, and sleep fragmentation are very prevalent among drinking and abstinent alcoholics (Colrain et al., 2014;

Knapp et al., 2014). Cohn and colleagues showed that more than 90% of alcoholic patients suffered from impaired sleep (Cohn et al., 2003). Perney et al. reported that around 70% of alcoholics experienced a certain extent of sleep problems, including around 20% with severe sleep disturbances (Perney et al., 2012). Brower et al. concluded that about 36 to 72 percent of alcohol-dependent patients admitted into any treatment programs reported insomnia, which was higher than the insomnia rates of the general population (17 to 30 percent) (Brower, 2001b; Brower, 2003a, 2001a). Insomnia alone could account for about 10 percent of total alcohol-related costs (Stoller, 1994).

Alcoholic patients also tend to have higher rates of obstructive sleep apnea (OSA) compared to non-alcoholics (Aldrich, 1999).

Sleep disturbances in AUD can severely impair the quality of life and can also lead to negative health consequences, rendering alcoholic patients vulnerable to physiological and psychiatric challenges in daily life (Foster et al., 1988; Roth, 2007).

Chronic sleep problems can lead to excessive daytime sleepiness, impair daytime performance, decrease daytime alertness, produce memory dysfunction, and increase the risk of major health problems, such as diabetes and cancer (Ancoli-Israel, 1999; Gillin,

1998; Landolt and Borbély, 2000; Roehrs and Roth, 2001). However, the underlying

11 mechanisms by which chronic alcohol consumption may disturb sleep still remains unclear, limiting the development of effective mass treatment options.

A. Acute Alcohol and Sleep

Many studies have reported the effects of acute alcohol consumption on sleep since the 1930s, most of which showed that the exact effects depended on the dosage and the time of alcohol administration (Roehrs, 1992; Stone, 1980; Williams et al., 1983;

Zwyghuizen-Doorenbos, 1990). Effects of acute alcohol directly relates to the blood and/or brain alcohol concentration: during the ascending phase of the blood alcohol concentration curve, alcohol is mildly stimulatory, which increases physiological alertness and prolongs sleep latency; during the descending phase of the curve, alcohol is often inhibitory and can reduce sleep latency (Papineau et al., 1998). However, late afternoon drinking (6 hours prior to bedtime) has been reported to disrupt afternoon sleep

(Rouhani et al., 1989) and even cause changes in nighttime sleep patterns, such as reduced REM sleep, reduced total sleep, decreased Stage 1 sleep, and increased wakefulness, though the blood alcohol concentration at bedtime has cleared to zero

(Landolt et al., 1996). Dijk et al also reported long-lasting changes in the spectral power distribution of NREM and REM sleep 24 hours after the alcohol consumption (Dijk et al.,

1992), suggesting that the effects of alcohol may well outlast the presence of the blood alcohol content.

Acute alcohol administration has profound effects on REM sleep in particular.

Acute alcohol ingestion in healthy humans often leads to REM suppression, especially during the first half of the night or immediately following the alcohol consumption

(Gresham et al., 1963; Knowles et al., 1968; Rouhani et al., 1989), usually with a rebound of REM sleep during the second half of the night (Feige et al., 2006). Some studies also reported changes in REM sleep particularly during the second half of the night

(Kobayashi et al., 1998; Yules et al., 1967) or throughout the whole night (Yules, 1966a).

REM suppression is often evidenced by decreased REM sleep time, increased REM latency, shortened REM episode duration, reduced REM density, and increased variation of latency to the first REM episode (Feige et al., 2006; Kobayashi et al., 1998; Lobo and

Tufik, 1997; Yules, 1966b). The dosage of alcohol consumed may correlate with the length of REM sleep for the first 5 hours of sleep (Williams et al., 1983). Repeated days of acute alcohol administration may continuously decrease REM sleep up to 2 days when

REM sleep returns to baseline levels, or may even increase REM sleep for 3 days to exceed the level of controls, and the REM sleep is often elevated for the first couple days after discontinuation of the alcohol treatment (Yules, 1966a; Yules et al., 1967). Acute alcohol by intraperitoneal injections in rats also suppresses REM sleep by reducing the number of REM sleep episodes and by increasing the intervals between REM sleep

(Mendelson and Hill, 1978).

In non-alcoholic subjects, acute alcohol often reduces sleep latency and increases

SW sleep in the first half of the night with a rebound in the second half of the night

(Feige, 2006). A low dose of alcohol in the night also increases total sleep time and improves the sleep efficiency index, but disturbs sleep patterns by decreasing SW sleep in the second half of the night and by reducing the REM/NREM ratio (Stone, 1980). In healthy subjects, acute alcohol decreases plasma testosterone, induces a surge of plasma luteinizing hormone (Mendelson et al., 1977), increases plasma prolactin (Ellingboe,

1980), and suppresses plasma growth hormone (GH) (Prinz et al., 1980), which may contribute to the effects of acute alcohol on sleep. In rats, acute alcohol has similar effects but differences exist depending on the method of alcohol administration.

Intraperitoneal injections of alcohol reduce REM sleep and increase NREM sleep in a dose-dependent manner (Mendelson, 1978). Gastric intubation of alcohol also reduces

SW sleep (Kubota, 2002). The effects of acute alcohol on sleep patterns are time- dependent: dark-onset administration decreases wake, increases SW sleep, suppresses

REM sleep, increases the time of vigilance cycling (VC) and changes the structure of

VC; light-onset administration increases wake, decreases SW sleep, suppresses REM sleep and causes fragmentation of VCs (Mukherjee, 2009).

Despite the inconsistency existing in the literature, alcohol is generally considered by the public as a sedative due to its ‘hypnotic-like’ effect. Insomnia patients often use alcoholic beverages to self-medicate as sleep aid (Ancoli-Israel and Roth, 1999; Brower,

2001a). However, alcohol use may in fact disrupt the restorative function of sleep, probably by inhibiting the parasympathetic nerve activity during sleep (Sagawa et al.,

2011). A recent study also found that low doses of alcohol did not promote sleep when administrated at an adverse circadian phase during a forced-desynchrony protocol, suggesting that some of the observed sleep-promoting effects of alcohol is very likely circadian-dependent (van Reen et al., 2011). In addition, moderate alcohol consumption before sleep can relax the upper airway dilator muscles and thus narrow the air passage, which may cause or worsen preexisting obstructive sleep apnea (OSA), especially if patients snore (Issa and Sullivan, 1982; Taasan et al., 1981).

B. Chronic Alcohol and Sleep

The effects of chronic alcohol consumption are different from those of acute alcohol administration. Human alcoholics often have an alcohol-consuming history of years or decades. Much research has focused on sleep patterns of abstinent alcoholics and the effects of withdrawal on their sleep. The amount of SW sleep of alcoholics varies from 0.7 to 44 percent, and the effect of alcohol administration on their SW sleep depends on their baseline SW sleep amount (Gross and Hastey, 1975). After spontaneous drinking, the sleep onset is fast, but the sleep duration is short and the sleep is mostly

NREM sleep (Roehrs, 2001). The sleep of alcoholics is fragmented and tends to distribute across the day (Mello, 1970). In recent abstinent alcoholics, insomnia is a common withdrawal symptom. Prolonged sleep latency, decreased total sleep time, decreased sleep efficiency index, decreased SW sleep, excessive REM sleep, decreased

REM sleep latency and change of the distribution of REMS have been reported (Brower,

2001b; Brower, 1998; Gillin, 1990; Imatoh, 1986; Johnson, 1970; Snyder, 1985;

Williams, 1981). In addition, sleep disturbances often persist for years after abstinence.

The fragmentation of sleep may last 1 to 2 years (Brower, 2001b). The sleep latency recovers after 5 to 9 months, and the total sleep time recovers after 1 to 2 years (Adamson and Burdick, 1973; Williams, 1981). Change in SW sleep may persist for 1 to 4 years

(Adamson and Burdick, 1973; Drummond, 1998). The abnormality of REM sleep usually persists longer, and the time to recover varies among different studies (Adamson and

Burdick, 1973; Williams, 1981).

In rats, low levels of alcohol increase SW sleep in the dark period without affecting REM sleep, while relatively high levels of alcohol affect both REＭ and SW

15 sleep in both light and dark periods (Kubota, 2002). Chronic alcohol increases NREM sleep, reduces the circadian variation of REM sleep, changes the distribution and the variation of amplitude of SW and REM sleep across the day and causes fragmentation of

VCs (Mukherjee, 2008; Mukherjee, 2009). However, after weeks of recovery, the sleep patterns of alcohol-treated rats usually return to baseline. Chronic alcohol is usually administrated to rats through drinking water, liquid diet or gastric intubation. Ethanol vapor is another chronic alcohol administration method, which can maintain a relatively high blood alcohol level for a relatively long period of time. In the ethanol vapor method, animals are housed in sealed chambers filled with ethanol vapor for a certain period of time each day (usually several hours each day). Ethanol vapor reduces the overall EEG spectral power and the power in the delta, theta, and beta frequencies, and this effect persists after five weeks of withdrawal (Ehlers and Slawecki, 2000).

C. Sleep Disturbances and Relapse

Sleep disturbances may predict relapse among alcoholic patients. Alcoholics with insomnia are more likely to relapse compared to alcoholics without insomnia (Gillin,

1994). The severity of sleep disturbances, especially of insomnia, sleep fragmentation, and REM sleep disruptions during withdrawal and early abstinence has been shown to predict relapse later in sobriety (Brower et al., 1998; Drummond et al., 1998; Gillin et al.,

1994). Many studies also indicate that sleep problems actually contribute to the initial development and the maintenance of alcohol use problems (Crum et al., 2004; Roehrs and Roth, 2015). In addition, due to the sedative-like, sleep-promoting effects of acute alcohol ingestion, alcohol is often reported to be used as a sleep aid in self-medication

16 when experiencing insomnia (Brower et al., 2001). According to Brower and colleagues, the relationship between alcohol and sleep disturbances is reciprocal (Brower, 2003): sleep disturbances may contribute to the initial and/or recurrent alcohol use, potentially by increasing the general risk of substance abuse, or by encouraging self-medication with alcohol; chronic alcohol use can lead to the development of alcohol use disorder, which in turn may cause new or exacerbate any preexisting sleep conditions; sleep disturbances often persist during abstinence, even up to several years after patients stop drinking; the persistent sleep disturbances in abstinence may lead to relapse to alcohol use possibly by increasing stress in life in general or by self-medication. It is thus important to understand the underlying mechanisms of sleep problems associated with AUD, which has the potential to contribute to the development of new or alternative therapeutic innervations to help maintain sobriety in AUD patients.

D. Treatment Options for Sleep Disturbances in AUD

Current treatment options for sleep problems in AUD are very limited. Common pharmacological treatment of insomnia may involve the use of cross-dependent sedatives such as benzodiazepine receptor agonists, and physicians are often reluctant to prescribe to patients with a history of substance abuse (Roth, 1995). Other medications, such as certain anti-convulsants and anti-depressants have also been explored in their potential to improve sleep in alcoholic patients, but the results are often inconsistent among studies and the safety and effectiveness of the long-term use of these drugs in cases of sleep disturbances need to be determined (Arnedt et al., 2007a). Thus, non-medication treatments and alternative therapy to correct sleep disturbances could be particularly

17 beneficial for patients with AUD. Cognitive-behavioral treatment is now commonly used to treat insomnia and to help maintain sobriety in patients with AUD, but whether its impact on improving sleep has any influence on continued abstinence remains to be further studied (Arnedt et al., 2007b). In the last decades, many advancements have been made in understanding sleep and circadian rhythms and new circadian medication is becoming more and more available (Hardeland, 2009; Lack and Wright, 2007;

Reynoldson et al., 2008). The potential application of chronotherapy to treat sleep disorders in AUD is promising. For example, melatonin agonists, which have been used to treat circadian sleep disorders especially in blind people, may be beneficial to patients with AUD (Turek and Gillette, 2004). Bright light therapy has also been tested to help alcoholics in acute withdrawal, although its actual clinical values as a treatment of AUD were unclear (Schmitz et al., 1997). However, in this study, the light therapy was only applied during acute withdrawal and whether it is also effective in helping maintain long- term abstinence is untested.

E. Possible Mechanisms

Many sleep-regulating neural circuits and neurotransmitters can be affected by alcohol consumption and may likely play a role in disturbing sleep to a certain degree.

The exact mechanism by which alcohol affects sleep is still not fully understood. The inhibitory effect of acute alcohol on REM sleep may be due to the reduction of acetylcholine (Valenzuela, 1997). The increased sleep latency and decreased total sleep time caused by alcohol administration to alcoholics may be mediated by the norepinephrine system (Allan, 1980). Administration of GABA agonist during abstinence

18 can improve sleep in rats, suggesting that the GABA system may be responsible for the persistent sleep disturbances after abstinence (Rouhani, 1998). Similar effects of GABA agonists on increasing total sleep time are also found in humans (Aubin, 1994). The decrease of SW sleep may be a result of the cerebral atrophy caused by chronic alcohol consumption as SW sleep improves when the atrophy recovers (Ishibashi, 1987). Alcohol also influences many sleep-regulating factors, including the growth-hormone-releasing- hormone (GHRH) (Emanuele, 1988), melatonin (Kuhlwein, 2003) and the corticotrophin- releasing factor (CRF) (Pich, 1995; Olive, 2002), which may all contribute to the sleep disturbances after chronic alcohol consumption. Knapp et al. suspects that chronic alcohol exposure leads to sleep-wake disturbances by increasing NMDA and kainate receptor sensitivity and by decreasing GABAB receptor sensitivity of cholinergic neurons in the pedunculopontine tegmentum, but actual experimental evidence to support this idea is still lacking (Knapp et al., 2014). In summary, due to the fact that alcohol affects multiple neural systems and transmitters, there is not likely one single mechanism that can explain all the observed sleep disturbances after chronic alcohol use. In fact, many alcohol-related problems can be system disorders. Thus, we believe it is very important and more plausible to use the fundamental concepts, such as the two-process model of sleep regulation discussed in the following chapter, to help us understand sleep problems in AUD, rather than trying to pinpoint one nucleus or a neural transmitter in the brain.

Sleep and Sleep Regulation

Sleep is a self-regulating state, characterized by decreased voluntary motor activity, decreased response to stimulation (or increased stimulation threshold), and stereotypic postures. Sleep is different from other unconscious states such as coma because sleep is easily reversible. Total sleep time, sleep patterns and sleep characteristics differ among species. Despite the fact that during sleep the vulnerability to predation increases, sleep has been preserved during evolution (Siegel, 2005). Sleep plays an important role in maintaining normal cognitive and physical functions in humans.

Sleep deprivation decreases daytime performance, impairs brain functions such as cognition, learning and memory, and has various negative effects on major physiological processes, including the immune system, the endocrine system, and processes involved in energy metabolism (Diekelmann and Born, 2010; Dworak et al., 2010; Moldofsky et al.,

1989; Redwine et al., 2000; Wulff et al., 2010). Severe sleep deprivation leads to death in rats (Everson et al., 1989). However, in certain species, such as in whales and dolphins, sleep deprivation seems to only have minor effects, possibly due to the fact that their brains are able to enter uni-hemispheric local sleep (Siegel, 2008). Although the importance and necessity of sleep are widely appreciated, the precise mechanisms by which sleep affects other physiological processes remains to be further studied.

A. Sleep Measurements

Several electrophysiological measurements are commonly used to monitor sleep: electroencephalography (EEG), electromyography (EMG), and electrooculography

(EOG). EEG measures voltage changes between two positions on the scalp (in human subjects) or near surface of the cerebral cortex (in animals), which reflects the integrated neuronal activity of cortical neurons. During active wakefulness, EGG mainly contains high-frequency and low-amplitude beta waves (15-30 Hz), as a result of active and desynchronized neuronal activities from various cortical regions for different cognitive functions. During resting wakefulness, EGG mainly contains low-amplitude alpha waves

(8-12 Hz). EGG of sleep is different from that of wakefulness, which helps distinguish them from each other. EMG monitors voltage differences between two positions on a certain skeletal muscle, which represents the activity of muscle fibers as a result of movements or changing of forces. In addition, electrooculography (EOG) can measure the resting potential of the retina, which represents eye movements. These measurements help better describe, characterize and further classify sleep into different stages.

B. Sleep Stages

Sleep is not a uniform process and can be categorized into two major states: rapid- eye-movement (REM) sleep and non-REM (NREM) sleep. REM sleep is characterized by rapid eye movements while NREM sleep has no rapid eye movements. In humans,

NREM can be further classified into four stages based on their different characteristics in

EEG recordings. Stage 1 represents the transition from wakefulness to NREM sleep and contains low-amplitude and mixed-frequency waves. Stage 2 is characterized by sleep spindles and K complexes. Stage 3 and 4 contain low-frequency and high-amplitude delta waves (0.5-2 Hz) and thus are often called slow-wave (SW) sleep. Delta waves reflect the synchronized neuronal activity of cortical neurons during deep sleep. During REM sleep,

21 neuronal activities resemble those during Stage 1 of NREM sleep (in humans) or those during wakefulness (in rodents), but nearly all skeletal muscle tone is lost. As a result,

EMG signals will be minimal during REM sleep. Most dreams occur during REM sleep, but the function of dreams is still not well understood. During NREM sleep, neuronal activity, brain temperature, metabolic rate, heart rate and blood pressure are low, but muscle tone and reflex are intact.

Sleep is not a simple process. During sleep, REM and NREM sleep cycle in a very organized manner. Within NREM sleep, stages also alternate cyclically. The pattern that wakefulness, REM sleep, and NREM sleep cycle forms the species-unique sleep structure or architecture. Chronotype and gender contribute to differences in sleep patterns, and sleep patterns change with aging (Lehnkering and Siegmund, 2007; Wolkove et al.,

2007).

C. Rodent Sleep vs. Human Sleep

A lot of animal models are used to study sleep, including rodents, cats, and monkeys. There are several important differences between human sleep and rodent sleep patterns, although rodents are the most commonly used animal model in sleep research.

First of all, humans and non-human primates are monophasic sleepers. They sleep for a relatively long period of time, usually several hours, each day. On the other hand, rodents and cats are polyphasic sleepers, and they sleep (or nap) many times during a day.

Moreover, humans are nocturnal sleepers so most of the sleep is during nighttime, while rodents are diurnal sleepers and they sleep more in the light period. Finally, the length of

REM-NREM cycles increases with brain size across species (Datta and Maclean, 2007).

Thus, rodent REM-NREM cycles are much shorter than those of humans. During each sleep episode, rats cycle rapidly through wakefulness, NREM sleep, and REM sleep within several minutes, while humans usually only have 4 to 6 cycles throughout the night. A vigilance cycle of rats is described as a period of time, during which rats cycle rapidly through wakefulness, NREM and REM without being interrupted by any long- duration wakefulness, e.g. more than 5 minutes of wakefulness (Simasko and Mukherjee,

2009). The sleep structure of rats within a vigilance cycle appears to be an analog to that of humans during the night.

D. Sleep Regulation

Sleep is a complicated process that relies on various regulatory inputs. Despite many case reports of head trauma and brain injury so far, no patients have experienced total loss of sleep, which suggests that sleep is most likely not the function of a single nucleus.

In fact, many brain circuits and neurotransmitters have been proposed to play a role in the regulation of sleep. Some of these circuits have been shown to play critical roles as evidenced by lesion studies in animal models via various methods. Sleep-promoting neural circuits, especially the ventrolateral preoptic nucleus (VLPO) neurons, and wake- promoting networks, mainly the ascending arousal systems, form a mutually inhibiting

“flip-flop” switch that gates the transitions between sleep and wake (Saper et al., 2010).

The transition between NREMS and REMS is likely modulated in a similar manner by

REMS-on and REMS-off neurons (Fuller et al., 2006). In addition, recent findings also suggest that sleep is a property of individual neural assemblies and is regulated within local circuits in a use-dependent manner (Krueger et al., 2008).

E. Two-Process Model of Sleep Regulation

Based on the classic two-process model of sleep regulation proposed by Borbely in the early 1980s, the timing of sleep-wake cycles is a result of the interaction of two major sleep-regulating processes: a homeostatic process and a circadian process (Borbély,

1982). The homeostatic process (process S) is a sleep-dependent process which represents the sleep pressure or sleep drive that builds up during waking and reduces during sleep. The circadian process (process C) is a sleep-independent process which represents the control from the circadian clocks that receive information from the external environment and integrates sleep timing with external cues. Process C defines the high

(H) and low (L) thresholds of process S to determine the onset and the termination of sleep. The periodicity of sleep-wake cycles remains to be near 24 hours even in a forced desynchrony protocol (Dijk and Czeisler, 1995). Later, Edgar and colleagues proposed an opponent-process model to further describe the relationship between process S and process C (Edgar et al., 1993). In the opponent-process model, process C plays an active role in initiating and maintaining wakefulness and in opposing sleep propensity. There are also various more complicated interpretations about the relationship between process

S and process C. Recently, Achermann and Borbely modified the two-process model and proposed a mathematical model to explain processes underlying sleep regulation. This new model includes process S, process C, and an ultradian process which occurs during sleep and represents the alternation of REM and NREM sleep (Achermann and Borbély,

2003). Despite various refinements and interpretations of the two-process model, process

S and process C are well accepted as the two major components of sleep regulation. The two-process model may be an over-simplified model because there are also many other

24 factors besides process S or C that influence sleep, such as cognition, stress, and pain, and the two-process model assumes these factors to influence sleep by affecting thresholds of process S (Beersma, 2003). Models which consider sleep-affecting variables as major contributors are much more complicated and remain to be further developed and tested .

Process S represents a homeostatic need for sleep, which accumulates during wakefulness and discharges during sleep. After a perturbation such as sleep deprivation, process S will try to return the system to a set point by compensating extra recovery sleep in theory. In most studies, the NREM-REM cycle is taken as a whole unit in the analysis of sleep intensity. The sleep intensity is often represented by the spectral power of delta waves during NREM sleep. However, REM sleep and NREM sleep may have separate homeostatic regulation mechanisms. The mechanisms underlying process S and structures and substances involved are still being explored. Adenosine has been proposed to be critical in process S (Landolt, 2008). Adenosine accumulates during wakefulness and suppresses the wake-promoting circuitry in the basal forebrain and activates the sleep-promoting circuitry (Saper et al., 2005b). Injection of adenosine or adenosine agonist promotes sleep while injection of adenosine antagonist decreases sleep and promotes wakefulness (Porkka-Heiskanen et al., 2002). Other potential mediators of sleep homeostasis include sleep-regulating factors such as nitric oxide, prostaglandin D2, and cytokines such as interleukin-1 and interleukin-6 (Kapás et al., 1994; Krueger et al.,

2001; Ueno et al., 1983). However, mechanisms involving these factors are less studied and not very well understood.

Process C represents a circadian contribution to sleep regulation. The role of circadian rhythms in regulating sleep has been studied in various protocols, and process C is found to play an important role in spontaneous sleep onset, sleep duration, sleep propensity and sleep patterns (Mistlberger, 2005; Saper et al., 2005a). The circadian regulation of sleep will be discussed in more details under the last section of this chapter.

Circadian Rhythms

Circadian rhythms play an indispensable role in preparing mammals to anticipate environmental changes and to optimize their physiological function. One of the key functions of the circadian system is to synchronize internal processes with the external light/dark cycle. However, even under conditions when the external light cues would have been removed, such as in constant darkness, the circadian system in mammals would still remain its normal functioning to coordinate different physiological processes.

A. Molecular Clocks and Circadian Genes

Core clock genes, such as Period, Clock, Bmal1 and Cryptochrome, constitute the molecular clock. Many other clock genes also help facilitate and complete the sophisticated function of the circadian system. The translational products of these genes are often transcription factors that bind to the promoter region of other genes. Clock and

Bmal1 form a heterodimer to bind to the promoter region of genes including Period and

Cryptochrome. Period and Cryptochrome then form another heterodimer after they are translated, and Per:Cry complex represses the action of Clock:Bmal1 complex as a negative feedback mechanism. Clock:Bmal1 complex also activates Rev-erb α and Rorα, which in turn modulates the transcription of Bmal1 both negatively and positively. The self-sustained transcription-translation feedback loop of these core clock genes takes approximately 24 hours, setting the periodicity of internal rhythms (King and Takahashi,

2000; Ko and Takahashi, 2006; Moore, 2013). It is also possible for clock genes to function through transcription-independent pathways to modulate cellular components,

27 but the exact mechanism and significance of these pathways remain to be further determined.

B. The Suprachiasmatic Nucleus (SCN)

Core clock genes, such as Period, Clock, Bmal1 and Cryptochrome, and their corresponding proteins constitute the molecular clock in the central pacemaker- the suprachiasmatic nucleus (SCN). The SCN is located in the anterior hypothalamus and is superior to the optic chiasm. As the master clock, the SCN is responsible for receiving environmental light-dark cues and serving as a master oscillator to entrain various physiological processes with circadian patterns such as the endocrine system and sleep/wake cycle. Pacemakers in the SCN receive the external light-dark cues and then reset and synchronize themselves every day, leading to the normal 24-hour periodicity.

When under constant conditions (constant light or dark), the clock at the SCN plays an important role in maintaining endogenous rhythms, although the periodicity is a little different from 24 hours, which is determined by the intrinsic periodicity of the molecular clocks. Electrical or physical lesions to the SCN can abolish rhythms of various behaviors and physiological processes, including free locomotion activity, water-drinking behavior, wheel running behavior, hormone secretion, body and brain temperature, and sleep

(Edgar et al., 1993; Wollnik and Turek, 1989).

C. Interaction between Alcohol, Sleep, and Circadian Rhythms

The circadian clocks from the SCN are important to maintain normal sleep-wake cycles. SCN-lesion has various effects on sleep, depending on the species. SCN-lesioned

28 squirrel monkeys have increased total sleep time, loss of sleep-wake consolidation and decreased sleep latencies (Edgar, 1993). On the other hand, in rats, SCN-lesion eliminates the sleep-wake rhythm throughout the day and flattens the distribution of sleep and wake, but does not affect total sleep time, or the time of REM and NREM sleep (Mistlberger,

1987). In some strains of mice, such as C57B1/6j mice, SCN-lesion increases total sleep time (Easton, 2004). In humans, lesions around the SCN cause severe sleep disruptions

(Cohan, 1991). The effect of SCN-lesion on sleep may be through directly affecting the endogenous pacemakers and clock genes at the SCN. The knockout or mutation of different circadian clock genes also has various effects on sleep. The Clock-mutant mouse has decreased total sleep time and less increase of REM sleep during recovery after sleep deprivation than wild type (Naylor, 2000). The cryptochrome-knockout mouse has increased NREM sleep and also less rebound of REM sleep during recovery (Wisor,

2002). The Bmal1-mutant mouse has sleep fragmentation and increased total sleep time

(Laposky, 2005). However, the Period-knockout mouse has normal total, NREM and

REM sleep time (Kopp, 2002; Shiromani, 2004). At the same time, the effect of SCN lesion on sleep may also be indirect by affecting secondary circadian oscillators or downstream systems which receive regulatory inputs from the SCN. The SCN projects to the wake-promoting circuitry in the basal forebrain and to the mesopontine tegmental nuclei which regulates REM generation (McCarley, 1992). The synthesis of sleep regulating hormones, such as hypocretin and melatonin, has strong circadian patterns, and

SCN lesion abolishes these normal rhythms and affects sleep (Perreau-Lenz, 2003;

Deboer. 2004).

Chronic alcohol use in humans can lead to profound circadian disruptions on both the physiological and molecular level. Various abnormalities in sleep/wake cycle and diurnal rhythms of blood pressure, body temperature, and hormone secretion have been observed in AUD patients (Imatoh et al., 1986; Kumagai et al., 1992; Wasielewski and

Holloway, 2001). The acrophase, or the time of the peak, of REMS in AUD patients is phase-advanced by several hours (Imatoh et al., 1986). Although inconsistency exists in the exact extent in which chronic alcohol use affects the rhythms of hormones, AUD patients tend to have normal, elevated, phase-shifted, or blunted cortisol rhythms, blunted diurnal testosterone levels, and normal or inverted melatonin rhythms (Bertello et al.,

1982; Majumdar and Miles, 1987; Murialdo et al., 1991; Stokes, 1973). The diurnal rhythms of plasma monoamine metabolites are also phase-advanced and elevated in AUD patients with severe withdrawal symptoms (Sano et al., 1992; Sano et al., 1993). On the molecular level, Huang and colleagues recently found reduced and blunted mRNA expression of several key clock genes, including Per, Bmal, and Cry, in the peripheral blood mononuclear cells of patients in withdrawal (Huang et al., 2010). McCarthy et al. also suggest that the periodicity of Per2 expression in cultured skin fibroblast cells from alcoholic patients inversely correlates with the severity of AUD (McCarthy et al., 2013).

Animal studies have revealed that chronic alcohol exposure could attenuate responses to photic and non-photic phase shifting, alter the periodicity of wheel-running behaviors under constant conditions, and reduce locomotor activities in the dark (active) period, with no effects on the entrainment to the normal LD cycle (Brager et al., 2010;

Rosenwasser et al., 2005b; Ruby et al., 2009; Seggio et al., 2009; Seggio et al., 2007). In mice, chronic alcohol reduces photic phase delays when facing phase-shifting challenges

30 and shortens the periodicity of wheel-running behaviors (Seggio et al., 2009). Similar shortening of the periodicity is also found in neonatal rats and Syrian hamsters (Dwyer and Rosenwasser, 1998; Mistlberger and Nadeau, 1992). In some other studies, both lengthening and shortening of the periodicity of wheel-running behaviors are observed

(Rosenwasser et al., 2005a), indicating that the effect of alcohol depends on the species, strain, and the method and dosage of the alcohol administration. The rhythms of drinking behavior are different between the AA (Alko Alcohol Preferring) and ANA (Alko Non-

Alcohol Preferring) rat lines, but the mechanistic explanation of this difference in phenotype is unclear (Aalto, 1986).

Whether chronic alcohol exerts its action directly at molecular clocks in the SCN is inconclusive. Chen and colleagues reported that chronic alcohol reduced the amplitude of rhythmic mRNA expression of rPeriod2 and abolished rhythmic rPeriod3 mRNA in the

SCN (Chen et al., 2004). On the contrary, Filiano and Zhou et al. found that chronic alcohol altered the diurnal expression of clock genes and clock-controlled genes associated with metabolism specifically in the liver but only slightly, if at all, in the SCN

(Filiano et al., 2013; Zhou et al., 2014).

In summary, the circadian systems are vital in regulating the sleep/wake cycle, and chronic alcohol exposure can lead to many significant circadian and sleep disruptions.

However, whether these alcohol-induced sleep and circadian disruptions share similar mechanisms or chronic alcohol disturbs sleep and circadian rhythms via independent pathways is unclear.

References

Aalto J (1986) Circadian drinking rhythms and blood alcohol levels in two rat lines developed for their alcohol consumption. Alcohol 3 (1): 73-75.

Achermann P and Borbély AA (2003) Mathematical models of sleep regulation. Front Bioscience, 8 Cited April 25 Cited April 25, 2003.

Adamson J and Burdick JA (1973) Sleep of dry alcoholics. Arch Gen Psychiatry 28 (1): 146-149.

Adinoff B, Ruether K, Krebaum S, Iranmanesh A and Williams MJ (2003) Increased salivary cortisol concentrations during chronic alcohol intoxication in a naturalistic clinical sample of men. Alcoholism: Clinical and Experimental Research 27 (9): 1420-1427.

Aldrich MS (1999) Neurobiology of Sleep. Sleep Medicine. New York: Oxford University Press.

Ancoli-Israel S and Roth T (1999) Characteristics of insomnia in the United States: results of the 1991 National Sleep Foundation Survey. I. Sleep 22 S347-53.

Arnedt JT, Conroy DA and Brower KJ (2007a) Treatment options for sleep disturbances during alcohol recovery. Journal of addictive diseases 26 (4): 41-54.

Arnedt JT, Conroy D, Rutt J, Aloia MS, Brower KJ and Armitage R (2007b) An open trial of cognitive-behavioral treatment for insomnia comorbid with alcohol dependence. Sleep Medicine 8 (2): 176-180.

Balsalobre A (2002) Clock genes in mammalian peripheral tissues. Cell and Tissue Research 309 (1): 193-199.

Baraona E and Lieber CS (1998) Alcohol and lipids. Recent Developments in Alcoholism 14 (1): 97-134.

Becker HC (1999) Alcohol withdrawal: neuroadaptation and sensitization. CNS Spectrums 4 (1): 38-40.

Beersma DGM (2003) Models of human sleep regulation. Sleep

Bertello P, Agrimonti F, Gurioli L, Frairia R, Fornaro D and Angeli A (1982) Circadian patterns of plasma cortisol and testosterone in chronic male alcoholics. Alcoholism: Clinical and Experimental Research 6 (4): 475-481.

Blum RH and Blum EM (1969) A cultural case study in drugs: society and drugs.

Boileau I, Assaad JM, Pihl RO, Benkelfat C, Leyton M, Diksic M and Dagher A (2003) Alcohol promotes dopamine release in the human nucleus accumbens. Synapse 49 (4): 226-231.

Borbély AA (1982) A two process model of sleep regulation. Human neurobiology.

Brager AJ, Ruby CL, Prosser RA and Glass JD (2010) Chronic ethanol disrupts circadian photic entrainment and daily locomotor activity in the mouse. Alcoholism: Clinical and Experimental Research 34 (7): 1266-1273.

Brower KJ, Aldrich MS and Hall JM (1998) Polysomnographic and subjective sleep predictors of alcoholic relapse. Alcoholism: Clinical and Experimental Research 22 (8): 1864-1871.

Brower KJ, Aldrich MS, Robinson EA, Zucker RA and Greden JF (2001) Insomnia,self- medication,and relapse to alcoholism. American Journal of Psychiatry 158 (3): 399-404.

Brower KJ (2003a) Insomnia, alcoholism and relapse. Sleep Medicine Reviews. (6): 523- 39.

Brower KJ, Aldrich MS and Hall JM (1998) Polysomnographic and subjective sleep predictors of alcohol relapse. Alcoholism: Clinical and Experimental Research (22): 1864-1871.

Brower KJ, Aldrich MS, Robinson EA, Zucker RA and Greden JF (2001a) Insomnia, self-medication, and relapse to alcoholism. American Journal of Psychiatry (158): 399-404.

Brower KJ (2001b) Alcohol's Effects on Sleep in Alcoholics. Alcohol Research & Health 25 (2): 110-125.

Brower KJ (2003b) Insomnia, alcoholism and relapse. Sleep Medicine Reviews 7 (6): 523-539.

Bujanda L (2000) The effects of alcohol consumption upon the gastrointestinal tract. The American journal of gastroenterology 95 (12): 3374-3382.

CDC (2006) Excessive Drinking Costs U.S. $223.5 Billion [Online]. Centers for Disease Control and Prevention (CDC). Available: http://www.cdc.gov/features/alcoholconsumption/.

Chastain G (2006) Alcohol, neurotransmitter systems, and behavior. The Journal of general psychology 133 (4): 329-35.

Chen CP, Kuhn P, Advis JP and Sarkar DK (2004) Chronic ethanol consumption impairs the circadian rhythm of pro-opiomelanocortin and period genes mRNA expression in the hypothalamus of the male rat. Journal of Neurochemistry 88 (6): 1547-1554.

Chen SH, Wang JW and Li YM (2010) Is alcohol consumption associated with gastroesophageal reflux disease? Journal of Zhejiang University - Science B 11 (6): 423-428.

Cohn TJ, Foster JH and Peters TJ (2003) Sequential studies of sleep disturbance and quality of life in abstaining alcoholics. Addiction biology 8 (4): 455-462.

Colrain IM, Nicholas CL and Baker FC (2014) Alcohol and the sleeping brain. Handbook of clinical neurology 125: 415-431.

Crum RM, Storr CL, Chan YF and Ford DE (2004) Sleep disturbance and risk for alcohol-related problems. American Journal of Psychiatry 161 (7): 1197-1203.

Datta S and Maclean RR (2007) Neurobiological mechanisms for the regulation of mammalian sleep-wake behavior: reinterpretation of historical evidence and inclusion of contemporary cellular and molecular evidence. Neuroscience & Biobehavioral Reviews 31 (5): 775-824.

Diekelmann S and Born J (2010) The memory function of sleep. Nature Reviews Neuroscience 11 (2): 114-126.

Dijk DJ and Czeisler CA (1995) Contribution of the circadian pacemaker and the sleep homeostat to sleep propensity, sleep structure, electroencephalographic slow waves, and sleep spindle activity in humans. The Journal of neuroscience 15 (5): 3526-3538.

Dijk DJ, Brunner DP, Aeschbach D, Tobler I and Borbely AA (1992) The effects of ethanol on human sleep EEG power spectra differ from those of benzodiazepine receptor agonists. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 7 (3): 225-232.

Dinwiddie SH and Reich T (1993) Genetic and family studies in psychiatric illness and alcohol and drug dependence. Journal of addictive diseases 12 (3): 17-27.

Drummond S, Gillin JC, Smith TL and DeModena A (1998) The sleep of abstinent pure primary alcoholic patients: natural course and relationship to relapse. Alcoholism: Clinical and Experimental Research 22 (8): 1796-1802.

Dworak M, McCarley RW, Kim T, Kalinchuk AV and Basheer R (2010) Sleep and brain energy levels: ATP changes during sleep. Journal of Neuroscience 30 (26): 9007- 16.

Dwyer SM and Rosenwasser AM (1998) Neonatal clomipramine treatment, alcohol intake and circadian rhythms in rats. Psychopharmacology (Berl) 138 (2): 176- 183. Edgar DM, Dement WC and Fuller CA (1993) Effect of SCN lesions on sleep in squirrel monkeys: evidence for opponent processes in sleep-wake regulation. The Journal of Neuroscience 13 (3): 1065-1079.

Ehlers CL and Slawecki CJ (2000) Effects of chronic ethanol exposure on sleep in rats. Alcohol 20 (2): 173-179.

Ellingboe J, Mendelson JH, Kuehnle JC, Skupny AST and Miller KD (1980) Effect of acute ethanol ingestion on integrated plasma prolactin levels in normal men. Pharmacology Biochemistry and Behavior 12 (2): 297-310.

Ettinger PO, Wu CF, De La Cruz C Jr, Weisse AB, Ahmed SS and Regan TJ (1978) Arrhythmias and the "Holiday Heart": Alcoholassociated cardiac rhythm disorders. American Heart Journal 95 (5): 555-562.

Everson CA, Bergmann BM and Rechtschaffen A (1989) Sleep deprivation in the rat: III. Total sleep deprivation. Sleep 12 (1): 13-21.

Fadda F and Rossetti ZL (1998) Chronic ethanol consumption: from neuroadaptation to neurodegeneration. Progress in neurobiology 56 (4): 385-431.

Fauchier L (2003) Alcoholic cardiomyopathy and ventricular arrhythmias. CHEST Journal 123 (4): 1320.

Feige B, Gann H, Brueck R, Hornyak M, Litsch S, Hohagen F and Riemann D (2006) Effects of alcohol on polysomnographically recorded sleep in healthy subjects. Alcoholism: Clinical and Experimental Research 30 (9): 1527-37.

Filiano AN, Millender-Swain T, Johnson R Jr., Young ME, Gamble KL and Bailey SM (2013) Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus. PLOS ONE 8 (8): e71684.

Forbes RJ (1970) A short history of the art of distillation: from the beginnings up to the death of Cellier Blumenthal, BRILL.

Foster JH, Marshall EJ, Hooper R and Peters TJ (1988) Quality of life measures in alcohol dependent subjects and changes with abstinence and continued heavy drinking. Addiction Biology 3 321-332.

French HV (1890) Nineteen Centuries of Drink in England: A History, London, National Temperance Publication Depot.

Fuller PM, Gooley JJ and Saper CB (2006) Neurobiology of the sleep-wake cycle: sleep architecture, circadian regulation, and regulatory feedback. Journal of Biological Rhythms 21 (6): 482-93. Garver E, Tu GC, Cao QN, Aini M, Zhou F and Israel Y (2001) Eliciting the low-activity aldehyde dehydrogenase Asian phenotype by an antisense mechanism results in an aversion to ethanol. The Journal of experimental medicine 194 (5): 571-580.

Gillin JC (1998) Are sleep disturbances risk factors for anxiety, depressive and addictive disorders? Acta Psychiatrica Scandinavica 98 (S393): 39-43.

Gillin JC, Smith TL, Irwin M, Butters N, Demodena A and Schuckit M (1994) Increased pressure for rapid eye movement sleep at time of hospital admission predicts relapse in nondepressed patients with primary alcoholism at 3-month follow-up. Archives of General Psychiatry 51 (3): 189-197.

Gillin JC, Smith TL, Irwin M, Kripke DF and Schuckit M (1990) EEG sleep studies in 'pure' primary alcoholism during subacute withdrawal: Relationships to normal controls, age, and other clinical variables Biological Psychiatry 27 (5): 477-88.

Goldman D, Oroszi G and Ducci F (2005) The genetics of addictions: uncovering the genes. Nature Reviews Genetics 6 521-532.

Gresham SC, Webb WB and Williams RL (1963) Alcohol and caffeine: effect on inferred visual dreaming. Science 140 (3572): 1226-1227.

Grewal P and Viswanathen VA (2012) Liver cancer and alcohol. Clinics in liver disease 16 (4): 839-850.

Gross MM and Hastey JM (1975) The relation between baseline slow wave sleep and the slow wave sleep response to alcohol in alcoholics. Advances in Experimental Medicine and Biology. (59): 467-75.

Hardeland R (2009) Tasimelteon, a melatonin agonist for the treatment of insomnia and circadian rhythm sleep disorders. Current opinion in investigational drugs (London, England: 2000) 10 (7): 691-701.

Hawley RJ, Nemeroff CB, Bissette G, Guidotti A, Rawlings R, Linnoila M (1994) Neurochemical correlates of sympathetic activation during severe alcohol withdrawal. Alcoholism: Clinical and Experimental Research 18 (6): 1312-1316.

Hellig M, Koob GF (2007) A key role for corticotropin-releasing factor in alcohol dependence. Trends Neurosci (30): 399-406.

Huang MC, Ho CW, Chen CH, Liu SC, Chen CC and Leu SJ (2010) Reduced expression of circadian clock genes in male alcoholic patients. Alcoholism: Clinical and Experimental Research 34 (11): 1899-1904.

Imatoh N, Nakazawa Y and Ohshima H (1986) Circadian rhythm of Rem sleep of chronic alcoholics during alcohol withdrawal. Drug and Alcohol Dependence 18 (1): 77-85. Issa FG and Sullivan CE (1982) Alcohol, snoring and sleep apnea. Journal of Neurology, Neurosurgery & Psychiatry 45 (4): 353-359

Johnson LC, Burdick JA, Smith J (1970) Sleep During Alcohol Intake and Withdrawal in the Chronic Alcoholic. Archives of General Psychiatry 22 (5): 406-418.

Kapás L, Fang J and Krueger JM (1994) Inhibition of nitric oxide synthesis inhibits rat sleep. Brain research 664 (1): 189-196.

Keshavarzian A, Holmes EW, Patel M, Iber F, Fields JZ and Pethkar S (1999) Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage. The American journal of gastroenterology 94 (1): 200-207.

King DP and Takahashi JS (2000) Molecular genetics of circadian rhythms in mammals. Annual review of neuroscience 23 (1): 713-742.

Klatsky AL, Armstrong MA, Friedman GD and Hiatt RA (1988) The relations of alcoholic beverage use to colon and rectal cancer. American journal of epidemiology 128 (5): 1007-1015.

Knapp CM, Ciraulo DA and Datta S (2014) Mechanisms underlying sleep-wake disturbances in alcoholism: Focus on the cholinergic pedunculopontine tegmentum. Behav Brain Res 274C 291-301.

Knowles JB, Laverty SG and Kuechler HA (1968) Effects on REM sleep. Quarterly Journal of Studies on Alcohol 29 (2): 342.

Ko CH and Takahashi JS (2006) Molecular components of the mammalian circadian clock. Human molecular genetics 15 (Suppl 2): R271-R277.

Kobayashi T, Misaki K, Nakagawa H, Okuda K, Ota T, Kanda I, Isaki K, Kosino Y and Fukuda H (1998) Alcohol effect on sleep electroencephalography by fast Fourier transformation. Psychiatry and Clinical Neuroscience 52 (2): 154-5.

Koob GF (2013) Neurocircuitry of alcohol addiction: synthesis from animal models. Handbook of clinical neurology 125: 33-54.

Koob GF, Le MM (2008) Addiction and the brain antireward system. Annual review of psychology 59: 29-53.

Krueger JM, Obál F, Fang J, Kubota T and Taishi P (2001) The role of cytokines in physiological sleep regulation. Annals of the New York Academy of Sciences 933 (1): 211-221.

Krueger JM, Rector DM, Roy S, Van Dongen HP, Belenky G and Panksepp J (2008) Sleep as a fundamental property of neuronal assemblies. Nature Reviews Neuroscience, 9(12): 910-919.

Kubota T, De A, Brown RA, Simasko SM and Krueger JM (2002) Diurnal Effects of Acute and Chronic Administration of Ethanol on Sleep in Rats. Alcoholism: Clinical and Experimental Research 26 (8): 1153-1161. Kumagai Y, Shiga T, Sunaga K, Fukushima C, Cornélissen G, Ebihara A and Halberg F (1992) Repeated alcohol intake changes circadian rhythm of ambulatory blood pressure. Chronobiologia 20 (1-2): 77-85.

Lack LC and Wright HR (2007) Treating chronobiological components of chronic insomnia. Sleep Medicine 8 (6): 637-644.

Landolt HP and Borbély AA (2000) Alcohol and sleep disorders. Ther Umsch. 57 (4): 241-45.

Landolt HP, Roth C, Dijk DJ and Borbely AA (1996) Late-afternoon ethanol intake affects nocturnal sleep and the sleep EEG in middle-aged men. Journal of Clinical Psychopharmacology 16 (6): 428-436.

Landolt HP (2008) Sleep homeostasis: a role for adenosine in humans? Biochemical pharmacology 75 (11): 2070-2079.

Lehnkering H and Siegmund R (2007) Influence of chronotype, season, and sex of subject on sleep behavior of young adults. Chronobiology International 24 (5): 875-888.

Li W, Li JF, Liu WM, Altura BT, Altura BM (2004) Alcohol-induced apoptosis of canine cerebral vascular smooth muscle cells: role of extracellular and intracellular calcium ions. Neuroscience Letters 354 (3).

Littleton J (1998) Neurochemical mechanisms underlying alcohol withdrawal. Alcohol Health & Research World 22 (1): 13-24.

Lobo LL and Tufik S (1997) Effects of alcohol on sleep parameters of sleep-deprived healthy volunteers. sleep 20 (1): 52-59.

Majumdar SK and Miles A (1987) Disturbed melatonin secretion in chronic alcoholism and withdrawal. Clinical Chemistry 33 (7): 1291.

Matsuo Y, Seki A (1972) Effects of alcohol on gastrin secretion. Horumon to Rinsho. Clinical Endocrinology 20 (7): 523-529.

McCarthy MJ, Fernandes M, Kranzler HR, Covault JM and Welsh DK (2013) Circadian clock period inversely correlates with illness severity in cells from patients with alcohol use disorders. Alcoholism: Clinical and Experimental Research 37 (8): 1304-10.

McGovern PE, Zhang J, Tang J, Zhang Z, Hall GR, Moreau RA, Nuñez A, Butrym ED, Richards M.P., Wang C.S. and Cheng G. (2004) Fermented beverages of pre-and proto-historic China. Proceedings of the National Academy of Sciences of the United States of America 101 (51): 17593-17598.

McGovern Patrick E (2003) Ancient Wine: The Search for the Origins of Viniculture, Princeton University Press.

Mello NK and Mendelson JK (1970) Behavioral studies of sleep patterns in alcoholics during intoxication and withdrawal. Journal of Pharmacology and Experimental Therapeutics. (175): 94-112.

Mendelson JH, Mello NK and Ellingboe J (1977) Effects of acute alcohol intake on pituitary-gonadal hormones in normal human males. JPET 202 (3): 676-682.

Mendelson WB and Hill SY (1978) Effects of the acute administration of ethanol on the sleep of the rat: a dose-response study. Pharmacology Biochemistry and Behavior 8 (6): 723-726.

Mendelson WB and Hill SY (1978) Effects of the acute administration of ethanol on the sleep of the rat: A dose-response study. Pharmacology Biochemistry and Behavior 8 (6): 723-6.

Migliori M, Pezzilli R, Tomassetti P, Gullo L (2004) Exocrine pancreatic function after alcoholic or biliary acute pancreatitis. Pancreas (28): 359-363.

Mistlberger RE (2005) Circadian regulation of sleep in mammals: role of the suprachiasmatic nucleus. Brain Research Reviews 49 (3): 429-54.

Mistlberger RE and Nadeau J (1992) Ethanol and circadian rhythms in the syrian hamster: effects on entrained phase, reentrainment rate, and period. Pharmacology Biochemistry and Behavior 43 (1): 159-165.

Moldofsky H, Lue FA, Davidson JR and Gorczynski R (1989) Effects of sleep deprivation on human immune functions. The FASEB journal : official

publication of the Federation of American Societies for Experimental Biology 3 (8): 1972-1977.

Moore RY (2013) The suprachiasmatic nucleus and the circadian timing system. Chronobiology: Biological timing in health and disease.

Mukherjee S and Simasko SM (2009) Chronic alcohol treatment in rats alters sleep by fragmenting periods of vigilance cycling in the light period with extended wakenings. Behavioural Brain Research. (198): 113-124.

Mukherjee S, Kazerooni M, Simasko SM (2008) Dose-response study of chronic alcohol induced changes in sleep patterns in rats. Brain Research. (1208): 120-127.

Murialdo G, Filippi U, Costelli P, Fonzi S, Bo P, Polleri A and Savoldi F (1991) Urine melatonin in alcoholic patients: a marker of alcohol abuse? Journal of endocrinological investigation 14 (6): 503-507.

NDATUS (1993) National drug and alcoholism treatment unit survey US Department of Health and Human Services.

NIAAA (2000) Tenth special report to the u.s. congress on alcohol and health. National Institute on Alcohol Abuse and Alcoholism.

NIAAA (2014) Alcohol Facts and Statistics. National Institute on Alcohol Abuse and Alcoholism (NIAAA)

Papineau KL, Roehrs TA, Petrucelli N, Rosenthal LD and Roth T (1998) Electrophysiological assessment (the multiple sleep latency test) of the biphasic effects of ethanol in humans. Alcoholism: Clinical and Experimental Research 22 (1): 231-235.

Perney P, Lehert P and Mason BJ (2012) Sleep disturbance in alcoholism: proposal of a simple measurement, and results from a 24-week randomized controlled study of alcohol-dependent patients assessing acamprosate efficacy. Alcohol and alcoholism 47 (2): 133-139.

Piano MR (2002) Alcoholic cardiomyopathy: incidence, clinical characteristics, and pathophysiology. CHEST 121 (5): 1638-1650.

Pohorecky LA (1978) Biphasic action of ethanol. Biobehavioral Reviews 1 (4): 231-240.

Porkka-Heiskanen T, Alanko L, Kalinchuk A and Stenberg D (2002) Adenosine and sleep. Sleep medicine reviews 6 (4): 321-332.

Prinz PN, Roehrs TA, Vitaliano PP, Linnoila M and Weitzman ED (1980) Effect of alcohol on sleep and nighttime plasma growth hormone and cortisol

concentrations*. The Journal of Clinical Endocrinology & Metabolism 51 (4): 759-764.

Redwine L, Dang J, Hall M and Irwin M (2003) Disordered sleep, nocturnal cytokines, and immunity in alcoholics. Psychosomatic medicine 65 (1): 75-85.

Redwine L, Hauger RL, Gillin JC and Irwin M (2000) Effects of sleep and sleep deprivation on interleukin-6, growth hormone, cortisol, and melatonin levels in humans 1. The Journal of Clinical Endocrinology & Metabolism 85 (10): 3597- 3603.

Reynolds K, Lewis B, Nolen JD, Kinney GL, Sathya B and He J (2003) Alcohol consumption and risk of stroke a meta-analysis. JAMA 289 (5): 579-588.

Reynoldson JN, Elliott E Sr. and Nelson LA (2008) Ramelteon: a novel approach in the treatment of insomnia. The Annals of pharmacotherapy 42 (9): 1262-71.

Richardson PJ, Wodak AD, Atkinson L, Saunders JB and Jewitt DE (1986) Relation between alcohol intake, myocardial enzyme activity, and myocardial function in dilated cardiomyopathy. Evidence for the concept of alcohol induced heart muscle disease. British heart journal. (56): 165-170.

Roehrs T and Roth T (2015) Sleep disturbance in substance use disorders. Psychiatric Clinics of North America In Press.

Roehrs T and Roth T (2001) Sleep, sleepiness, sleep disorders and alcohol use and abuse. Sleep Medicine Reviews 5 (4): 287-297.

Roehrs T, Zwyghuizen‐Doorenbos A, Knox M, Moskowitz H and Roth T (1992) Sedating effects of ethanol and time of drinking. Alcoholism: Clinical and Experimental Research 16 (3): 553-7.

Rosenwasser AM, Fecteau ME and Logan RW (2005a) Effects of ethanol intake and ethanol withdrawal on free-running circadian activity rhythms in rats. Physiology & Behavior 84 (4): 537-542.

Rosenwasser AM, Logan RW and Fecteau ME (2005b) Chronic ethanol intake alters circadian period-responses to brief light pulses in rats. Chronobiology International 22 (2): 227-236.

Rossetti ZL, Melis F, Carboni S, Diana M and Gessa GL (1992) Alcohol withdrawal in rats is associated with a marked fall in extraneuronal dopamine. Alcoholism: Clinical and Experimental Research 16 (3): 529-532.

Roth T (2007) Insomnia: definition, prevalence, etiology, and consequences. Journal of clinical sleep medicine 3 (5 Suppl): S7.

Roth T, Roehrs T and Vogel G (1995) Zolpidem in the treatment of transient insomnia: a double-blind, randomized comparison with placebo. Sleep 18 (4): 246-51.

Roueche B (1963) Alcohol in Human Culture, New York, McGraw-Hill.

Rouhani S, Tran G, Leplaideur F, Durlach J and Poenaru S (1989) EEG effects of a single low dose of ethanol on afternoon sleep in the nonalcohol-dependent adult. Alcohol 6 (1): 87-90.

Ruby CL, Brager AJ, DePaul MA, Prosser RA and Glass JD (2009) Chronic ethanol attenuates circadian photic phase resetting and alters nocturnal activity patterns in the hamster. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 297 (3): R729-R737.

Sagawa Y, Kondo H, Matsubuchi N, Takemura T, Kanayama H, Kaneko Y, Kanbayashi T, Hishikawa Y and Shimizu T (2011) Alcohol has a dose‐related effect on parasympathetic nerve activity during sleep. Alcoholism: Clinical and Experimental Research 35 (11): 2093-2100.

SAMHSA (2015) Behavioral health trends in the United States: Results from the 2014 National Survey on Drug Use and Health. The Substance Abuse and Mental Health Services Administration (SAMHSA).

Sander M, Irwin M, Sinha P, Naumann E, Kox W and Spies C (2002) Suppression of interleukin-6 to interleukin-10 ratio in chronic alcoholics: association with postoperative infections. Intensive care medicine 28 (3): 285-292.

Sano H, Suzuki Y, Ohara K, Yazaki R, Ishigaki T, Yokoyama T and Ohara K (1992) Circadian variation in plasma homovanillic acid level during and after alcohol withdrawal in alcoholic patients. Alcoholism: Clinical and Experimental Research 16 (6): 1047-1051.

Sano H, Suzuki Y, Yazaki R, Tamefusa K, Ohara K, Yokoyama T, Miyasato K and Ohara K (1993) Circadian variation in plasma 5-hydroxyindoleacetic acid level during and after alcohol withdrawal: phase advances in alcoholic patients compared with normal subjects. Acta Psychiatrica Scandinavica 87 (4): 291-296.

Saper CB, Cano G and Scammell TE (2005a) Homeostatic, circadian, and emotional regulation of sleep. Journal of Comparative Neurology 493 (1): 92-8.

Saper CB, Scammell TE and Lu J (2005b) Hypothalamic regulation of sleep and circadian rhythms. Nature 437 (7063): 1257-63.

Saper CB, Fuller PM, Pedersen NP, Lu J and Scammell TE (2010) Sleep state switching. Neuron 68 (6): 1023-1042.

Schmitz M, Frey R, Pichler P, Röpke H, Anderer P, Saletu B and Rudas S (1997) Sleep quality during alcohol withdrawal with bright light therapy. Progress in Neuro- Psychopharmacology and Biological Psychiatry 21 (6): 965-977.

Schuckit MA (1985) Studies of populations at high risk for alcoholism. Psychiatric developments 3 (1): 31-63.

Seggio JA, Fiixaris MC, Reed JD, Logan RW and Rossenwasser AM (2009) Chronic ethanol intake alters circadian phase shifting and free-running period in mice. Journal of Biological Rhythms 24 (4): 304-312.

Seggio JA, Logan RW and Rosenwasser AM (2007) Chronic ethanol intake modulates photic and non-photic circadian phase responses in the Syrian hamster. Pharmacology Biochemistry and Behavior 87 (3): 297-305.

Siegel JM (2005) Clues to the functions of mammalian sleep. Nature 437 (7063): 1264- 71.

Siegel JM (2008) Do all animals sleep? Trends Neurosci 31 (4): 208-13.

Simasko SM and Mukherjee S (2009) Novel analysis of sleep patterns in rats separates periods of vigilance cycling from long-duration wake events. Behavioural brain research 196 (2): 228-36.

Snyder S, Karacan I (1985) Sleep patterns of sober chronic alcoholics. Neuropsychobiology (13): 97-100.

Stokes PE (1973) Adrenocortical activation in alcoholics during chronic drinking. Annals of the New York Academy of Sciences 215 (1): 77-83.

Stoller MK (1994) Economic effects of insomnia. Clinical Therapeutics. 16 (5): 873-97.

Stone BM (1980) Sleep and low doses of alcohol. Electroencephalography and Clinical Neurophysiology 48 (6): 706-9.

Summa KC, Voigt RM, Forsyth CB, Shaikh M, Cavanaugh K, Tang Y, Vitaterna MH, Song S, Turek FW and Keshavarzian A (2013) Disruption of the circadian clock in mice increases intestinal permeability and promotes alcohol-induced hepatic pathology and inflammation. PLoS One 8 (6): e67102.

Taasan VC, Block AJ, Boysen PG and Wynne JW (1981) Alcohol increases sleep apnea and oxygen desaturation in asymptomatic men The American Journal of Medicine 71 (2): 240-5.

Turek FW and Gillette MU (2004) Melatonin, sleep, and circadian rhythms: rationale for development of specific melatonin agonists. Sleep medicine 5 (6): 523-532.

Ueno R, Honda K, Inoue S and Hayaishi O (1983) Prostaglandin D2, a cerebral sleep- inducing substance in rats. Proceedings of the National Academy of Sciences 80 (6): 1735-1737. van Reen E, Tarokh L, Rupp TL, Seifer R and Carskadon MA (2011) Does timing of alcohol administration affect sleep? Sleep 34 (2): 195.

Walker DW, Hunter BE (1978) Short-term memory impairment following chronic alcohol consumption in rats. Neuropsychologia 16 (5): 545-553.

Wasielewski JA and Holloway FA (2001) Alcohol's interactions with circadian rhythms. Alcohol Research & Health 25 94-100.

Weinshenker D and Schroeder JP (2007) There and back again: a tale of norepinephrine and drug addiction. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 32 (7): 1433-51.

WHO (2014) Global status report on alcohol and health World Health Organization (WHO).

Williams DL, MacLean AW and Cairns J (1983) Dose-response effects of ethanol on the sleep of young women. Journal of studies on alcohol 44 (3): 515-523.

Williams HL, Rundell OH Jr. (1981) Altered sleep physiology in chronic alcoholics: Reversal with abstinence. Alcoholism: Clinical and Experimental Research (5): 318-325.

Wolkove N, Elkholy O, Baltzan M and Palayew M (2007) Sleep and aging: 2. Management of sleep disorders in older people. Canadian Medical Association Journal 176 (10): 1449-1454.

Wollnik F and Turek FW (1989) SCN lesions abolish ultradian and circadian components of activity rhythms in LEW/Ztm rats. American Journal of Physiology- Regulatory, Integrative and Comparative Physiology 256 (5): R1027-R1039.

Wulff K, Gatti S, Wettstein JG and Foster RG (2010) Sleep and circadian rhythm disruption in psychiatric and neurodegenerative disease. Nature Reviews Neuroscience 11 (8): 589-599.

Yules RB, Freedman DX and Chandler KA (1966a) The effect of ethyl alcohol on man's electroencephalographic sleep cycle. Electroencephalography and clinical neurophysiology 20 (2): 109-111.

Yules RB, Lippman ME and Freedman DX (1967) Alcohol administration prior to sleep: the effect on EEG sleep stages. Archives of General Psychiatry 16 (1): 94-97.

Yules RB, Freedman DX and Chandler KA (1966b) The effect of ethyl alcohol on man's electroencephalographic sleep cycle. Electroencephalography and Clinical Neurophysiology 20 (2): 109-11.

Zhou P, Ross RA, Pywell CM, Liangpunsakul S and Duffield GE (2014) Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis. Scientific Reports - Nature 4 (3725): 1-11.

Zhou SL, Gordon RE, Bradbury M, Stump D, Kiang CL and Berk PD (1998) Ethanol up- regulates fatty acid uptake and plasma membrane expression and export of mitochondrial aspartate aminotransferase in HepG2 cells. Hepatology 27 (4): 1064-74.

Zwyghuizen‐Doorenbos A, Roehrs T, Timms V and Roth T (1990) Individual differences in the sedating effects of ethanol. Alcoholism: Clinical and Experimental Research 14 (3): 400-404.

CHAPTER TWO

CHRONIC ALCOHOL CONSUMPTION DELAYS AND FRAGMENTS

RECOVERY SLEEP AFTER SLEEP DEPRIVATION IN RATS *

Rong Guo, Heiko T. Jansen, and Steven M. Simasko

Programs in Neuroscience

Department of Integrative Physiology and Neuroscience

College of Veterinary Medicine

Washington State University, Pullman, Washington, USA, 99164

______* This work is to be submitted to Drug and Alcohol Dependence for review. Guo R, Jansen HT and Simasko SM (2016).

Abstract

Background: Sleep disturbances such as difficulty falling asleep and frequent awakenings are common among individuals suffering from alcohol use disorder (AUD).

However, to what extent alcohol-induced sleep disturbances are due to alterations of factors that influence homeostatic control of sleep remains unclear.

Methods: Adult male Sprague-Dawley rats were exposed to chronic alcohol via liquid diet for 10 weeks. We then probed their sleep response to a homeostatic challenge by subjecting them to 6 hours of acute sleep deprivation via gentle handling. We examined the quantity and the quality of the recovery sleep during the 24 hours after the challenge.

Results: Both control and alcohol-treated rats showed robust rebound sleep following the sleep deprivation challenge. However, alcohol-treated rats had delayed sleep onset, slightly increased total wake time over the 24 hours following the challenge, and their sleep was more fragmented. The total amount of rapid-eye movement sleep

(REMS) and non-REMS (NREMS) did not significantly differ between groups over the

24 hours following sleep deprivation. The rebound in SWA was similar in both groups.

Conclusions: Our study demonstrates that chronic alcohol consumption delays and fragments recovery sleep that follows a homeostatic challenge, but does not interfere with the animal’s ability to eventually recover lost sleep following sleep deprivation.

These results do not support the hypothesis that chronic alcohol treatment dramatically impairs the homeostatic build-up of sleep pressure during sleep deprivation.

Introduction

Sleep disturbances are common comorbidities in patients with alcohol use disorder (AUD) (Brower, 2001; Colrain et al., 2014; Knapp et al., 2014). Around 70% of people with AUD have been reported to experience sleep problems, including more than

20% with severe symptoms (Perney et al., 2012). Active alcoholics often show more night awakenings, frequent transitions among sleep stages, suppression and fragmentation of rapid eye movement sleep (REMS), and a decrease of slow wave sleep

(SWS) (Gross et al., 1973; Gross and Hastey, 1975; Johnson et al., 1970; Mello and

Mendelson, 1970). Abstinent alcoholics continue to suffer from prolonged sleep latency, poor sleep efficiency with sleep fragmentation, reduced total sleep time, decreased slow wave sleep, and increased REMS pressure (Allen et al., 1971; Colrain et al., 2014;

Colrain et al., 2009; Gann et al., 2001; Gross et al., 1973; Gross and Hastey, 1975;

Johnson et al., 1970). Poor sleep can severely impair the quality of life (Foster et al.,

1988) and lead to negative health consequences (Roth, 2007), rendering individuals with

AUD vulnerable to physiological and psychiatric challenges. Sleep problems may also contribute to the initial development and the maintenance of excessive alcohol use (Crum et al., 2004; Roehrs and Roth, 2015). The severity of sleep disturbances during withdrawal and early abstinence has been shown to predict relapse later in life (Brower et al., 1998; Drummond et al., 1998; Gillin et al., 1994). Despite decades of research, the underlying mechanisms of sleep disturbances in AUD are still not well understood.

A fruitful conceptual framework for understanding the regulation of the sleep- wake cycle is the two-process model proposed by Archermann and Borbély (Borbély,

1982), in which the timing of sleep and wake results from the interaction of a homeostatic process and a circadian process. The homeostatic process represents sleep pressure that builds up during wake and dissipates during sleep, possibly via sleep-promoting substances such as adenosine (Landolt, 2008). On the other hand, the circadian process is sleep-independent and involves circuits and factors controlled by the endogenous circadian clocks that integrate internal physiology with external environmental cues

(Mistlberger, 2005). Thus, it is possible that chronic alcohol consumption impairs sleep either through the homeostatic process, the circadian process, or both. Surprisingly, little attention has been given to the use of this framework to understand sleep disturbances in

AUD. The importance of this approach is that although the exact manner in which these two processes control sleep have yet to be fully resolved, enough is known to conclude they have some fundamental differences. Knowing which process is altered by chronic alcohol use would be helpful both in directing research into understanding the fundamental mechanisms by which chronic alcohol disrupts sleep and in possibly generating new insights for rehabilitating healthy sleep in patients with AUD.

Our prior work in this area using a rodent model suggests that chronic alcohol exposure disrupts control of sleep via the circadian process. We found that rats treated for at least 6 weeks with 6% ethanol in liquid diet had a redistribution of sleep/wake time, such that sleep (both REMS and non-REMS (NREMS)) was more equally distributed between the light and dark periods, while the total amount of wake, NREMS, and REMS

49 over a 24-hour period did not differ between control and treated rats (Mukherjee and

Simasko, 2009). These observations are consistent with a relatively intact homeostatic process of sleep regulation as indicated by the constant amount of total sleep over 24 hours. But because the distribution of sleep between light and dark periods was altered, these results are consistent with the hypothesis that chronic alcohol interferes with the circadian process that controls sleep. We have further found that through direct analysis of several circadian parameters (temperature, locomotor activity, plasma corticosterone, and Period 1 gene expression), chronic alcohol administered by liquid diet does not prevent rhythms from maintaining a 24-hour periodicity, but rather causes desynchrony in the timing of these circadian processes in relation to the central pacemaker at the suprachiasmatic nucleus (Guo et al., 2016). However, whether chronic alcohol exposure in our model directly interferes with normal recovery from a homeostatic challenge to sleep remains unknown.

To answer this question, we examined the effects of our chronic alcohol exposure protocol on the recovery sleep following 6 hours of acute sleep deprivation induced by gentle handling. Mild sleep deprivation is a common approach to probe sleep homeostasis, which induces a robust rebound in both the amount of sleep and the depth of sleep as measured by the slow wave amplitude (SWA) in the electroencephalogram

(EEG) recordings during NREMS (Franken et al., 1991). If the chronic alcohol treatment interferes with the homeostatic control of sleep, we would expect to find alterations in the amount, depth, or both of sleep during recovery following sleep deprivation. In this study, both control and alcohol-treated rats showed robust rebound sleep following sleep

50 deprivation. Although sleep onset was slightly delayed and the recovery sleep that occurred was more fragmented in the alcohol-treated animals, their total amount of

NREMS and REMS time was not different from controls over the 24 hours following sleep deprivation (total wake was slightly increased). The rebound in SWA was identical in the two groups. These results demonstrate that chronic alcohol consumption delays and fragments recovery sleep that follows a homeostatic challenge, but they do not support the hypothesis that chronic alcohol treatment dramatically impairs the build-up of a homeostatic factor during sleep deprivation which would have led to less recovery sleep or blunted SWA during rebound after the homeostatic challenge.

Materials and Methods

Animals

Male Sprague-Dawley rats (~ 300 g, Harlan, Indianapolis, IN) were randomly assigned to either receive ethanol-containing liquid diet (#F1436SP; Bio-Serv,

Flemington, NJ) or to be pair-fed with the control liquid diet (#F1268SP; Bio-Serv). Rats were housed individually in acrylic enclosures in a temperature- and humidity- controlled environment on a 12:12 Light:Dark (L:D) cycle (Mukherjee and Simasko, 2009).

Zeitgeber Time (ZT) 0: lights-on; ZT 12: lights-off. Experiments were completed using three cohorts of rats, with n = 3 to 6 for each group in each cohort. All experimental procedures were approved by the Institutional Animal Care and Use Committee of

Washington State University (protocol #4116).

Chronic Alcohol Exposure and Pair-feeding

(ethanol calories replaced with maltose dextrin) based on the average group volume that was consumed by the alcohol-treated rats one day before. All rats had ad lib access to water throughout all experiments. We refilled liquid diet feeding tubes daily under dim red illumination (<5 lux) at random times in the dark period to prevent the potential

52 entrainment to any disturbances. Rats had continuous access to diet during sleep recordings and sleep deprivation sessions. We weighed rats weekly at times of cage change and did not observe significant differences between groups (data not shown).

Sleep Recordings, Sleep Deprivation, and Sleep Data Analysis

We used a stereotactic apparatus to implant two electroencephalogram (EEG) electrodes with stainless-steel screws (#E363/20/2.4/SP; PlasticsOne Inc., Roanoke, VA,

USA) over the right frontal and the left parietal cortices under isoflurane anesthesia

(Kubota et al., 2002). Another two stainless-steel screws (#0-80X3/32; PlasticsOne Inc.) were mounted on the skull at the corresponding contralateral positions to facilitate anchoring the dental cement. Two electromyography (EMG) electrodes were tied into the nuchal neck muscles. We made EMG electrodes from multi-stranded Teflon-insulated stainless steel wire (#212-50F- 357-0; New England Wire, Lisbon, NH) (Phillips et al.,

2011), with 4 mm of exposed wire on one end and a gold-plated stainless steel socket contact lead (#E363/0; PlasticsOne Inc.) soldered on the other end. All leads from electrodes were fed into a plastic pedestal (#MS363; PlasticsOne Inc.). Electrodes and pedestal were secured and sealed by dental composite resin (Prime-Dent, Prime Dental

Manufacturing Inc., Chicago, IL, USA). Rats were allowed at least 1 week of recovery before the baseline recordings started.

Before sleep recording started, rats in their enclosures were moved into environmental chambers (4 rats/chamber) and tethered to swivel commutators (#SL6C;

PlasticsOne Inc.) by 6-channel cables (#363-363; PlasticsOne Inc.) to allow unrestricted movement. Rats were allowed 3 days of acclimation. Signals were filtered (-6 dB

53 low/high-frequency filters) and amplified by high-performance AC pre-amplifiers (Model

P511; Grass Technologies, Natus Neurology Incorporated, Warwick, RI). EEG signals were filtered below 0.3 Hz and above 30 Hz and amplified 5000 times; EMG signals were filtered below 30 Hz and above 300 Hz and amplified 2000 times. Signals were digitalized and collected at 128 Hz via Vital Recorder (Kissei Comtec Co., LTD.,

Matsumoto, Japan).

Two sleep deprivation sessions (6-hour each) were administered by gentle handling from ZT 0 (lights-on) to ZT 6 during week 9 and week 10, with 6-8 days of recovery between sessions. Sleep recordings continued during sleep deprivation sessions and during the week after sleep deprivation.

Vigilance states (wake, NREMS and REMS) were determined off-line in 10 sec epochs from visually scored EEG/EMG records in SleepSign for Animal (Kissei Comtec

Co., LTD.) based on standard criteria (Kubota et al., 2002). Data from the two sleep deprivation sessions were averaged as technical replicates.

Fast fourier transform (FFT) was performed within each individual epoch.

Spectral power analyses during wake, NREMS, and REMS epochs were performed for 1

- 20 Hz frequencies within each state. We measured slow wave amplitude (SWA) as the averaged spectral power in the delta wave frequencies (0.5 to 4 Hz) during NREMS epochs. SWA following sleep deprivation was normalized to the 24-hr average of SWA obtained 1 day prior to the sleep deprivation challenge.

We compared the number and duration of long duration wake (LDW) episodes, brief wake (BW) episodes, NREMS episodes, NREMS bouts, micro-wake (MW) episodes within NREMS bout, and REMS episodes. LDW and BW episodes were

54 determined based on published criteria (Simasko and Mukherjee, 2009). LDW episodes are wake episodes that are longer than or equal to 300 seconds, and any NREMS episodes that are in between LDW episodes and are shorter than or equal to 30 seconds would be included into and considered as part of the LDW episode. BW episodes are wake episodes that are shorter than 300 seconds and the same 30s NREMS criterion applies to

BW episodes. Any wake episodes that are in between NREMS episodes and are shorter than or equal to 30 seconds would be defined as MW episodes within NREM bouts. MW and NREMS episodes are both included into and considered as parts of NREMS bouts.

We examined the latency to NREMS and REMS after sleep deprivation. The latency to NREMS was determined as the time from the end of the sleep deprivation session (ZT 6) to the start of the first long NREMS bout (≥ 5 minutes). The latency to

REMS determined as the time to the first long REMS episode (≥ 1 minute). We used the above bout/episode durations as the criteria for the measurement of latency to avoid brief, unstable transitions and thus focus on the time until the transition into recovery sleep was stable. Analyses using the criteria of ≥ 3 minutes for NREMS bouts yielded similar results.

Core Body Temperature and Locomotor Activity Counts

We continuously monitored core body temperature and spontaneous locomotor activity counts via a telemetry system (MiniMitter, Philips Respironics Inc., Bend, OR) as previously published (Guo et al., 2016). Transmitters (PDT-4000 E-Mitter) were implanted into the peritoneal cavity of rats at the same time when EEG/EMG electrodes were implanted. Receivers (ER-4000 series) were located beneath each cage.

Temperature and activity data were collected by VitalView 4.2 (MiniMitter Inc.) at 1-min intervals and analyzed in 2-hour bins. Temperature and locomotor recordings continued during sleep recordings.

Statistical Analyses

We expressed data as mean ± SEM unless otherwise indicated. Two-tailed student t-tests and mixed linear model analysis of variance (ANOVA) (SPSS Statistics 17.0;

Polar Engineering and Consulting, Nikiski, AK) were used for statistical analyses. Mixed model ANOVA includes fixed main effects, fixed interactions between main effects, and a random effect of subjects (rats) (Van Dongen et al., 2004). Significant interactions were followed by Bonferroni posthoc tests. P ≤ 0.05 was considered statistically significant.

Results

Chronic Alcohol Treatment Delays Rebound Sleep after Sleep Deprivation

We first examined the amount of time spent in wake, NREMS, and REMS during sleep deprivation sessions and over the next 24 hours (Figure 2-1). During the sleep deprivation session rats were kept awake for an average of ~ 97% of the time (Con: 96.5

± 0.7 %; Alc: 97.6 ± 0.5 %). Following the sleep deprivation, both control and alcohol- treated rats showed robust rebound sleep, especially in the 6-hr light period immediately following sleep deprivation. We observed more wake time, less REMS time, and a trend for less NREMS time in the treated rats immediately following SD. In both groups, wake time increased and NREMS time decreased when rats entered the dark period. However, the peak of the rebound REMS in alcohol-treated rats was delayed into the first half of the dark period, while controls showed a decrease of REMS time after entering the dark period. We then analyzed the total amount of wake, NREMS, and REMS time over the

24-hour recovery period after sleep deprivation. Alcohol-treated rats had more 24-hr wake time (Con: 7.372 ± 0.166 hrs; Alc: 7.905 ± 0.170 hrs), a trend for slightly less

NREMS time (Con: 12.924 ± 0.181 hrs; Alc: 13.399 ± 0.179 hrs, p = 0.079), and a similar amount of REMS time compared to controls. Sleep onset, as measured by the latency to the first long NREMS bout (≥ 5 minutes) and to the first long REMS episode

(≥ 1 minute), was delayed in alcohol-treated rats (Figure 2-2). NREMS latency: 16.73 ±

1.32 mins in Con vs. 22.22 ± 1.87 mins in Alc; REMS latency: 38.77 ± 2.39 mins in Con vs. 58.13 ± 5.63 mins in Alc.

Chronic Alcohol Treatment Fragments Rebound Sleep after Sleep Deprivation

We did detailed bout analysis on sleep parameters and examined the episode/bout number and duration in the first 6-hr light period and the subsequent 12-hr dark period after sleep deprivation (Table 2). Wake episodes were further classified into long duration wake (LDW) and brief wake (BW) episodes for analysis. Prior studies have suggested that BW and LDW represent different levels of stability in the wake state, and that BW episodes are part of a period of vigilance cycling (during which states oscillate rapidly from one to the other) compared to the relative stability exhibited in LDW episodes (Simasko and Mukherjee, 2009). During the light period following sleep deprivation, alcohol-treated rats had longer LDW episodes and more BW episodes compared to controls. These observations are consistent with an inability of the alcohol- treated rats to enter into general sleep (i.e., vigilance cycling periods), and the sleep that does occur is interrupted by the appearance of brief wake episodes. NREMS of alcohol- treated rats, whether examined as episodes uninterrupted by any wake, or as bouts in which micro wake (MW) is included, were fragmented in the light period, as evidenced by shortened and more frequented NREMS episodes/bouts and more frequent MW episodes within NREMS bouts. REMS episodes were also shortened in the alcohol- treated rats.

On the other hand, during the subsequent dark period, alcohol-fed rats continued to have longer LDW episodes but now had fewer BW episodes. They also had fewer

NREMS episodes, a trend for longer NREMS episode/bout duration, and fewer MW episodes within NREMS bouts, all of which indicate more consolidated and less fragmented NREM sleep in the dark period. In addition, the normal light/dark differences

58 in BW episode number, NREMS episode/bout number, MW episode number, and REMS episode duration observed in controls were eliminated in the alcohol-treated rats.

Chronic Alcohol Treatment Has No Effects on SWA after Sleep Deprivation

We compared the slow wave amplitude (SWA) over the 24 hours after sleep deprivation between control and alcohol-treated rats and found no significant differences between groups (Figure 2-3). Since SWA is normalized to SWA in the 24 hours prior to the sleep deprivation challenge, it is possible that differences in pre-deprivation SWA exist between groups, however, we were not able to answer this question due to decreases in the absolute magnitude of the EEG signals over the 10 weeks of treatment. In other words, the signals decreased from pre-treatment baseline levels to various degrees with large individual differences in the amount of decrease at the time when the SD sessions were performed. Thus we were not able to normalize the pre-deprivation SWA to the baseline data measured 10 weeks ahead. However, over a 24-hour period, the loss of signal amplitude was minimal, so we were able to normalize the post-deprivation SWA to the signal obtained in the 24 hours prior to sleep deprivation. Regardless of whether the

SWA amplitude in alcohol-treated rats was changed prior to the sleep deprivation challenge, our observation demonstrates that alcohol-treated rats increased SWA amplitude in response to sleep deprivation to a magnitude similar to the magnitude of controls.

We also took a close look at the EEG data and performed analyses on spectral power distribution in wake, NREMS, and REMS epochs in the 6-hr light period and in the subsequent 12-hr dark period after sleep deprivation (Figure 2-4). We observed minor

59 changes in the delta and theta frequency bands in Wake epochs (Figure 2-4A and 2-4D) and a slight shift to slower theta frequencies in REMS epochs (Figure 2-4C and 2-4F).

However, there was no change in the power distribution between 1 to 20 Hz in NREMS epochs (Figure 2-4B and 2-4E). If alcohol-treated rats were compromised on their ability to increase delta power during NREMS in response to sleep deprivation, one would expect that there would be a selective decrease in the delta frequencies of NREMS in this analysis. Thus the results of our power distribution analysis further supports the conclusion that the alcohol treatment does not reduce SWA rebound as reported in Figure

2-3.

Body Temperature and Locomotor Activity before and after sleep deprivation

We measured core body temperature and locomotor activity counts continuously at 1-min intervals and examined the effects of chronic alcohol treatment and acute SD on these two parameters. The body temperature and locomotor activity from 1 day before to

3 days after the sleep deprivation challenge are plotted in 2-hr blocks in Figure 2-5.

Chronic alcohol treatment decreased body temperature (Figure 2-5A) but had little effect on locomotor activity except for the first 2 hours of the dark period (Figure 2-5B). Both body temperature and locomotor activity increased dramatically during acute SD but the locomotor activity of alcohol-treated animals increased to a less degree. During the 6-hr recovery in the light period, both parameters decreased to reach similar levels despite that alcohol-treated rats had lower body temperature in the light period before sleep deprivation. Temperature and locomotor activity remained dampened through the

60 subsequent dark period but returned to the baseline values 2-3 days after the sleep deprivation challenge.

Discussion

In this study we found that chronic alcohol treatment significantly altered the structure of rebound sleep in response to modest sleep deprivation, but had no effects on the SWA rebound and minor effects on the total sleep/wake time over the 24 hours following sleep deprivation. In the first 6 hours following sleep deprivation, alcohol- treated rats had more total wake time, an effect primarily due to the loss of REMS, although the slight drop in NREMS also contributed in producing this increase of wake time. However, the decrease in REMS was mostly compensated in the first 2 hours of the subsequent dark period. In agreement with the delay in the recovery of REMS, we also found that the latency to consolidated periods of REMS, as well as NREMS, were prolonged in alcohol-treated animals. On the other hand, detailed bout analysis demonstrated that the sleep that occurred after the sleep deprivation challenge was fragmented and showed signs of relative state instability. Taken together, our results do not support the idea that chronic alcohol exposure dramatically disrupts the ability to respond to increased homeostatic sleep drive, but rather suggest that chronic alcohol treatment alters the stability of vigilance states (i.e., NREMS and REMS), and that this change in sleep timing and stability may account for most of the effects we observed in response to the sleep deprivation challenge.

Acute alcohol consumption increases NREMS (Kubota et al., 2002), possibly by inhibiting basal forebrain wake-promoting cholinergic neurons via pathways involving adenosine A1 receptors (Thakkar et al., 2014), but very limited evidence suggests that

62 chronic alcohol consumption can impair sleep homeostasis (Colrain et al., 2014). Irwin et al. studied recently abstinent alcoholics and found lower slow wave activity (SWA) and blunted rebound stage 4 sleep after 4 hours of SD (Irwin et al., 2002). Brower, Armitage, and colleagues also reported lower baseline SWA and blunted SWA response after a 3- hour sleep delay in abstinent alcoholics (Armitage et al., 2012; Brower et al., 2011).

Results obtained from abstinent alcoholics may be confounded by age, race, gender, history of alcohol use, and duration of withdrawal or abstinence. Thakkar et al. studied acute withdrawal from chronic binge drinking in mice and found that these mice failed to accumulate adenosine in response to sleep deprivation, as evidence of disrupted sleep homeostasis (Thakkar et al., 2014). Mice in acute withdrawal from chronic ethanol vapor exposure also showed reduced overall spectral power, especially in the delta and theta frequencies (Ehlers and Slawecki, 2000). However, these studies focused on patients and animals in withdrawal or early abstinence while our study maintained continued access to the alcohol treatment during and after sleep deprivation, which may explain some of the inconsistency between these reports and our observations of SWA. In fact, acute alcohol has been shown to increase delta activity in NREMS (Lands, 1999). In addition, these reports either did not examine (Thakkar et al., 2014) or failed to find any (Armitage et al.,

2012; Brower et al., 2011; Ehlers and Slawecki, 2000; Irwin et al., 2002) significant differences in total sleep time after partial sleep deprivation or moderate sleep delay, consistent with our findings but contradicting the expectation of alcohol-induced impairments in the homeostatic rebound sleep in response to sleep loss. In general, our data are consistent with previous findings and the difference in SWA might be explained by the continuity of alcohol treatment in our study.

Sleep fragmentation is one of the most commonly reported sleep problems by alcoholic patients during active drinking, acute withdrawal, and abstinence, and often times is also the last symptom to resolve after prolonged sobriety (Mello and Mendelson,

1970; Rundell et al., 1977). In this study, we observed profound sleep fragmentation in both NREMS and REMS after acute sleep deprivation, suggesting that the stability of sleep episodes might have been compromised by chronic alcohol exposure. Sleep- promoting neural circuits, especially the ventrolateral preoptic nucleus (VLPO) neurons, and wake-promoting networks, mainly the ascending arousal systems, are mutually inhibiting, which forms a “flip-flop” switch that gates the transitions between sleep and wake (Saper et al., 2010). The transition between NREMS and REMS is likely modulated in a similar manner by REMS-on and REMS-off neural populations in the brain (Fuller et al., 2006). The thresholds of these switches are under control of both the homeostatic sleep drive and the circadian systems, and it is possible that chronic alcohol exposure decreases the thresholds of these switches, leading to unstable states and more frequent transitions. Previous work in our group suggests that chronic alcohol fragments sleep in the light period with extended long duration wake episodes (i.e., inability to enter into sleep periods) while the sleep of alcohol-treated rats in the dark period becomes more consolidated, potentially to compensate the poor sleep quality during the inactive period

(Mukherjee and Simasko, 2009). In this study, we observed similar trends after acute sleep deprivation; immediately following sleep deprivation, alcohol-treated rats showed fragmented NREMS and REMS; later in the dark period, the NREMS of alcohol-treated rats was more consolidated. This reversed distribution of sleep stability between light and dark periods is consistent with disruptions in the circadian regulation of the “flip-flop”

64 switch rather than disrupting the response to homeostatic factors that build-up during wake.

Alcohol consumption, especially chronic alcohol use, has been shown to induce disruptions on various circadian parameters. Although studies focusing on circadian rhythms of active alcoholics are rare, abnormalities in diurnal rhythms of blood pressure, body temperature, and hormone secretion (e.g. cortisol, melatonin and testosterone) are often reported in abstinent alcoholics (Bertello et al., 1982; Kawano et al., 2002;

Majumdar and Miles, 1987; Murialdo et al., 1991; Stokes, 1973; Wasielewski and

Holloway, 2001). In alcoholics who have prolonged sleep latencies, the nocturnal peak of melatonin is delayed and the diurnal rhythm of circulation cortisol is advanced and blunted (Kühlwein et al., 2003). Furthermore, animal studies using various models reveal that chronic alcohol exposure can attenuate responses to photic and non-photic stimuli, alter the periodicity of wheel-running behavior under constant conditions, flatten the diurnal rhythm of corticosterone (CORT) levels, and disrupt the gene expression rhythms of many key clock genes, including Per1 and Per2, in sites peripheral to the SCN, especially in the liver (Brager et al., 2010; Filiano et al., 2013; Kakihana and Moore,

1976; Seggio et al., 2009; Seggio et al., 2007). Our previous study also shows that one of the primary effects of chronic alcohol on the circadian systems is to cause an internal desynchrony between the central and peripheral clocks and among various peripheral rhythms (Guo et al., 2016). Whether the delay of rebound sleep and the reversed distribution of sleep stability observed in this study are caused by circadian disruptions after chronic alcohol exposure should be determined in future studies.

Sleep disturbances can severely impair the quality of life, increase irritability, negative emotion affect, and stress, and contribute to risks of relapse during abstinence

(Brower et al., 2001; Foster et al., 1999; Minkel et al., 2012). Due to the sedative-like, sleep-promoting effects of alcohol, acute alcohol is also often used as a sleep aid in self- medication when experiencing insomnia (Brower et al., 2001). However, current treatment options for sleep problems in AUD are very limited and may involve the use of cross-dependent sedatives such as benzodiazepine receptor agonists (Roth, 1995). Thus, non-medication treatments and alternative therapy to correct sleep disturbances could be particularly beneficial for patients with AUD. Cognitive-behavioral treatment is now commonly used to treat insomnia and to help maintain sobriety in patients with AUD, but whether its impact on improving sleep has any influence on continued abstinence remains to be further studied (Arnedt et al., 2007). As reviewed above, the available literature strongly indicates circadian desynchrony is a primary effect of chronic alcohol exposure, which suggests that a conscious effort to restore consistent circadian synchrony may be of great value in reducing the negative impact of AUD-associated sleep problems on general health. Bright light therapy has been used to help alcoholics in acute withdrawal, although its actual clinical values as a treatment of AUD were unclear (Schmitz et al.,

1997). However, in this study the light therapy was only applied during acute withdrawal and whether it is also effective in helping maintain long-term abstinence is untested. In the last decades, many advancements have been made in understanding sleep and circadian rhythms and new circadian medication is becoming more and more available

(Hardeland, 2009; Lack and Wright, 2007; Reynoldson et al., 2008). Incorporating a

66 more rigorous approach to restore circadian rhythmicity may be very promising in the treatment of sleep disturbances associated with AUD.

In conclusion, this study shows that our chronic alcohol treatment dose not interfere with the animal’s ability to mount a homeostatic sleep rebound after sleep deprivation, but rather the timing of the rebound sleep is delayed and the recovery sleep that occurs is highly fragmented. The mechanistic explanation of these observations requires additional studies, but these results provides an important guiding framework for future research. The relevance of these results to applications of sleep medicine in general and to maintaining sobriety in AUD should be further explored.

W a k e

A B

1 2 0 ) 9

C o n D

)

r * s

A l c e

H r

8 0 6

( u

4 0 *** ** 3 o

e **

(

i *

T m

SD i

0 T 0 0 6 1 2 1 8 2 4 3 0 C o n A l c

T im e (H o u r) T r e a t m e n t

N R E M S le e p

C D

9 0 ) 1 5

SD D

)

2 6 0 1 0

u (

3 0 5

(

m i

0 T 0 0 6 1 2 1 8 2 4 3 0 C o n A l c

T im e (H o u r) T r e a t m e n t

R E M S le e p

E F

3 0 ) 4

SD D

)

r s

r ***

u f

o * a

2 2 0

t **

4 2

M u ( ***

1 0

o e

H 1

(

m ***

m i

0 T 0 0 6 1 2 1 8 2 4 3 0 C o n A l c

T im e (H o u r) T r e a t m e n t

Figure 2-1. State time in Wake, NREMS, and REMS during Sleep Deprivation and during the 24 Hours after. White-gray shadings indicate the 12h:12h light:dark cycle in this and all subsequent graphs. Asterisks: significantly different from controls by t-test or by Bonferroni post-hoc tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001. Con: n = 11; Alc: n = 14. Panels A, C, and E are plotted in 2-hr time blocks with the black bars indicating the period of the 6-hr SD session. Analysis by mixed model 2-way ANOVA showed significant Group (Con vs. Alc) X Time interactions in wake (F (14, 322) = 3.105, p <

0.001) and REMS (F (14, 322) = 5.959, p < 0.001), while analysis of NREMS showed a main Time effect (F (14, 322) = 483.324, p < 0.001) and a trend of a main Group effect (F (1,

23) = 4.053, p = 0.056). Panels B, D, and F summarize the total amount of wake and sleep time in the 24 hrs after SD. Alcohol-treated rats had similar amounts of NREMS and

REMS and slightly more wake in the 24 hours after SD compared to control rats

(unpaired t-test with Welch’s correction, two-tailed, t (22.75) = 2.248, p = 0.035).

A B N R E M S R E M S

4 0 1 0 0

) e

t 3 0 7 5

n i

M * * (

y 2 0 5 0

e t

a 1 0 2 5 L

0 0 C o n A lc C o n A lc

Figure 2-2. Latency to NREMS and REMS after Sleep Deprivation. Con: n = 11;

Alc: n = 14. Alcohol-treated rats had longer NREMS latency (unpaired t-test, two-tailed, t

(23) = 2.278, p = 0.032) and longer REMS latency (unpaired t-test with Welch’s correction, two-tailed, t (17.37) = 3.168, p = 0.006). *: p < 0.05; **: p < 0.01.

P o s t - D e p r iv a t io n

) 1 9 0 e

e C o n

a r

u A l c

v i

l 1 6 0

r 1 3 0

1 0 0

. S 0 SD ( 7 0 0 6 1 2 1 8 2 4 3 0

T im e (H o u r)

Figure 2-3. Slow Wave Amplitude (SWA) after Sleep Deprivation. SWA is plotted in

2-hr time blocks and normalized to the 24-hr average of the pre-deprivation day. Con: n

= 11; Alc: n = 14. Analysis by mixed model 2-way ANOVA showed a significant main time effect, F (11, 253) = 207.386, p < 0.001. There was no difference in SWA between groups.

A B C

r W a k e N R E M S R E M S

5 5 5 e

) C o n

o H

4 A l c 4 4

f r

t 3 * * 3 3

e c

r * * * *

P w

* * * * * *

p t

o 2 * * 2 2

v e

i 1 1 1

( l

e 0 0 0 R 0 4 8 1 2 1 6 2 0 0 4 8 1 2 1 6 2 0 0 4 8 1 2 1 6 2 0

F r e q u e n c y (H z ) D E F

r W a k e N R E M S R E M S

5 5 5

)

z o

H * * *

4 4 4

f r

t 3 3 3

e e

P * * * * * * w

* * * p

o 2 * * * 2 2

k S

P *

r * * *

e i

D 1 1 1

( l

e 0 0 0 R 0 4 8 1 2 1 6 2 0 0 4 8 1 2 1 6 2 0 0 4 8 1 2 1 6 2 0

F r e q u e n c y (H z )

Figure 2-4. Spectral Power Distribution in Wake, NREMS, and REMS after Sleep

Deprivation. Spectral power was normalized to the average power of all frequency bands

(1-20 Hz) within each state during the light period (6 hrs) and during the dark period (12 hrs) after sleep deprivation. Con: n = 11; Alc: n = 12. Analysis by mixed model 3-way

ANOVA showed significant Group (Con vs. Alc) X Frequency (1 – 20 Hz) interactions in wake (F (19, 840) = 4.57, p < 0.001) and REMS (F (19, 840) = 5.07, p < 0.001) and significant Light/Dark X Frequency interactions in wake (F (19, 840) = 6.73, p < 0.001),

NREMS (F (19, 840) = 90.34, p < 0.001), and REMS (F (19, 840) = 2.83, p < 0.001). Alcohol- treated rats had slightly less delta (1-4 Hz) power and more theta (5-8 Hz) power during wake. Their theta power during REMS shifted slightly to the lower frequency bands.

There was no difference in spectral power distribution during NREMS between groups.

Asterisks: significantly different from controls by Bonferroni post-hoc tests. *: p < 0.05;

**: p < 0.01; ***: p < 0.001.

A B o d y T e m p e r a t u r e 3 8 .6 C o n

3 8 .3 A l c

)

C (

3 8 .0

e r

u * *** ** * t

a 3 7 .7 *** r * e ** ** p *** * 3 7 .4 *** ** * m * ***

e ** *** T 3 7 .1 ** * * *** *** * SD 3 6 .8 0 1 2 2 4 3 6 4 8 6 0 7 2 8 4 9 6

T im e (H o u r)

B L o c o m o t o r A c t iv it y 3 0

) 2 5

t u

n ***

i M

2 0

/ **

n u

o 1 5

(

y t i 1 0

v * **

i *

t * c

A 5 **

SD 0 0 1 2 2 4 3 6 4 8 6 0 7 2 8 4 9 6

T im e (H o u r)

Figure 2-5. Body Temperature and Locomotor Activity before and after Sleep

Deprivation. Data are plotted in 2-hr time blocks. Con: n = 11; Alc: n = 11. Analysis by mixed model 2-way ANOVA showed significant Group (Con vs. Alc) X Time (Hour) interactions in body temperature (F (47, 940) = 2.284, p < 0.001) and locomotor activity

(F (47, 940) = 2.368, p < 0.001). Asterisks: significantly different from controls by

Bonferroni post-hoc tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Table 2. Sleep Parameters after Sleep Deprivation

Light Period (6 Hrs) Dark Period (12 Hrs)

Control Alcohol Control Alcohol Parameters p-value p-value (n = 11) (n = 14) (n = 11) (n = 14) Wake Long Duration Wake (LDW, ≥ 300s) LDW Episode # (/ 6Hrs) 2.6 ± 0.2 3.4 ± 0.3 NS 6.8 ± 0.5 ### 5.7 ± 0.3 ### < 0.05 LDW Episode Duration (s) 622 ± 29 883 ± 68 < 0.001 989 ± 53 ### 1,226 ± 76 ### < 0.001 Brief Wake (BW, < 300s) BW Episode # (/ 6Hrs) 61 ± 2 72 ± 3 < 0.01 87 ± 3 ### 75 ± 3 < 0.01 BW Episode Duration (s) 31 ± 2 32 ± 1 NS 31 ± 1 a 28 ± 1 a NS NREM Sleep NREMS Episode # (/6Hrs) 70 ± 3 84 ± 3 < 0.001 101 ± 3 ### 89 ± 3 < 0.01 NREMS Episode Duration 215 ± 10 169 ± 8 < 0.001 103 ± 3 ### 112 ± 5 ### NS (s) NREMS Bout NREMS Bout # (/ 6Hrs) 41 ± 1 46 ± 2 < 0.05 50 ± 1 ### 47 ± 1 NS NREMS Bout Duration (s) 368 ± 15 318 ± 14 < 0.01 205 ± 5 ### 214 ± 9 ### NS ` Micro Wake within NREMS Bout (MW, ≤ 30s) MW Episode # (/ 6Hrs) 24 ± 2 33 ± 2 < 0.001 43 ± 2 ### 34 ± 2 < 0.001 MW Episode Duration (s) 14.8 ± 0.3 15.9 ± 0.2 NS a 14.9 ± 0.2 14.9 ± 0.3 a NS REM Sleep REMS Episode # (/ 6Hrs) 38 ± 2 36 ± 2 NS 41 ± 1 ### 43 ± 2 ### NS REMS Episode Duration (s) 100 ± 4 83 ± 4 < 0.01 70 ± 2 ### 79 ± 3 NS

Data analyzed by mixed model ANOVA with 2 fixed factors: Group (Con vs.

Alc) and Light Condition (Light vs. Dark) with Bonferroni post-hoc tests. Episode and

bout numbers expressed as averaged values per 6 hours. Significant Group X Light

interactions were found in LDW # (F (1, 23) = 8.019, p < 0.01), BW # (F (1, 23) = 23.333, p <

0.001), NREMS # (F (1, 23) = 25.311, p < 0.001), NREMS Duration (F (1, 23) = 21.536, p <

0.001), NREMS Bout # (F (1, 23) = 10.080, p < 0.01), NREMS Bout Duration (F (1, 23) =

10.065, p < 0.01), MW # (F (1, 23) = 36.463, p < 0.001), MW Duration (F (1, 23) = 6.956, p <

0.05), and REMS Duration (F (1, 23) = 15.237, p < 0.001). LDW Duration showed a significant main Group effect (F (1, 23) = 15.259, p < 0.001) and a main Light effect (F (1, 23)

= 31.822, p < 0.001.). Main Light effects were also found in BW Duration (F (1, 23) =

4.431, p = 0.04) and REMS # (F (1, 23) = 13.278, p < 0.001).

Column of p values: significant difference between groups; NS: not significantly different. a: statistically significant differences between groups. However, because sleep recording was scored in 10-s epochs, these differences are likely statistical artifacts.

Pound signs: significant difference between the light and dark period within a group; #: p

< 0.05; ##: p < 0.01; ###: p < 0.001

References

Allen RP, Wagman A, Faillace LA and McIntosh M (1971) Electroencephalographic (EEG) sleep recovery following prolonged alcohol intoxication in alcoholics. The Journal of Nervous and Mental Disease 153 (6): 424-433.

Armitage R, Hoffmann R, Conroy DA, Arnedt JT and Brower KJ (2012) Effects of a 3- hour sleep delay on sleep homeostasis in alcohol dependent adults. Sleep 35 (2): 273.

Arnedt JT, Conroy D, Rutt J, Aloia MS, Brower KJ and Armitage R (2007) An open trial of cognitive-behavioral treatment for insomnia comorbid with alcohol dependence. Sleep Medicine 8 (2): 176-180.

Borbély AA (1982) A two process model of sleep regulation. Human Neurobiology 1 (3): 195-220.

Brower KJ, Aldrich MS and Hall JM (1998) Polysomnographic and subjective sleep predictors of alcoholic relapse. Alcoholism: Clinical and Experimental Research 22 (8): 1864-1871.

Brower KJ, Aldrich MS, Robinson EA, Zucker RA and Greden JF (2001) Insomnia, self- medication, and relapse to alcoholism. American Journal of Psychiatry 158 (3): 399-404.

Brower KJ, Hoffmann R, Conroy DA, Arnedt JT and Armitage R (2011) Sleep homeostasis in alcohol-dependent, depressed and healthy control men. European Archives of Psychiatry and Clinical Neuroscience 261 (8): 559-566.

Brower KJ (2001) Alcohol's effects on sleep in alcoholics. Alcohol Research and Health 25 (2): 110-125.

Colrain IM, Nicholas CL and Baker FC (2014). Alcohol and the Sleeping Brain, ELSEVIER.

Colrain IM, Turlington S and Baker FC (2009) Impact of alcoholism on sleep architecture and EEG power spectra in men and women. Sleep 32 (10): 1341.

Crum RM, Storr CL, Chan YF and Ford DE (2004) Sleep disturbance and risk for alcohol-related problems. American Journal of Psychiatry 161 (7): 1197-1203.

Ehlers CL and Slawecki CJ (2000) Effects of chronic ethanol exposure on sleep in rats. Alcohol 20 (2): 173-179.

Filiano AN, Millender-Swain T, Johnson Jr. R, Young ME, Gamble KL and Bailey SM (2013) Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus. PLOS ONE 8 (8): e71684.

Foster JH, Marshall EJ, Hooper R and Peters TJ (1988) Quality of life measures in alcohol dependent subjects and changes with abstinence and continued heavy drinking. Addiction Biology 3 (3): 321-332.

Foster JH, Powell JE, Marshall EJ and Peters TJ (1999) Quality of life in alcohol- dependent subjects-a review. Quality of Life Research 8 (3): 255-261.

Franken P, Dijk DJ, Tobler I and Borbely AA (1991) Sleep deprivation in rats: effects on EEG power spectra, vigilance states, and cortical temperature. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 261 (1): R198-R208.

Fuller PM, Gooley JJ and Saper CB (2006) Neurobiology of the sleep-wake cycle: sleep architecture, circadian regulation, and regulatory feedback. Journal of Biological Rhythms 21 (6): 482-93.

Gann H, Feige B, Hohagen F, van Calker D, Geiss D and Dieter R (2001) Sleep and the cholinergic rapid eye movement sleep induction test in patients with primary alcohol dependence. Biological Psychiatry 50 (5): 383-390.

Gross MM and Hastey JM (1975) The relation between baseline slow wave sleep and the slow wave sleep response to alcohol in alcoholics. Advances in Experimental Medicine and Biology (59): 467-75.

Gross MM, Goodenough DR, Hastey J and Lewis E (1973) Experimental study of sleep in chronic alcoholics before, during, and after four days of heavy drinking, with a nondrinking comparison. Annals of the New York Academy of Sciences 215 (1): 254-265.

Guo R, Simasko SM. and Jansen HT (2016) Chronic Alcohol Consumption in Rats Leads to Desynchrony in Diurnal Rhythms and Molecular Clocks. Alcoholism: Clinical and Experimental Research 40 (2): 291-300.

Hardeland R (2009) Tasimelteon, a melatonin agonist for the treatment of insomnia and circadian rhythm sleep disorders. Current Opinion in Investigational Drugs 10 (7): 691-701.

Irwin M, Gillin JC, Dang J, Weissman J, Phillips E and Ehlers CL (2002) Sleep deprivation as a probe of homeostatic sleep regulation in primary alcoholics. Biological Psychiatry 51 (8): 632-641.

Johnson LC, Burdick JA and Smith J (1970) Sleep during alcohol intake and withdrawal in the chronic alcoholic. Archives of General Psychiatry 22 (5): 406-418.

Kakihana R and Moore JA (1976) Circadian rhythm of corticosterone in mice: the effect of chronic consumption of alcohol. Psychopharmacologia 46 (3): 301-305.

Kawano Y, Pontes CS, Abe H, Takishita S and Omae T (2002) Effects of alcohol consumption and restriction on home blood pressure in hypertensive patients: serial changes in the morning and evening records. Clinical and Experimental Hypertension 24 (1-2): 33-39.

Knapp CM, Ciraulo DA and Datta S (2014) Mechanisms underlying sleep-wake disturbances in alcoholism: Focus on the cholinergic pedunculopontine tegmentum. Behavioural Brain Research 274: 291-301.

Kubota T, De A, Brown RA, Simasko SM and Krueger JM (2002) Diurnal effects of acute and chronic administration of ethanol on sleep in rats. Alcoholism: Clinical and Experimental Research 26 (8): 1153-1161.

Kühlwein E, Hauger RL and Irwin MR (2003) Abnormal nocturnal melatonin secretion and disordered sleep in abstinent alcoholics. Biological Psychiatry 54 (12): 1437- 1443.

Lack LC and Wright HR (2007) Treating chronobiological components of chronic insomnia. Sleep Medicine 8 (6): 637-644.

Landolt HP (2008) Sleep homeostasis: a role for adenosine in humans? Biochemical Pharmacology 75 (11): 2070-2079.

Lands WE (1999) Alcohol, slow wave sleep, and the somatotropic axis. Alcohol 18 (2): 109-122.

Majumdar SK and Miles A (1987) Disturbed melatonin secretion in chronic alcoholism and withdrawal. Clinical Chemistry 33 (7): 1291.

Mello NK and Mendelson JH (1970) Behavioral studies of sleep patterns in alcoholics during intoxication and withdrawal. Journal of Pharmacology and Experimental Therapeutics 175 (1): 94-112.

Minkel JD, Banks S, Htaik O, Moreta MC, Jones CW, McGlinchey EL, Simpson NS and Dinges DF (2012) Sleep deprivation and stressors: evidence for elevated negative affect in response to mild stressors when sleep deprived. Emotion 12 (5): 1015.

Mistlberger RE (2005) Circadian regulation of sleep in mammals: role of the suprachiasmatic nucleus. Brain Research Reviews 49 (3): 429-54.

Murialdo G, Filippi U, Costelli P, Fonzi S, Bo P, Polleri A and Savoldi F (1991) Urine melatonin in alcoholic patients: a marker of alcohol abuse? Journal of Endocrinological Investigation 14 (6): 503-507.

Phillips DJ, Schei JL, Meighan PC and Rector DM (2011) State-dependent changes in cortical gain control as measured by auditory evoked responses to varying intensity stimuli. Sleep 34 (11): 1527-1537.

Reynoldson JN, Elliott E, Sr. and Nelson LA (2008) Ramelteon: a novel approach in the treatment of insomnia. The Annals of Pharmacotherapy 42 (9): 1262-1271.

Roehrs TA and Roth T (2015) Sleep Disturbance in Substance Use Disorders. Psychiatric Clinics of North America 38 (4): 793-803.

Roth T (2007) Insomnia: definition, prevalence, etiology, and consequences. Journal of Clinical Sleep Medicine 3 (5 Suppl): S7.

Roth T, Roehrs TA and Vogel G (1995) Zolpidem in the treatment of transient insomnia: a double-blind, randomized comparison with placebo. Sleep 18 (4): 246-51.

Rundell OH, Williams HL and Lester BK (1977) Sleep in alcoholic patients: longitudinal findings. Alcohol Intoxication and Withdrawal—IIIb. Springer.

Saper CB, Fuller PM, Pedersen NP, Lu J and Scammell TE (2010) Sleep state switching. Neuron 68 (6): 1023-1042.

Simasko SM and Mukherjee S (2009) Novel analysis of sleep patterns in rats separates periods of vigilance cycling from long-duration wake events. Behavioural Brain Research 196 (2): 228-36.

Stokes PE (1973) Adrenocortical activation in alcoholics during chronic drinking. Annals of the New York Academy of Sciences 215 (1): 77-83.

Thakkar MM, Sharma R and Sahota P (2014) Alcohol disrupts sleep homeostasis. Alcohol 49 (4): 299-310.

Van Dongen HP, Olofsen E, Dinges DF and Maislin G (2004) Mixed-model regression analysis and dealing with interindividual differences. Methods in Enzymology 384: 139-171.

Wasielewski JA and Holloway FA (2001) Alcohol's interactions with circadian rhythms. Alcohol Research and Health 25: 94-100.

CHAPTER THREE

CHRONIC ALCOHOL CONSUMPTION IN RATS LEADS TO DESYNCHRONY IN

DIURNAL RHYTHMS AND MOLECULAR CLOCKS*

Rong Guo, Steve M. Simasko, and Heiko T. Jansen

Programs in Neuroscience

Department of Integrative Physiology and Neuroscience

College of Veterinary Medicine

Washington State University, Pullman, Washington, USA, 99164

______* This work has been published in Alcoholism: Clinical and Experimental Research 40 (2): 291- 300; Guo R, Simasko SM, and Jansen HT (2016).

Abstract

Circadian rhythms are essential for adapting to the environment. Chronic alcohol consumption often leads to sleep and circadian disruptions, which may impair the life quality of individuals with alcohol use disorders and contribute to the morbidity associated with alcoholism. We used a pair-feeding liquid diet alcohol exposure protocol (6 weeks duration) in

PER1::LUC transgenic rats to examine the effects of chronic alcohol exposure on: (i) diurnal rhythms of core body temperature and locomotor activity, (ii) plasma corticosterone (CORT) concentrations, and (iii) rhythms of ex vivo Period1 (Per1) expression in the suprachiasmatic nucleus (SCN), pituitary, and adrenal glands. We followed multiple circadian outputs not only to examine individual components, but also to assess the relative phase relationships among rhythms. In this study we found that chronic alcohol consumption: (i) reduced 24-hour body temperature and locomotor activity counts in the dark period, (ii) advanced the acrophase of diurnal rhythms of body temperature and locomotor activity, (iii) abolished the phase difference between temperature and activity rhythms, (iv) blunted and advanced the diurnal CORT rhythm, and (v) advanced Per1 expression in the adrenal and pituitary glands but not in the SCN. We found that chronic alcohol altered the phase relationships among diurnal rhythms and between the central (SCN) and peripheral (adrenal and pituitary) molecular clocks. Our findings suggest that desynchrony among internal rhythms is an important and overlooked aspect of alcohol- induced circadian disruptions. The misalignment of phases among rhythms may compromise normal physiological functions and put individuals with chronic alcohol use at greater risk for developing other physical and mental health issues. How this desynchrony occurs and the extent to which it participates in alcohol-related pathologies requires further investigation

Introduction

Circadian rhythms allow animals to optimize their performance by anticipating periodic changes in the environment and adjusting their physiological functions accordingly. This process depends on a molecular clock consisting of genes such as Period (Per), Clock, Bmal1 and

Cryptochrome, and their corresponding proteins. The clock comprises a self-sustained transcription-translation feedback loop that repeats every 24 hours and thus sets the periodicity of internal rhythms (Reppert and Weaver, 2002). Mammalian circadian rhythms are organized in a hierarchical fashion with the master pacemaker residing in the suprachiasmatic nucleus (SCN) of the hypothalamus and entraining internal rhythms to the external light/dark cycle. However, molecular clocks also exist in virtually all peripheral organs. Maintaining an appropriate phase relationship among clocks in various organs is vital in achieving synchronized functions (Dibner et al., 2010). Circadian desynchrony can occur when proper phase relationships among oscillators are altered, which may contribute to the development of certain diseases (Voigt et al.,

2013).

Chronic alcohol consumption can disrupt sleep and circadian rhythms, which often persists during abstinence and increases the risk of relapse (Brower, 2003; Spanagel et al., 2005).

Sleep problems such as insomnia and frequent night waking are common among alcoholics, and have been observed in animal studies (Brower, 2003; Mukherjee and Simasko, 2009).

Abnormalities in diurnal rhythms of blood pressure, body temperature and hormone secretion

(e.g. cortisol, melatonin and testosterone) have been reported in alcoholics (Bertello et al., 1982;

Kawano et al., 2002; Wasielewski and Holloway, 2001). Disrupted diurnal rhythms of corticosterone (CORT) after chronic alcohol have also been observed in rodents (Kakihana and

Moore, 1976). The sleep and circadian dysfunctions after chronic alcohol may result from disturbed sleep homeostasis or disrupted circadian rhythms or very likely a combination of both, but the exact mechanism remains unclear at present.

Based on the close interactions among alcohol consumption, the hypothalamic-pituitary- adrenal (HPA) axis, sleep and circadian rhythms, it is likely that the HPA axis is involved in mediating some of the negative effects of chronic alcohol on sleep and circadian rhythms

(Brower, 2003; Buckley and Schatzberg, 2005; Sarkar, 2012). The HPA axis exhibits prominent rhythms in hormone secretion and tissue responsiveness (Kalsbeek et al., 2012) and appears to be particularly susceptible to alcohol-induced disruptions (Clarke et al., 2008). Centrally, chronic alcohol can alter levels of corticotropin-releasing hormone (CRH) and vasopressin and also can reduce the expression of glucocorticoid receptors in the paraventricular nucleus (PVN) of the hypothalamus (Roy et al., 2002; Silva et al., 2002). Peripherally, chronic alcohol has been shown to impair the adrenal sensitivity to adrenocorticotropin (ACTH) and the pituitary responsiveness to CRH (Allen et al., 2011). Moreover, withdrawal from drugs of abuse is often stressful. which activates the HPA axis, whereupon the up-regulation of brain CRH activity contributes to the negative affect associated with alcoholism (Koob, 2008).

The goal of the present study was to further explore the manner in which chronic alcohol affects the integrity of biological rhythms and specifically the molecular clock. To this end we used a PER1::LUC transgenic rat model (Yamazaki et al., 2000) and examined effects of chronic alcohol exposure on: 1) diurnal rhythms of body temperature and locomotor activity, 2) plasma corticosterone (CORT) concentrations, and 3) ex vivo Per1 expression in the master pacemaker

(SCN) and in peripheral clocks within the HPA axis (pituitary and adrenal glands). We followed multiple circadian outputs thereby allowing us to examine the effects on individual components and, more importantly, to assess the phase relationships of rhythms among different organs. We found that 6 weeks of alcohol exposure in liquid diet shifted the phase relationship between the rhythms of body temperature and locomotor activity, advanced and blunted the plasma CORT rhythm, and phase-advanced the molecular clock (Per1) in peripheral HPA clocks (adrenal and pituitary) but not in the central pacemaker (SCN).

Materials and Methods

Animals

We used in-house bred male Wistar PER1::LUC transgenic rats (initial breeding pair gifted by Dr. Michael Menaker at the University of Virginia). Rats weighed 250 ~ 350 g at the beginning of experiments and were randomly assigned to receive either alcohol exposure (Alc) or pair-feeding (Con). We housed rats individually in a temperature- and humidity- controlled environment on a 12:12 Light:Dark (L:D) cycle. Zeitgeber Time (ZT) 0: lights-on; ZT 12: lights- off. All experimental procedures were approved by the Institutional Animal Care and Use

Committee of Washington State University (protocol #4116).

Chronic Alcohol Exposure and Pair-feeding

Following a 2-week baseline period with ad-lib access to the control liquid diet

(#F1268SP; Bio-Serv, Flemington, NJ), the alcohol exposure protocol was initiated. Rats receiving alcohol were acclimated to ethanol-containing liquid diet (#F1436SP; Bio-Serv) for 2 days at 2% ethanol followed by 2 days at 4%, achieving 6% ethanol at day 5 and continuing for at least 6 weeks. Control rats were pair-fed with the control liquid diet (ethanol calories replaced with maltose dextrin) in a group-based manner: the volume that controls received each day was the same as the mean volume consumed by the alcohol-fed rats the day before (Mukherjee and

Simasko, 2009). All rats had ad lib access to water. To prevent the entrainment to any potential disturbance caused by the action of liquid diet refilling, we refilled feeding tubes under dim-red illumination (<5 lux) at random times late in the dark period (ZT 18 - 23). The alcohol treatment was a minimum of 6 weeks and continued while our measurements were taken until rats were

87 euthanized for tissue collection at the end of the alcohol treatment paradigm. Rats were weighed weekly at times of cage changes.

Diurnal Rhythms of Core Body Temperature and Locomotor Activity Counts

We used a telemetry system (MiniMitter, Philips Respironics Inc., Bend, OR) to continuously monitor core body temperature and spontaneous locomotor activity counts as previously published (Jansen et al., 2012). Transmitters (PDT-4000 E-Mitter) were implanted in the peritoneal cavity of rats (n = 10 for Con; n = 10 for Alc) under isoflurane anesthesia at least one week before the recordings started. Receivers (ER-4000 series) were located beneath each cage. Temperature and activity data were collected by VitalView 4.2 (MiniMitter Inc.) at 1-min intervals. Actograms of body temperature and locomotor activity were generated by Clocklab software (Actimetrics, Evanston, IL) run in MATLAB R2015a (Mathworks Co., Natick, MA).

The daily acrophase was estimated and plotted on the actograms by Clocklab. Seven days of continuous temperature and activity data during baseline and at the last week of the alcohol treatment were used to determine light/dark differences and rhythm parameters (period, amplitude, and acrophase; sine-wave fits estimated by Cosinor Periodogram (Refinetti, 2006)).

The correlation between temperature and activity was determined using the Pearson correlation coefficient of hourly averaged data. Fifteen days of temperature and activity data (15-min binned) during baseline and at the last two weeks of the alcohol treatment were used for the discrete wavelet transform (DWT) analysis in MATLAB R2015a with custom scripts written by

Tanya Leise (Leise and Harrington, 2011). We estimated the circadian energy and variability within the range of circadian periods (16 - 32 hrs).

Blood Sampling, Blood Alcohol Concentration (BAC). and Plasma Corticosterone (CORT)

Concentration

We used another two cohorts of rats (n = 24 for Con; n = 24 for Alc) for blood sampling and measurements of blood alcohol and plasma CORT concentrations. Blood samples were collected at 4-hr intervals (ZT 3, 7, 11, 15, 19, and 23) into cold heparin-Li-coated tubes

(#HC0540713C; Fisher Scientific Co.) during the last week (Wk 6) of the 6-week alcohol treatment. Two samples were obtained from each rat, both under isoflurane anesthesia: one from the tail vein, and another one week later by cardiac puncture at the time of euthanasia. The two samples from each rat were collected at different time points of the day. All samples were collected within 30 mins (tail bleeds) to 1 hr (cardiac bleeds) of the assigned time point. For both groups, each time point included n = 4 from tail bleeds and n = 3 - 5 from cardiac bleeds.

Samples were stored on ice until centrifuged at 1300xg, 4 °C, for 20 min. The plasma aliquots were stored at -20 °C.

We measured blood alcohol concentration (BAC) of plasma samples from alcohol-treated rats using a kit for alcohol determination (NAD-ADH Reagent Multiple Test Vial, Sigma N7160;

Sigma Aldrich, Saint Louis, MO).

The plasma corticosterone (CORT) concentration was measured via a commercially available rat corticosterone double-antibody radioimmunoassay kit (MP Biomedicals,

Orangeburg, NY). Because preliminary analysis showed no difference in blood sampling methods within cohorts, data from tail and cardiac bleeds were pooled. However, a significant difference in cohort averages was found and thus CORT levels were normalized to cohort averages for analysis.

Ex vivo PER1::LUC Tissue Luminescence

Rats whose body temperature and locomotor activity had been monitored were used in experiments to measure PER1::LUC tissue luminescence. Alcohol-treated rats had continued alcohol treatment until they were euthanized for tissue collection at the end of the alcohol treatment (at the end of Wk 6). Rats were euthanized between ZT 7 - 11 for tissue collection, because it has been shown that culture preparations between ZT 5 - 11 will not reset the SCN

(Yoshikawa et al., 2005). Whole brains, pituitary and adrenal glands were quickly collected into

Hank’s balanced salt solution (#14065; Gibco, Thermo Fisher Scientific Inc.) on ice (Yamazaki and Takahashi, 2005). The SCN was microdissected from 300 µm coronal brain slices collected using a vibratome (Leica VT1200S; Leica Microsystems, Nussloch, Germany). The pituitary gland was flattened and hand-sliced into halves. The adrenal gland was hand-sliced into 1 - 2 mm cubes. All tissues were placed on Millicell-CM culture membranes (#PICMORG50;

Millipore, Bedford, MA) in 35-mm culture dishes containing 1.2 ml of Dulbecco's modified

Eagle's medium (#D2902; Sigma Aldrich, St. Louis, MO) with 0.1 mM luciferin (#115144359;

Gold Bio-technology Inc., St. Louis, MO) (Yamazaki and Takahashi, 2005). Glass coverslips were placed over the culture dishes and sealed with sterile vacuum grease. Explants were maintained in constant darkness and temperature (~ 36.5 °C). We continuously monitored the luminescence from individual dishes using a LumiCycle 32 system (ActiMetrics, Wilmette, IL).

Data were collected at 1-min intervals using LumiCycle software (ActiMetrics). All recordings started by ZT 12.

Cultures that did not exhibit rhythmic expression of luminescence for at least 3 days were excluded from analysis (LumiCycle Analysis; ActiMetrics). The raw data from rhythmic explants were then detrended using a 24-hr running average and smoothed using a 3-hr running

90 average, followed by the estimation of acrophase (time of the peak luminescence) using Origin

9.0.0 (OriginLab Corp., Northampton, MA). The first 20 hours of recording were excluded due to transient fluctuations in luminescence after tissue preparation, and thus only the acrophase of the first full cycle was measured. Vector data (acrophase in degrees) were analyzed by

Rayleigh's test for uniformity and Rao's spacing test for random distribution in Oriana 4.02

(Kovach Computing Services, Pentraeth, Wales, United Kingdom).

Statistical Analyses

Data are expressed as mean ± SEM, unless otherwise indicated. We used the mixed linear model analysis of variance (ANOVA) in SPSS Statistics 17.0 (Polar Engineering and Consulting,

Nikiski, AK) for statistical analyses (Van Dongen et al., 2004). Factors included fixed main effects, fixed interactions between main effects, and a random effect of subjects (rats).

Significant interactions were followed by Bonferroni post-hoc tests. P ≤ 0.05 was considered statistically significant.

Results

Chronic Alcohol Exposure and Pair-feeding

After the first week of alcohol exposure/pair-feeding, liquid diet consumption decreased by 25 - 30% leading to a reduction in body weight gain (Figure 3-1). However, the consumption returned to baseline levels by Wk 3 (data not shown), and both groups of rats had similar rates of weight gain at the time when our measurements were taken (~Wk 6) (Figure 3-1), indicating that they were in a similar metabolic state. Measurements of blood alcohol concentration (BAC) showed average BAC of 0.06% - 0.09% during the light period and 0.10% - 0.11% during the dark period. However, due to high variances in BACs among individual rats, the light/dark differences were not statistically significant.

Chronic Alcohol Exposure Alters Diurnal Variations of Body Temperature and Locomotor

Activity

To determine the effects of chronic alcohol exposure on general physiological and behavioral circadian outputs, we recorded core body temperature and locomotor activity continuously during experiments. Representative actograms of body temperature and locomotor activity from a control rat (Figure 3-2A) and an alcohol-treated rat (Figure 3-2B) are plotted.

Simple visual inspection of the actograms reveals an instability and a trend to be advanced of the daily acrophases in the alcohol-treated rat after the alcohol treatment started. The light/dark characteristics are summarized in Table 3-1. During the baseline period temperature and activity did not differ between groups (Table 3-1, Figure 3-2 and Figure 3-3). Rats showed normal diurnal variations: less activity and lower body temperature in the light period; more activity and higher body temperature in the dark period. The mean temperature (light, dark, and 24-hour) of

92 alcohol-fed rats decreased significantly after the alcohol exposure. Controls showed a small reduction in the mean temperature in the dark period leading to a small reduction in the 24-hour mean temperature (0.05℃). However, alcohol-treated rats showed a much more dramatic reduction in body temperature compared to controls (Con: 0.07℃ in the dark period; Alc: 0.27℃ in the light period and 0.36℃ in the dark period). The summed locomotor activity counts in the dark period of both groups decreased after treatment, leading to reduced overall counts.

However, alcohol-fed rats exhibited a reduction in activity counts more than twice as large as controls (Con: 1258 counts/day; Alc: 2636 counts/day).

Further analysis of the data at 1-hr intervals enabled us to examine details within in the light and dark periods more closely. We found significant effects of the alcohol treatment on diurnal rhythms of temperature and locomotor activity (Figure 3-3). Alcohol-treated rats showed lower body temperature between ZT 0 - 10 and between ZT 14 - 24 (Figure 3-3A) and lower activity counts in the late dark and very early light period compared to controls (Figure 3-3B), and the normal peak of activity at around ZT 15 - 17 was blunted in alcohol-fed rats (Figure 3-

3B).

We compared rhythm parameters (period, amplitude and acrophase) for temperature and activity during the baseline period and after the treatment. The time difference between acrophases and the correlation between temperature and activity counts were examined as indicators of phase relationships. The results are summarized in Table 3-2. During the baseline period, there were no differences between groups in any of the parameters measured. Before and after the alcohol/pair-feeding treatment, both groups showed a normal 24-hr period in temperature and activity. However, while the amplitude of temperature rhythm did not change in either group, the acrophase of temperature rhythm was advanced significantly in alcohol-treated

93 rats. In contrast, the amplitude of activity rhythm was reduced in both groups after treatment, and the reduction in alcohol-treated rats was more dramatic, consistent with their larger decrease of activity counts in the dark after alcohol exposure. Alcohol-treated rats also showed an advancement in the acrophase of activity rhythm, but it was less than the advance in the acrophase of temperature rhythm. As a result, the normal phase relationship in which the acrophase of activity rhythm preceded the acrophase of temperature rhythm was abolished in alcohol-fed rats after the treatment (Table 3-2, Acrophase Difference). These changes resulted in decreases in the correlation between activity and temperature in both groups of rats (Table 3-2,

Act X Temp Correlation), but the decrease was greater in alcohol-treated rats (Con: 0.034; Alc:

0.094).

We used the discrete wavelet transform (DWT) analysis to estimate the circadian energy and circadian variability of temperature and activity as indicators of rhythm stability. Results are summarized in Table 3-3. No differences between groups were observed during the baseline period. However, chronic alcohol exposure decreased the circadian energy of temperature and locomotor activity, and increased the circadian variability of activity without changing this parameter for temperature. Low circadian energy combined with high circadian variability in alcohol-fed rats indicates that their rhythms are less robust, coherent and stable.

Effects of chronic alcohol exposure on plasma corticosterone (CORT) levels

We measured plasma CORT concentrations at 4-hr intervals to determine the effects of chronic alcohol on the activity of the HPA axis. The CORT levels before lights-off (ZT 11 - 12) were similar in both groups of rats (Con: 334.0 ± 22.5 ng/ml; Alc: 333.8 ± 20.9 ng/ml), but alcohol-treated rats had significantly elevated CORT levels at ZT 3 - 4 and decreased CORT

94 levels at ZT 15 – 16 (Figure 3-4). Due to the limitation of our sampling schedule, we were not able to use cosinor analysis to determine rhythm parameters. Thus, illustrative sine-wave fits were generated based on averaged data for each group (Figure 3-4). Visual inspection of fitted curves suggests that the diurnal rhythm of CORT was blunted (peak of fits: Con, 198.0 %; Alc,

172.5 %) and advanced (acrophase of fits: Con, ZT 13.5; Alc, ZT 11.8) in alcohol-fed rats.

Chronic alcohol exposure advanced the acrophase of Per1 expression in the adrenal and pituitary glands but not in the SCN

To determine whether chronic alcohol affected the molecular clock, and at which level chronic alcohol might have exerted its effects, we measured ex vivo Period1 (Per1) expression in the master pacemaker (SCN) and at two peripheral sites within the HPA axis (pituitary and adrenal glands). Representative traces of detrended and smoothed luminescence of the SCN and the pituitary and adrenal glands from a control rat and an alcohol-fed rat are plotted in Figure 3-

5A. We found that the acrophase of Per1 expression in alcohol-treated rats was advanced in the pituitary gland (Con: 13.044 ± 0.744 hr; Alc: 8.944 ± 0.572 hr) and adrenal gland (Con: 14.442

± 0.459 hr; Alc: 9.565 ± 1.326 hr) compared to controls while the acrophases in the SCN were not different between alcohol-treated and control rats (Con: 6.822 ± 0.992 hr; Alc: 6.755 ± 0.642 hr) (Figure 3-5B). In addition, chronic alcohol advanced the acrophase of the adrenal clock slightly more than that of the pituitary clock (pituitary: ~4.1 hrs; adrenal: ~4.9 hrs). As a result, the phase differences between the master pacemaker and peripheral clocks and between the adrenal and pituitary clocks were reduced by chronic alcohol.

Discussion

In the present study, we observed major alterations in rhythms of body temperature, locomotor activity, plasma corticosterone (CORT) levels, and Per1 expression in rats after 6 weeks of alcohol exposure. With the exception of Per1 expression in the SCN, the rhythms of all other parameters we measured (temperature, locomotor activity, CORT, Per1 expression in the adrenal and pituitary) were advanced by chronic alcohol. In addition, the normal phase difference between diurnal rhythms of temperature and activity was abolished by the alcohol exposure. Taken together, our results suggest that the primary effect of chronic alcohol on circadian rhythms is to cause desynchrony among rhythms and molecular clocks. Although this desynchrony may appear to be relatively modest, the cumulative effect of desynchrony over an extended period of time can potentially cause profound negative consequences on health, such as metabolic syndromes and cognitive impairments (Cho et al., 2000; Voigt et al., 2013).

Changes in rhythmic physiological parameters after chronic alcohol exposure are common in both rodent and human studies. For example, alcoholics often show disturbed sleep- wake cycles and disrupted diurnal rhythms of body temperature and hormone secretion (Bertello et al., 1982; Brower, 2003; Wasielewski and Holloway, 2001). Several studies in rodents reported that chronic alcohol decreased body temperature both in the light and dark period and decreased locomotor activity in the dark similarly to our findings, but these studies either did not examine the relationship between temperature and activity or found no effects on the acrophase of temperature (Damaggio and Gorman, 2014; Taylor et al., 2006). In our study we found that chronic alcohol advanced the acrophase of the diurnal rhythms of temperature and activity, abolished their phase lag and reduced the correlation between these two parameters, which all

96 suggest that an important aspect of alcohol-induced circadian disruption is changing the relationship between outputs. Moreover, we found that chronic alcohol advanced the molecular clock (Per1) in the adrenal and pituitary glands but not in the SCN, consistent with the previous report that chronic alcohol did not affect the molecular clock in the SCN (as measured by Per2 expression) while advancing clocks in the liver (Filiano et al., 2013). Other groups have reported alterations in the rhythmic expression of clock genes in the SCN after alcohol (Chen et al., 2004,

2006; Farnell et al., 2008). However, these studies either used prenatal or neonatal alcohol exposure in which the effects could be developmental or used adult rats with two weeks of alcohol exposure where changes in only Per2 and Per3 but not Per1were observed. In addition, samples in these studies were collected at 4-hr intervals thereby providing limited temporal resolution. Our results combined with those of others suggest that the observed desynchrony after chronic alcohol is likely to be an effect on the synchronization between central and peripheral clocks instead of disruptions on the central molecular clock in the SCN per se.

Much effort has gone into determining whether alcohol affects the SCN using various in vivo and in vitro approaches (Prosser and Glass, 2014). Previous studies suggested that chronic alcohol had little effect on the ability of the SCN to entrain to external L:D cycles (Brager et al.,

2010), consistent with our results that rats exhibited robust 24-hr periodicities in temperature and activity under a 12:12 L:D cycle. Although we found minimal effects on the molecular clock in the SCN as measured by Per1 expression in our paradigm, other animal studies suggested that chronic alcohol exposure could interfere with normal functions of the SCN. For example, the number of neurons containing vasopressin, vasoactive intestinal polypeptide, and several other neuropeptides in the SCN was shown to be decreased after chronic alcohol (Madeira et al.,

1997). Chronic alcohol also disrupts the responsiveness of the SCN to both photic and non- photic phase resetting (Seggio et al., 2007). In addition, under constant conditions without the strong Zeitgeber of light, chronic alcohol shortens the periodicity of wheel-running behaviors

(Seggio et al., 2009). Although the advanced acrophases of the diurnal rhythms we observed are generally consistent with a shortened period under constant conditions, our studies were performed under a 12:12 L:D condition and therefore were not able to address this issue directly.

It is well documented that chronic alcohol induces circadian alterations at many sites other than the SCN. Huang and colleagues reported blunted rhythms of clock gene expression in human peripheral blood mononuclear cells during alcohol withdrawal (Huang et al., 2010). In animal studies, prenatal alcohol exposure and chronic alcohol in adulthood disrupted the expression of period genes and pro-opiomelanocortin in the arcuate nucleus of rats (Chen et al.,

2004, 2006), while neonatal alcohol exposure advanced the rhythm of Per2 in the cerebellum

(Farnell et al., 2008). Chronic alcohol was also shown to decrease natural killer (NK) cell activity by disrupting rhythms of granzyme B, perforin and interferon-γ (IFN-γ) in the spleen

(Arjona et al., 2004). Diurnal rhythms of blood glucose, lactic acid and cholesterol were also shifted following chronic alcohol (Rajakrishnan et al., 1999). Lastly, changes in the expression of clock and clock-controlled genes in the liver have been observed after chronic alcohol (Farnell et al., 2008; Filiano et al., 2013; Zhou et al., 2014). In the present study, we found multiple changes in rhythms within the HPA axis after chronic alcohol. First, the diurnal rhythm of plasma CORT was blunted and advanced in alcohol-treated rats. In alcoholics, normal, overall elevated and sporadic diurnal rhythms of cortisol have all been reported (Bertello et al., 1982;

Margraf et al., 1967; Stokes, 1973). In mice, blunted diurnal CORT rhythms along with

98 dampened amplitude have been seen after chronic alcohol (Kakihana and Moore, 1976; Sipp et al., 1993), findings consistent with our results in rats. Moreover, we found that chronic alcohol in rats advanced the molecular clock (Per1) in the adrenal and pituitary glands, yet the SCN was unaffected. Our findings, along with previous reports on many organs, suggest that peripheral clocks are susceptible to disruptions caused by chronic alcohol exposure.

The exact mechanism whereby alcohol induces desynchrony remains to be determined.

However, two processes seem to be promising candidates. First, the changes in the diurnal rhythm of body temperature after chronic alcohol could contribute to the system-wide desynchrony. Body temperature exhibits prominent circadian rhythms and reflects the integrated influence of locomotor activity, sleep/wake cycles and metabolism (Refinetti and Menaker,

1992). The molecular clock in the SCN is temperature-compensated and therefore is resistant to the normal daily fluctuations of body temperature (Ruby et al., 1999). However, for peripheral clocks which are not temperature compensated, temperature can serve as a universal resetting cue (Buhr et al., 2010). In the context of the current study where temperature acrophase was advanced, the other clocks may have followed. However, in our measurements the acrophase of the temperature rhythm was only advanced about 30 minutes, whereas the molecular clocks in the pituitary and adrenal were advanced by 4-5 hours. Besides shifting the acrophase, we also found chronic alcohol reduced body temperature across the day, suggestive that chronic alcohol lowered the set-point of thermoregulation. Whether this also contributes to the alcohol-induced desynchrony remains unclear. Another potential contributor to alcohol-induced desynchrony is the peripheral clock in the HPA axis, particularly the CORT rhythm. Glucocorticoids such as

CORT can also act as potent resetting cues to entrain peripheral clocks via glucocorticoid

99 response elements found in many genes (Balsalobre et al., 2000). Although the HPA axis is under multisynaptic control by the SCN, the adrenal also has its own clock that gates the rhythmic secretion of glucocorticoids (Son et al., 2011). Therefore, chronic alcohol exposure may have led to alterations in the CORT rhythm via peripheral effects at which point the changes could have been propagated to other organs via an endocrine action.

Chronic alcohol consumption leads to internal desynchrony among rhythms in multiple organs. The consequence of this disruption is likely to compromise the physiological integrity of individuals with alcohol use disorders and degrade their ability to efficiently engage with the environment. Precisely how the desynchrony occurs and the extent to which it contributes to alcohol-related pathologies requires future investigation. Circadian disruptions are also manifested in mental disorders such as depression (Bunney and Potkin, 2008). Thus, circadian therapeutic interventions focused on reinstating proper phase relationships of rhythmic physiological systems may be beneficial in a variety of clinical settings.

100

Figure 3-1. Rate of Body Weight Change. BSL: baseline; AC: acclimation; Wk 1 - 6: 6 weeks of alcohol exposure/pair-feeding. Analysis by mixed model 2-way ANOVA showed a significant main effect of Time (BSL, Wk 1 to Wk 6) (F (6, 119) = 31.325, p < 0.001) but no differences between groups. Controls showed a non-significant trend to gain more weight at Wk 2 than alcohol-treated rats.

101

Figure 3-2. Representative Actograms of Body Temperature and Locomotor Activity from a Control and an Alcohol-treated Rat. Temperature: left; Locomotor: right. Actograms are double-plotted. While-black bars on top of actograms indicate the 12h:12h Light:Dark cycle. For the control rat, black arrows indicate the start of the pair-feeding treatment with the days marked blue on the actograms. For the alcohol-treated rat, black arrows indicate the start of the alcohol treatment protocol with the days marked red. Red dots on actograms indicate the daily acrophase estimated by Clocklab.

102

Figure 3-3. Diurnal Rhythms of Body Temperature and Locomotor Activity during the

Baseline Period and at the End of the 6-week Alcohol Exposure/Pair-feeding Treatment.

Temperature: A; Locomotor: B. Baseline: left; After treatment: right. X-axis: zeitgeber (ZT) time. White-grey shading: the 12:12 L:D cycle. Illustrative sine-wave fits are imposed on the averaged data of each panel. Analysis by mixed model 3-way ANOVA showed significant interactions between Group (Con vs. Alc), Time (ZT Time) and Treatment (Baseline vs. 6 weeks of alcohol exposure/pair-feeding) in body temperature (F (23, 846) = 3.690, p < 0.001) and locomotor activity (F (23, 846) = 3.523, p < 0.001). During baseline there were no differences between groups. +: significantly different from controls by Bonferroni post-hoc tests.

103

Figure 3-4. Effects of Chronic Alcohol Exposure on Plasma Corticosterone (CORT).

Results within each cohort were normalized to the average of all measurements within a cohort.

Analysis by mixed model 2-way ANOVA showed a significant Group (Con vs. Alc) X Time (ZT

Time) interaction (F (5, 67.87) = 3.173, p = 0.012). Illustrative sine-wave fits based on the averaged data of each group are imposed on the figure. Asterisks indicate points significantly different from controls by Bonferroni post-hoc tests. *: p < 0.05.

104

Figure 3-5. Effects of Chronic Alcohol Exposure on Per1 Expression in the SCN and the

Pituitary and Adrenal Glands. (A) Representative detrended and smoothed luminescence traces from a control rat and an alcohol-treated rat in the SCN (left) and the pituitary (middle) and adrenal glands (right). Gray shadings: the Light:Dark cycle before tissue collection. In each panel, traces are offset on the Y-axis to prevent overlapping. Triangles indicate the acrophases on the first full cycle. Analysis of acrophases by mixed model 2-way ANOVA showed a significant

Group (Con vs. Alc) X Tissue (SCN vs. Pituitary vs. Adrenal) interaction (F (2, 14.385) = 5.448, p =

0.017). Bonferroni post-hoc tests showed that the Per1 acrophase was significantly advanced in the pituitary gland (p = 0.001) and adrenal gland (p < 0.001) in alcohol-treated rats compared to

105 controls. (B) Vector plots of Per1 acrophases. Each dot represents the acrophase measured from an individual rat. The clock hands point to the mean vector (θ, in degrees) for each group. The length of the hand (r) represents how closely the data are clustered around the mean vector; for perfect alignment, r = 1. SCN: Con, θ = 101.082°, r = 0.839; Alc, θ = 100.362°, r = 0.919.

Pituitary: Con, θ = 195.771°, r = 0.872; Alc, θ = 135.022°, r = 0.925. Adrenal: Con, θ =

216.433°, r = 0.937; Alc, θ = 150.423°, r = 0.657. Rayleigh's test indicated modal directions in all measurements. SCN: Con, Z = 4.22, p < 0.01; Alc: Z = 5.91, p < 0.001. Pituitary: Con, Z =

6.09, p < 0.001; Alc, Z = 6.85, p < 0.001. Adrenal: Con, Z = 8.78, p < 0.001; Alc, Z = 3.45, p <

0.05. Rao's spacing test indicated non-random distributions in all measurements except for the adrenal Per1 acrophase in alcohol-treated rats which showed a tendency to non-random distribution. SCN: Con, U = 213.7, p < 0.01; Alc: U = 238.171, p < 0.01. Pituitary: Con, U =

229, p < 0.01; Alc, U = 242.5, p < 0.01. Adrenal: Con, U = 252.2, p < 0.01; Alc, U = 170, p <

0.1.

106

Table 3-1. Light vs. Dark Characteristics of Body Temperature and Locomotor Activity

Body Temperature (°C) Locomotor Activity (Counts)

Treatment Parameters Control (n = 10) Alcohol (n = 10) p Value Control (n = 10) Alcohol (n = 10) p Value

Light 37.38 ± 0.03 37.39 ± 0.02 ns 2,535 ± 79 2,858 ± 110 ns

Baseline Dark 38.15 ± 0.04 38.14 ± 0.02 ns 9,561 ± 221 9,533 ± 537 ns

Total 37.77 ± 0.03 37.77 ± 0.02 ns 12,096 ± 250 12,391 ± 626 ns

Light 37.35 ± 0.02 37.12 ± 0.03 ### <0.001 2,307 ± 96 2,566 ± 87 ns After 6-wk Alcohol or Dark 38.08 ± 0.03 # 37.78 ± 0.02 ### <0.001 8,303 ± 212 ### 6,897 ± 368 ### <0.001 Pair-feeding Total 37.72 ± 0.03 # 37.45 ± 0.02 ### <0.001 10,611 ± 281 ### 9,463 ± 412 ### ns

Body temperature expressed as mean body temperature in the light period (Light), the dark period (Dark), and the total 24 hour (Total); locomotor activity expressed as summed activity counts in corresponding periods.

For Light versus Dark comparisons, mixed model ANOVA included 3 fixed factors:

Light Condition (Light vs. Dark), Group (Con vs. Alc) and Treatment (Baseline vs. 6 weeks of alcohol exposure/pair-feeding). For Total comparison, mixed model ANOVA included 2 factors:

Group and Treatment. Light/Dark body temperature: significant main effect of Light Condition,

F(1, 54) = 1949.868, p < 0.001, and interaction between Group and Treatment, F(1, 54) = 64.423, p <

0.001. Total body temperature: significant Group X Treatment interaction, F(1, 18) = 89.441, p <

0.001. Light/Dark locomotor activity: significant Light Condition X Group X Treatment interaction, F(1, 54) = 5.270, p = 0.026. Total locomotor activity: significant Group X Treatment interaction, F(1, 18) = 11.659, p = 0.003. Column of p values: Bonferroni post-hoc tests between groups; ns: not significantly different. #: significant difference between baseline and after treatment within a group; #p < 0.05; ##p < 0.01; ###p < 0.001.

107

Table 3-2. Rhythm Parameters for Body Temperature and Locomotor Activity

Body Temperature Locomotor Activity p p Treatment Parameters Control (n = 10) Alcohol (n = 10) Control (n = 10) Alcohol (n = 10) Value Value Period 24.0 ± 0.03 24.0 ± 0.02 ns 24.0 ± 0.02 24.0 ± 0.03 ns (hour) Amplitude 0.55 ± 0.03 0.54 ± 0.02 ns 6.6 ± 0.2 6.2 ± 0.4 ns Acrophase (ZT Time, 18.08 ± 0.11 17.87 ± 0.09 ns 17.66 ± 0.14 17.60 ± 0.10 ns hour) Baseline Acrophase Difference (Act- — — — -25.6 ± 5.5 -16.6 ± 6.8 ns Temp, minute) Act X — — — Temp 0.887 ± 0.007 0.889 ± 0.009 ns Correlation Period 24.0 ± 0.02 24.0 ± 0.06 ns 24.0 ± 0.04 24.0 ± 0.04 ns (hour) Amplitude 0.54 ± 0.02 0.49 ± 0.03 ns 5.6 ± 0.2 ### 4.0 ± 0.4 ### <0.01 Acrophase (ZT Time, 18.37 ± 0.15 16.96 ± 0.14 ### <0.001 17.75 ± 0.15 17.03 ± 0.14 ## <0.01 After 6-wk hour) Alcohol or Acrophase Pair- Difference feeding (Act- — — — -37.0 ± 7.2 4.2 ± 7.3 # <0.001 Temp, minute) Act X — — — Temp 0.853 ± 0.008 ### 0.795 ± 0.001 ### <0.001 Correlation

For analysis, mixed model ANOVA included 2 fixed factors: Group (Con vs. Alc) and

Treatment (Baseline vs. 6 weeks of alcohol exposure/pair-feeding). Body temperature: no

effects in Period or Amplitude; a significant Group X Treatment interaction in Acrophase,

F(1,18) = 30.888, p < 0.001. Locomotor activity: no effects in Period; significant Group X

Treatment interaction in Amplitude, F(1, 18) = 14.800, p = 0.001, and in Acrophase, F(1, 18) =

8.903, p = 0.008. Acrophase Difference (Act-Temp): significant Group X Treatment

interaction, F(1, 18) = 7.799, p = 0.012. Act X Temp correlation: significant Group X Treatment

# interaction, F(1, 18) = 16.957, p = 0.001. Column of p values and : same as in Table 3-1.

108

Table 3-3. Results of Discrete Wavelet Transform (DWT) Analysis of Body Temperature

and Locomotor Activity

Body Temperature Locomotor Activity

Treatment Parameters Control (n = 10) Alcohol (n = 10) p Value Control (n = 10) Alcohol (n = 10) p Value

Circadian 70 ± 2 68 ± 2 ns 42 ± 1 38 ± 2 ns Energy (%) Baseline Circadian Variability 0.54 ± 0.05 0.49 ± 0.04 ns 0.51 ± 0.02 0.57 ± 0.04 ns (hour) Circadian 70 ± 1 59 ± 2 ### <0.001 41 ± 1 25 ± 2 ### <0.001 After 6-wk Energy (%) Alcohol or Circadian Pair-feeding Variability 0.50 ± 0.03 0.56 ± 0.03 ns 0.48 ± 0.02 0.66 ± 0.04 # <0.001 (hour)

Mixed model ANOVA included 2 fixed factors: Group (Con vs. Alc) and Treatment

(Baseline vs. 6 weeks of alcohol exposure/pair-feeding). Body temperature: significant Group

X Treatment interaction in circadian energy, F(1, 18) = 16.106, p = 0.001; no effects in

circadian variability. Locomotor activity: significant Group X Treatment interaction in

circadian energy, F(1, 18) = 51.081, p < 0.001, and in circadian variability, F(1, 18) = 4.800, p

= 0.042. Column of p values and #: same as in Table 3-1.

109

References

Allen CD, Lee S, Koob GF and Rivier C (2011) Immediate and prolonged effects of alcohol exposure on the activity of the hypothalamic-pituitary-adrenal axis in adult and adolescent rats. Brain, Behavior, and Immunity 25 (Suppl 1): S50-60.

Arjona A, Boyadjieva N and Sarkar DK (2004) Circadian rhythms of Granzyme B, Perforin, IFN- , and NK cell cytolytic activity in the spleen: Effects of chronic ethanol. The Journal of Immunology 172 (5): 2811-2817.

Balsalobre A, Brown SA, Marcacci L, Tronche F, Kellendonk C, Reichardt HM, Schütz G and Schibler U (2000) Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science 289 (5488): 2344-2347.

Brower KJ (2003) Insomnia, alcoholism and relapse. Sleep Medicine Reviews 7 (6): 523-539.

Buckley TM and Schatzberg AF (2005) On the interactions of the hypothalamic-pituitary- adrenal (HPA) axis and sleep: normal HPA axis activity and circadian rhythm, exemplary sleep disorders. The Journal of Clinical Endocrinology and Metabolism 90 (5): 3106- 3114.

Buhr ED, Yoo SH and Takahashi JS (2010) Temperature as a universal resetting cue for mammalian circadian oscillators. Science 330 (6002): 379-385.

Bunney JN and Potkin SG (2008) Circadian abnormalities, molecular clock genes and chronobiological treatments in depression. British Medical Bulletin 86 (1): 23-32.

Chen CP, Kuhn P, Advis JP and Sarkar DK (2006) Prenatal ethanol exposure alters the expression of period genes governing the circadian function of beta-endorphin neurons in the hypothalamus. Journal of Neurochemistry 97 (4): 1026-1033.

110

Cho K, Ennaceur A, Cole JC and Suh CK (2000) Chronic jet lag produces cognitive deficits. The Journal of Neuroscience 20 (6): RC66.

Clarke TK, Treutlein J, Zimmermann US, Kiefer F, Skowronek MH, Rietschel M, Mann K and Schumann G (2008) HPA-axis activity in alcoholism: examples for a gene-environment interaction. Addiction Biology 13 (1): 1-14.

Damaggio AS and Gorman MR (2014) The circadian timing system in ethanol consumption and dependence. Behavioral Neuroscience 128 (3): 371-386.

Dibner C, Schibler U and Albrecht U (2010) The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annual Review of Physiology 72 (1): 517-549.

Farnell YZ, Allen GC, Nahm S, Neuendorff N, West JR, Chen WA and Earnest DJ (2008) Neonatal alcohol exposure differentially alters clock gene oscillations within the suprachiasmatic nucleus, cerebellum, and liver of adult rats. Alcoholism: Clinical and Experimental Research 32 (3): 544-552.

Huang MC, Ho CW, Chen CH, Liu SC, Chen CC and Leu SJ (2010) Reduced expression of circadian clock genes in male alcoholic patients. Alcoholism: Clinical and Experimental Research 34 (11): 1899-1904.

Jansen HT, Sergeeva A, Stark G and Sorg BA (2012) Circadian discrimination of reward: evidence for simultaneous yet separable food-and drug-entrained rhythms in the rat. Chronobiology International 29 (4): 454-468.

Kakihana R and Moore JA (1976) Circadian rhythm of corticosterone in mice: the effect of chronic consumption of alcohol. Psychopharmacologia 46 (3): 301-305.

Kalsbeek A, van der Spek R, Lei J, Endert E, Buijs RM and Fliers E (2012) Circadian rhythms in the hypothalamo-pituitary-adrenal (HPA) axis. Molecular and Cellular Endocrinology 349 (1): 20-29. Kawano Y, Pontes CS, Abe H, Takishita S and Omae T (2002) Effects of alcohol consumption and restriction on home blood pressure in hypertensive patients: serial changes in the morning and evening records. Clinical and Experimental Hypertension 24 (1-2): 33-39.

Koob GF (2008) A role for brain stress systems in addiction. Neuron 59 (1): 11-34.

Leise TL and Harrington ME (2011) Wavelet-based time series analysis of circadian rhythms. Journal of Biological Rhythms 26 (5): 454-463.

111

Madeira MD, Andrade JP, Lieberman AR, Sousa N, Almeida OFX and Paula-Barbosa MM (1997) Chronic alcohol consumption and withdrawal do not induce cell death in the suprachiasmatic nucleus, but lead to irreversible depression of peptide immunoreactivity and mRNA levels. The Journal of Neuroscience 17 (4): 1302-1319.

Margraf HW, Moyer CA, Ashford LE and Lavalle LW (1967) Adrenocortical function in alcoholics. Journal of Surgical Research 7 (2): 55-62.

Prosser RA and Glass JD (2014) Assessing ethanol's actions in the suprachiasmatic circadian clock using in vivo and in vitro approaches. Alcohol 49 (4): 321-339.

Rajakrishnan V, Subramanian P, Viswanathan P and Menon VP (1999) Effect of chronic ethanol ingestion on biochemical circadian rhythms in Wistar rats. Alcohol 18 (2): 147-152.

Refinetti R 2006. Circadian Physiology, New York, CRC Press.

Refinetti R and Menaker M (1992) The circadian rhythm of body temperature. Physiology & Behavior 51 (3): 613-637.

Reppert SM and Weaver DR (2002) Coordination of circadian timing in mammals. Nature 418 (6901): 935-41.

Roy A, Mittal N, Zhang H and Pandey SC (2002) Modulation of cellular expression of glucocorticoid receptor and glucocorticoid response element-DNA binding in rat brain during alcohol drinking and withdrawal. Journal of Pharmacology and Experimental Therapeutics 301 (2): 774-784.

Ruby NF, Burns DE and Heller HC (1999) Circadian rhythms in the suprachiasmatic nucleus are temperature-compensated and phase-shifted by heat pulses in vitro. The Journal of Neuroscience 19 (19): 8630-8636.

Sarkar DK (2012) Circadian genes, the stress axis, and alcoholism. Alcohol Research: Current Reviews 34 (3): 362-366.

112

Silva SM, Paula-Barbosa MM and Madeira MD (2002) Prolonged alcohol intake leads to reversible depression of corticotropin-releasing hormone and vasopressin immunoreactivity and mRNA levels in the parvocellular neurons of the paraventricular nucleus. Brain Research 954 (1): 82-93.

Sipp TL, Blank SE, Lee EG and Meadows GG (1993) Plasma corticosterone response to chronic ethanol consumption and exercise stress. Experimental Biology and Medicine 204 (2): 184-190.

Son GH, Chung S and Kim K (2011) The adrenal peripheral clock: glucocorticoid and the circadian timing system. Frontiers in Neuroendocrinology 32 (4): 451-465.

Spanagel R, Rosenwasser AM, Schumann G and Sarkar DK (2005) Alcohol consumption and the body's biological clock. Alcoholism: Clinical and Experimental Research 29 (8): 1550-1557.

Stokes PE (1973) Adrenocortical activation in alcoholics during chronic drinking. Annals of the New York Academy of Sciences 215 (1): 77-83.

Taylor AN, Tio DL, Bando JK, Romeo HE and Prolo P (2006) Differential effects of alcohol consumption and withdrawal on circadian temperature and activity rhythms in Sprague- Dawley, Lewis, and Fischer male and female rats. Alcoholism: Clinical and Experimental Research 30 (3): 438-447.

Van Dongen HP, Olofsen E, Dinges DF and Maislin G (2004) Mixed-model regression analysis and dealing with interindividual differences. Methods in Enzymology 384 139-171.

Voigt RM, Forsyth CB and Keshavarzian A (2013) Circadian disruption: potential implications in inflammatory and metabolic diseases associated with alcohol. Alcohol Research: Current Reviews 35 (1): 87-96.

Wasielewski JA and Holloway FA (2001) Alcohol's interactions with circadian rhythms. Alcohol Research & Health 25 94-100.

Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M and Tei H (2000) Resetting central and peripheral circadian oscillators in transgenic rats. Science 288 (5466): 682-685.

Yamazaki S and Takahashi JS (2005) Real-time luminescence reporting of circadian gene expression in mammals. Methods in Enzymology 393 288-301.

Yoshikawa T, Yamazaki S and Menaker M (2005) Effects of preparation time on phase of cultured tissues reveal complexity of circadian organization. Journal of Biological Rhythms 20 (6): 500-512.

113

Zhou P, Ross RA, Pywell CM, Liangpunsakul S and Duffield GE (2014) Disturbances in the murine hepatic circadian clock in alcohol-induced hepatic steatosis. Scientific Reports - Nature 4 (3725): 1-11.

114

CHAPTER FOUR

CONSTANT DARKNESS ALLEVIATES CHRONIC ALCOHOL-INDUCED

CIRCADIAN AND SLEEP DISRUPTIONS IN RATS

Rong Guo, Heiko T. Jansen, and Steven M. Simasko

Programs in Neuroscience

Department of Integrative Physiology and Neuroscience

College of Veterinary Medicine

Washington State University, Pullman, Washington, USA, 99164

115

Abstract

Introduction: Chronic alcohol consumption leads to sleep and circadian disruptions, which often persist during abstinence and may contribute to relapse. Chronic alcohol treatment in rats can blunt the diurnal distribution of sleep without affecting the total amount of sleep time, suggesting that the circadian regulation of sleep may be compromised.

Methods: Male Sprague-Dawley rats (n = 8 for each group) were exposed to chronic alcohol treatment (6% ethanol in liquid diet) for 9 weeks under 12h:12h Light:Dark (L:D), followed by 4 weeks under constant darkness (D:D). The alcohol treatment was then removed for another 4 weeks under D:D, followed by 3 weeks under L:D. We recorded sleep and monitored body temperature and locomotor activity.

Results: Alcohol-treated rats showed blunted diurnal distribution of sleep-wake time and dampened and destabilized daily rhythms of body temperature and locomotor activity during chronic alcohol treatment under L:D. Chronic alcohol-treated rats under D:D showed normalized distribution of sleep-wake time between the peak active and inactive periods. The changes in circadian parameters were also restored in D:D, although the body temperature in the active period of alcohol-treated rats was still lower than that of the controls. Rats in immediate withdrawal from the alcohol treatment under D:D showed reduced sleep and increased wake, along with increased body temperature in the inactive (resting) period.

Conclusions: Chronic alcohol exposure blunted the diurnal distribution of sleep and weakened the daily rhythms of temperature and locomotor under normal L:D condition, which were alleviated by placing rats under constant darkness. Our results suggest that the observed

116 sleep disturbances caused by chronic alcohol treatment may be a result of the desynchrony between the external L:D cycle and the internal circadian control of sleep timing.

117

Introduction

Circadian rhythms play an indispensable role in preparing animals to anticipate periodic environmental changes and to adapt their physiological processes accordingly. Mammalian circadian rhythms are organized in a hierarchy with the master clock located in the suprachiasmatic nucleus (SCN). Core clock genes, such as Period, Clock, Bmal1 and

Cryptochrome, and their corresponding proteins constitute the molecular clock in the central pacemaker, while other clock genes and proteins also participate in various aspects of the circadian system. The auto-regulatory transcription-translation feedback loop of core clock genes takes approximately 24 hours, setting the periodicity of internal rhythms (King and Takahashi,

2000; Ko and Takahashi, 2006; Moore, 2013). Clock genes are also expressed in all tissues and organs, composing various peripheral clocks downstream of the SCN (Balsalobre, 2002). One of the key functions of the SCN is to reset and synchronize internal physiological processes and peripheral clocks, including sleep, with the external light/dark cycle. If the internal rhythms and the external light Zeitgeber are not synchronized with each other, such as in cases of jet-lag and shift-worker disorders, many health problems may develop (Voigt et al., 2013).

Circadian and sleep disruptions are commonly seen in individuals recovering from alcohol use disorder (AUD), including abnormal sleep/wake cycles and disrupted diurnal rhythms of blood pressure, body temperature, and hormone secretion (Imatoh et al., 1986;

Kumagai et al., 1992; Wasielewski and Holloway, 2001). Individuals with AUD often suffer from persistent sleep disturbances, such as insomnia, sleep fragmentation, disrupted rapid eye movement sleep (REMS), and decreased slow wave sleep (SWS) (Colrain et al., 2014). The

118 acrophase of REMS in AUD patients has also been reported to be phase-advanced by several hours (Imatoh et al., 1986). Despite the fact that circadian rhythms play an important role in regulating sleep timing, whether the chronic alcohol-induced sleep and circadian disruptions share common mechanisms and whether chronic alcohol use disturbs sleep via circadian regulatory processes remain unclear.

Findings from our previous work in rats indicate that chronic alcohol exposure is likely to disrupt sleep timing via the circadian process (Mukherjee and Simasko, 2009). In this previous study, we found that 6 weeks of chronic alcohol treatment in rats blunted the diurnal distribution of sleep/wake time without affecting the total amount of wake, non-REMS (NREMS), and

REMS over a 24-hour period. Alcohol-treated rats had equally distributed sleep and wake time between the light and dark periods The constant amount of total sleep/wake time over 24 hours after chronic alcohol treatment is consistent with relatively intact sleep homeostasis and potential disruptions in the circadian control of sleep timing. Moreover, we next challenged both alcohol- treated and control rats to an acute sleep deprivation and found that both groups of rats showed relatively normal and robust rebound in both sleep time and sleep quality (as measured by slow wave amplitude), further suggesting that the ability of animals to respond to the build-up of sleep pressure is mostly intact after chronic alcohol treatment (Guo et al, 2015). We also thoroughly examined the effects of our chronic alcohol treatment directly on several circadian parameters, including body temperature, locomotor activity, plasma corticosterone, and Period 1 gene expression, and found that chronic alcohol exposure did not change the 24-hour periodicity of rhythms, but rather caused an internal desynchrony among various circadian processes and between the central and peripheral molecular clocks (Guo et al., 2016). However, whether the

119 circadian disruptions caused by chronic alcohol exposure in our model directly contributes to the observed sleep disturbances, especially the re-distribution of sleep/wake time, remains unknown.

To answer this question, we placed control and alcohol-treated rats under conditions of constant darkness (D:D) and measured sleep, body temperature and locomotor activity. Under conditions when the external light cues would have been removed, such as in D:D, the circadian system should still retain its ability to coordinate different physiological processes, albeit in a free running mode. Thus, without the light Zeitgeber, we should be able to determine whether the desynchrony we observed were due to alcohol-induced disruptions in the SCN’s ability to process and integrate external lights Zeitgeber or due to changes within the intrinsic processes of the circadian systems. We found that alcohol-treated rats showed blunted sleep/wake time and destabilized daily rhythms of temperature and locomotor activity during chronic alcohol treatment under L:D, although the 24-hr periodicity of both temperature and locomotor rhythms remained unchanged. On the contrary, when alcohol-treated rats were held under D:D, they showed a normalized distribution of sleep-wake time between the peak active and inactive periods. The changes in circadian parameters induced by chronic alcohol treatment under L:D were also eliminated by D:D, although the overall body temperature of alcohol-treated rats was still lower than that of the controls in the active period. Then, when rats were in early abstinence under D:D, we observed reduced sleep and increased wake in the inactive (resting) period, along with increased inactive body temperature and unstable rhythms. After prolonged recovery, when rats were eventually returned to the L:D conditions, the diurnal rhythms of body temperature became stable and robust again. These results suggest that the observed sleep disturbances, especially the blunted distribution of sleep/wake time during chronic alcohol treatment is likely

120 caused by an external desynchrony between the external L:D cycle and the internal circadian control of sleep after prolonged alcohol use.

121

Materials and Methods

Animals

Male Sprague-Dawley rats (~ 300 g, Harlan, Indianapolis, IN) were randomly assigned to either receive ethanol-containing liquid diet (#F1436SP; Bio-Serv, Flemington, NJ) or to be pair- fed with the control liquid diet (#F1268SP; Bio-Serv). Rats were housed individually in acrylic enclosures in a temperature- and humidity- controlled environment on a 12:12 Light:Dark (L:D) cycle (Mukherjee and Simasko, 2009) or under constant darkness (D:D). Zeitgeber Time (ZT) 0: lights-on; ZT 12: lights-off. All experimental procedures were approved by the Institutional

Animal Care and Use Committee of Washington State University (protocol #4116).

Chronic Alcohol Exposure and Pair-feeding

We used a previously published protocol of chronic alcohol exposure in liquid diet (Guo et al., 2016). All rats had ad-lib access to the control diet during the 2-week baseline period. The alcohol exposure protocol started with gradual acclimations to the ethanol-containing diet (2 days at 2% followed by 2 days at 4% ethanol) and was maintained at 6%. Control rats were pair- fed with the control diet (ethanol calories replaced with maltose dextrin) based on the average group volume that was consumed by the alcohol-treated rats one day before. Alcohol-treated rats were kept for 9 weeks under 12h:12h Light:Dark (L:D), followed by 4 weeks under constant darkness (D:D). We then removed the alcohol from liquid diet and kept rats for another 4 weeks under D:D, followed by 3 weeks under L:D. All rats had ad lib access to water throughout all experiments. We refilled liquid diet feeding tubes daily under dim red illumination (<5 lux) at random times in the dark period to prevent the potential entrainment to any disturbances. Rats

122 had continuous access to diet during sleep recordings. We weighed rats weekly at times of cage change.

Core Body Temperature

We continuously monitor core body temperature via a telemetry system (MiniMitter,

Philips Respironics Inc., Bend, OR) as previously published (Guo et al., 2016). Transmitters

(PDT-4000 E-Mitter) were implanted into the peritoneal cavity of rats at the same time when

EEG/EMG electrodes were implanted. Receivers (ER-4000 series) were located beneath each cage. Data were collected by VitalView 4.2 (MiniMitter Inc.) at 1-min intervals and analyzed in

2-hour bins. Temperature recordings continued during sleep recordings. Fifteen days of temperature data (15-min binned) were used for the discrete wavelet transform (DWT) analysis in MATLAB R2015a with custom scripts written by Tanya Leise (Leise and Harrington, 2011).

We estimated the circadian energy and variability within the range of circadian periods (16 - 32 hrs). Seven days of body temperature data were averaged to generate the daily profile. Data under D:D were aligned to Time 0 which is the acrophase of the daily temperature rhythm estimated by sine wave fitting.

Sleep Recordings and Sleep Data Analysis

We used a stereotactic apparatus to implant two electroencephalogram (EEG) electrodes with stainless-steel screws (#E363/20/2.4/SP; PlasticsOne Inc., Roanoke, VA, USA) over the right frontal and the left parietal cortices under isoflurane anesthesia (Kubota et al., 2002).

Another two stainless-steel screws (#0-80X3/32; PlasticsOne Inc.) were mounted on the skull at the corresponding contralateral positions to facilitate anchoring the dental cement. Two

123 electromyography (EMG) electrodes were tied into the nuchal neck muscles. We made EMG electrodes from multi-stranded Teflon-insulated stainless steel wire (#212-50F- 357-0; New

England Wire, Lisbon, NH) (Phillips et al., 2011), with 4 mm of exposed wire on one end and a gold-plated stainless steel socket contact lead (#E363/0; PlasticsOne Inc.) soldered on the other end. All leads from electrodes were fed into a plastic pedestal (#MS363; PlasticsOne Inc.).

Electrodes and pedestal were secured and sealed by dental composite resin (Prime-Dent, Prime

Dental Manufacturing Inc., Chicago, IL, USA). Rats were allowed at least 1 week of recovery before the baseline recordings started.

Before sleep recording started, rats in their enclosures were moved into environmental chambers (4 rats/chamber) and tethered to swivel commutators (#SL6C; PlasticsOne Inc.) by 6- channel cables (#363-363; PlasticsOne Inc.) to allow unrestricted movement. Rats were allowed

3 days of acclimation. Signals were filtered (-6 dB low/high-frequency filters) and amplified by high-performance AC pre-amplifiers (Model P511; Grass Technologies, Natus Neurology

Incorporated, Warwick, RI). EEG signals were filtered below 0.3 Hz and above 30 Hz and amplified 5000 times; EMG signals were filtered below 30 Hz and above 300 Hz and amplified

2000 times. Signals were digitalized and collected at 128 Hz via Vital Recorder (Kissei Comtec

Co., LTD., Matsumoto, Japan).

Vigilance states (wake, NREMS and REMS) were determined off-line in 10 sec epochs from visually scored EEG/EMG records in SleepSign for Animal (Kissei Comtec Co., LTD.) based on standard criteria (Kubota et al., 2002). Several continuous days of sleep recordings under D:D were averaged before analysis. Sleep recordings under D:D were aligned to time 0 in the active and inactive period. Time 0 in the active period is the acrophase of the daily body temperature rhythm estimated by sine wave fitting. Time 0 in the inactive period is the trough of

124 the daily temperature rhythm estimated by sine wave fitting. Fast fourier transform (FFT) was performed within each individual epoch. We measured slow wave amplitude (SWA) as the averaged spectral power in the delta wave frequencies (0.5 to 4 Hz) during NREMS epochs.

Statistical Analyses

We expressed data as mean ± SEM unless otherwise indicated. Two-tailed student t-tests and mixed linear model analysis of variance (ANOVA) (SPSS Statistics 17.0; Polar Engineering and Consulting, Nikiski, AK) were used for statistical analyses. Mixed model ANOVA includes fixed main effects, fixed interactions between main effects, and a random effect of subjects (rats)

(Van Dongen et al., 2004). Significant interactions were followed by Bonferroni posthoc tests. P

≤ 0.05 was considered statistically significant.

125

Results

Chronic Alcohol Exposure under L:D Destabilized the Diurnal Rhythms of Body Temperature and Blunted the Diurnal Distribution of Sleep and Wake Time

To determine the effects of chronic alcohol exposure on physiological rhythms, we recorded core body temperature continuously during experiments. Representative actograms from a control and an alcohol-treated rat are plotted in Figure 4-1. Simple visual inspection of the actograms reveals a less stable and dampened rhythm of the alcohol-treated rat after the alcohol treatment started. Under L:D, our chronic alcohol treatment decreased body temperature in the dark (active) period, leading to a trend of decreased overall 24-hr body temperature

(Figure 4-2B). We further compared various rhythm parameters, including rhythm periodicity, amplitude, robustness, circadian energy and circadian variability. The results are summarized in

Table 4. Alcohol-treated rats showed smaller amplitude, reduced rhythm robustness, increased circadian variability and reduced circadian energy, which all suggest less stable and robust daily rhythms. Both groups of rats showed periodicity of around 24 hours under the normal 12h:12h

L:D cycles. We also measured sleep under L:D during the chronic alcohol exposure (Figure 4-3).

In the light (inactive) period, alcohol- treated rats showed more wake and less NREMS and

REMS; in the dark (active) period, alcohol-treated rats showed less wake and more NREMS and

REMS compared to controls. We did not find significant difference in the total amount of wake and REMS within 24 hours, although in this study we observed more NREMS in alcohol-treated rats as a result of non-significant decreased in wake and REMS time.

126

Constant Darkness Stabilized the Daily Rhythms of Body Temperature and Normalized the

Distribution of Sleep and Wake Time during Chronic Alcohol Exposure

After 9 week of chronic alcohol treatment under L:D, we placed rats under constant darkness for 4 weeks. The 4th week of body temperature data were used to generate the daily rhythm profile (Figure 4-4). Under D:D, the body temperature of alcohol-treated rats still remained lower in the active period, leading to a trend of lower overall body temperature (Figure

4-4B). However, when comparing the rhythm parameters (Table 4), the differences we observed between alcohol-treated and control rats in rhythm amplitude, rhythm robustness, circadian variability, and circadian energy were eliminated by constant darkness, suggesting that there was no difference in rhythm stability between the two groups of rats under D:D. The periodicities of free-running body temperature rhythm were not significantly different between groups, although alcohol-treated rats showed a trend for longer periodicity (Table 4). Because the body temperature rhythm under D:D was stable and robust, we were able to use the daily acrophase of temperature rhythm to align several days of sleep recordings under D:D. In terms of sleep time, there were no differences in wake and NREMS (Figure 4-5). Both groups of rats showed similar amount of wake and NREMS between the inactive and the active period. However, the REMS of alcohol-treated rats was suppressed during the inactive period, leading to decreased amount of total REMS within the 8 hours we measured. The observed REMS suppression is consistent with the effect of acute alcohol exposure or the presence of alcohol in the blood and tissues (Knowles et al., 1968).

Withdrawal from Alcohol under D:D Dampened the Daily Rhythms of Body Temperature and

Disrupts Sleep in the Inactive Period

127

After 4 week of chronic alcohol treatment under D:D, we discontinued the alcohol treatment from the liquid diet and kept the rats under constant darkness for another 3 weeks. The body temperature data within the first 2 week of withdrawal were used to generate the daily profiles. The acute withdrawal from our alcohol treatment caused a robust rebound of body temperature, especially during the inactive period in rats previously treated with alcohol, leading to an increased overall body temperature (Figure 4-6). Rats in withdrawal also showed lower rhythm amplitude, rhythm robustness and circadian energy, indicating less robust rhythms (Table

4). There was no difference in the circadian variability or periodicity between groups. We measured sleep during the first couple days immediately following the removal of the alcohol treatment. Rats in acute withdrawal showed increased wake and reduced sleep (both NREMS and REMS) during the inactive period when the majority of sleep should have happened (Figure

4-7). The total amount of wake was also higher and the amount of NREMS was lower in rats during alcohol withdrawal, suggesting disrupted sleep during acute withdrawal from alcohol.

Diurnal Rhythms of Body Temperature Return to Normal after Prolonged Abstinence

After 7 weeks in total under the constant darkness we moved rats back to a 12h:12h L:D cycle for another 3-4 weeks till they showed stable entrainment to the assigned light/dark cycle.

At the end of the L:D treatment, rats after prolonged abstinence showed normal rhythms of body temperature (Figure 4-8B) although the body temperature during the early light period and the late dark period were still higher than that of the control rats (Figure 4-8A). There were no differences between groups in any of the rhythm parameters we measured (Table 4).

128

Discussion

In the present study, we observed major alterations in the sleep/wake cycle and the diurnal rhythms of body temperature during our chronic alcohol treatment under 12h:12h L:D cycles, which were alleviated by constant darkness. The normal distribution of sleep and wake time was blunted and the rhythms of temperature were destabilized by chronic alcohol exposure in L:D. Constant darkness normalized the blunted distribution of sleep/wake time and restored the stability of daily rhythms in alcohol-treated rats. Taken together, our results suggest that the chronic alcohol exposure may cause a desynchrony between the external L:D cycle and the internal circadian control of sleep timing, which is at least partially responsible for the observed sleep disturbances after prolonged alcohol use.

Several studies using animal models have shown that chronic alcohol exposure attenuates responses to photic phase shifting, alters the periodicity of wheel-running behaviors under constant conditions, and reduces locomotor activities in the dark (active) period, but has no effect on the ability to entrain to normal LD cycles (Brager et al., 2010; Rosenwasser et al., 2005b;

Ruby et al., 2009; Seggio et al., 2009; Seggio et al., 2007). In our study, the diurnal rhythms of body temperature and locomotor activity maintained normal 24-hour periodicity under L:D, consistent with previous findings. However, we also found no difference in free-running periodicity under D:D between alcohol-treated and control rats, which contradicts with findings from some previous reports. Several groups reported that chronic alcohol treatment shortened periodicity of wheel-running behaviors in mice, neonatal rats, and Syrian hamsters, while in some other studies, both lengthening and shortening of the periodicity of wheel-running behaviors have been observed in rats after chronic alcohol exposure (Dwyer and Rosenwasser,

129

1998; Mistlberger and Nadeau, 1992; Rosenwasser et al., 2005a; Seggio et al., 2009). The rhythms of drinking behavior are different between the AA (Alko Alcohol) and ANA (Alko

Non-Alcohol) rat lines, but the mechanistic explanation of this difference in phenotype is unclear (Aalto, 1986). It is possible that we did not observe any differences in periodicity because we measured body temperature and general locomotor activity instead of wheel-running behavior or drinking behavior. This inconsistency may also be explained by species, animals strain, and the method and dosage of the alcohol administration.

It is well established that chronic alcohol use can induce profound circadian disruptions on both the physiological and molecular level. Although inconsistency exists in the exact extent in which chronic alcohol use affects the rhythms of hormones, AUD patients tend to have normal, or elevated, or phase-shifted, or blunted cortisol rhythms, blunted diurnal testosterone levels, and normal or inversed melatonin rhythms (Bertello et al., 1982; Majumdar and Miles,

1987; Murialdo et al., 1991; Stokes, 1973). The diurnal rhythms of plasma monoamine metabolites are also phase-advanced and elevated in AUD patients with severe withdrawal symptoms (Sano et al., 1992; Sano et al., 1993). On the molecular level, Huang and colleagues recently find reduced and blunted mRNA expression of several key clock genes, including Per,

Bmal, and Cry, in the peripheral blood mononuclear cells of patients in withdrawal (Huang et al.,

2010). McCarthy et al. also suggest that the periodicity of Per2 expression in cultured skin fibroblast cells from alcoholic patients inversely correlates with the severity of AUD (McCarthy et al., 2013). The mechanism in which chronic alcohol exposure leads to this desynchrony needs to be further explored.

130

Figure 4-1. Representative Actograms of Body Temperature from a Control and an

Alcohol-treated Rat. Con: left; Alc: right. Actograms are double-plotted. White-grey shadings beneath actograms indicate the Light:Dark cycle (12:12 L:D and D:D). For the control rat, black arrows indicate the start and end of the pair-feeding treatment; for the alcohol-treated rat, black arrows indicate the start and end of the alcohol treatment protocol.

131

A B o d y T e m p e r a t u r e 1 0 1 C o n tr o l (n = 9 )

A lc o h o l (n = 8 )

)

n a

a 1 0 0

9 9

d (

o + + + + + + + + B

9 8 0 6 1 2 1 8 2 4

Z e it g e b e r T im e (H o u r)

B B o d y T e m p e r a t u r e 1 0 1 1 0 1 C o n

A lc

)

a e

u 1 0 0 * * 1 0 0

s a

m 9 9 9 9

% T

(

9 8 9 8 L ig h t D a r k T o t a l

Figure 4-2. Body Temperature in L:D during Chronic Alcohol Exposure.

(A). Daily Rhythms of Body Temperature. X-axis: Zeitgeber time. White/Grey shadings:

light/dark schedule. Two weeks of data were normalized to baseline to generate the daily

profiles (1-hr bin). Crosses (+): significantly different from controls by Bonferroni post-hoc

tests. (B). Mean Body Temperature during the Light/Dark Period, and the Total 24 hrs.

Asterisks: significantly different from controls by Bonferroni post-hoc tests, * p < 0.05, ** p

< 0.01, *** p < 0.001.

132

A W a k e N R E M S le e p R E M S le e p 1 0 0 1 0 0 2 5 C o n tro l (n = 8)

A lco h o l (n = 8)

8 0 8 0 2 0

)

s s

h h

+ + + + + h + + +

6 0 6 0 1 5

(

e e

4 0 4 0 e 1 0

T T 2 0 2 0 T 5 + + + + + + + + + 0 0 0 0 4 8 1 2 1 6 2 0 2 4 0 4 8 1 2 1 6 2 0 2 4 0 4 8 1 2 1 6 2 0 2 4 Z e it g e b e r T im e (H o u r) Z e it g e b e r T im e (H o u r) Z e it g e b e r T im e (H o u r)

B W a k e N R E M S le e p R E M S le e p 9 1 2 9 1 5 2 3

C o n

) )

) A lc **

r r

r *

o o

o 6 *** 8 6 1 0 *** 2

H H

H ***

( (

( ***

** 1

e e

3 4 3 5 1

T T T 0 0 0 0 0 0 L ig h t D a r k T o ta l L ig h t D a r k T o ta l L ig h t D a r k T o ta l

Figure 4-3. Sleep in L:D during Chronic Alcohol Exposure.

(A). Wake, NREM and REM Sleep Time. X-axis: Zeitgeber time. White/Grey shadings:

light/dark schedule. Crosses (+): significantly different from controls by Bonferroni post-hoc

tests. (B). Mean Wake, NREM and REM Sleep Time during the Light / Dark period, and the

Total 24-hr Period. Asterisks: significantly different from controls by Bonferroni post-hoc

tests, * p < 0.05, ** p < 0.01, *** p < 0.001.

133

A B o d y T e m p e r a t u r e 1 0 1 C o n tr o l (n = 8 )

A lc o h o l (n = 8 )

)

n a

a 1 0 0

9 9

(

o B

9 8 - 6 0 6 1 2 1 8

T im e f r o m A c r o p h a s e (H o u r)

B B o d y T e m p e r a t u r e

1 0 1 1 0 1 C o n

A lc

)

a e

u 1 0 0 * 1 0 0

s a

m 9 9 9 9

T (

9 8 9 8 In a c t i v e A c t iv e T o t a l

Figure 4-4. Body Temperature in D:D during Chronic Alcohol Exposure.

(A). Daily Rhythms of Body Temperature. X-axis: Time from the Acrophase of the body

temperature rhythms. Data under D:D were aligned to the acrophase (Time 0) of the

temperature rhythms estimated by sine-wave fitting. While/Grey shadings: light/dark

schedule. Two weeks of data were normalized to baseline to generate the daily profiles (1-hr

bin). Crosses (+): significantly different from controls by Bonferroni post-hoc tests. (B).

Mean Body Temperature during the Light/Dark Period, and the Total 24 hrs. Asterisks:

significantly different from controls by Bonferroni post-hoc tests, * p < 0.05, ** p < 0.01,

*** p < 0.001.

134

A W a k e N R E M S le e p R E M S le e p 1 0 0 1 0 0 2 5 C o n tro l (n = 4)

A lco h o l (n = 8)

8 0 8 0 2 0

) ) )

s s s

h h h

2 2 2

/ / /

6 0 6 0 1 5

n n n

i i i

m m m

( ( (

e e e 4 0 4 0 1 0

+ +

m m m

i i i

T T T 2 0 2 0 5

In a c tiv e P e rio d A c tiv e P e rio d In a c tiv e P e rio d A c tiv e P e rio d In a c tiv e P e rio d A c tiv e P e rio d 0 0 0 -4 0 4 -4 0 4 -4 0 4 -4 0 4 -4 0 4 -4 0 4 T im e (H o u r) T im e (H o u r) T im e (H o u r)

W a k e N R E M S le e p R E M S le e p 6 9 6 1 2 1 .5 2 .4

C o n

) )

A lc )

u u u **

o 4 o

6 4 8 o 1 .0 1 .6 *

( (

( *

e e 2

m 3 2 4 0 .5 0 .8

T T

0 0 0 0 0 .0 0 .0 In a c tiv e A c tiv e T o ta l In a c tiv e A c tiv e T o ta l In a c tiv e A c tiv e T o ta l

Figure 4-5. Sleep in D:D during Chronic Alcohol Exposure.

(A). Wake, NREM and REM Sleep Time. Data under D:D were aligned to the acrophase

(Time 0 of active period) and trough (Time 0 of inactive period) of the temperature rhythms

estimated by sine-wave fitting. While/Grey shadings: light/dark schedule. Crosses (+):

significantly different from controls by Bonferroni post-hoc tests. (B). Mean Wake, NREM

and REM Sleep Time during the Light / Dark period, and the Total 24-hr Period. Asterisks:

significantly different from controls by Bonferroni post-hoc tests, * p < 0.05, ** p < 0.01,

*** p < 0.001.

135

A B o d y T e m p e r a t u r e 1 0 1 C o n tr o l (n = 8 )

N o A lc o h o l (n = 8 )

)

n a

a 1 0 0

9 9

(

o B

9 8 - 6 0 6 1 2 1 8

T im e f r o m A c r o p h a s e (H o u r)

B B o d y T e m p e r a t u r e

1 0 1 1 0 1 C o n

N o A lc

)

a e

u 1 0 0 1 0 0 t

M *

i l

e * *

s a

m 9 9 9 9

T (

9 8 9 8 In a c t i v e A c t iv e T o t a l

Figure 4-6. Body Temperature in D:D during Withdrawal of Alcohol.

(A). Daily Rhythms of Body Temperature. X-axis: Time from the Acrophase of the body temperature rhythms. Data under D:D were aligned to the acrophase (Time 0) of the temperature rhythms estimated by sine-wave fitting. While/Grey shadings: light/dark schedule. Two weeks of data were normalized to baseline to generate the daily profiles (1-hr bin). Crosses (+): significantly different from controls by Bonferroni post-hoc tests. (B). Mean Body Temperature during the Light/Dark Period, and the Total 24 hrs. Asterisks: significantly different from controls by Bonferroni post-hoc tests, * p < 0.05, ** p < 0.01, *** p < 0.001.

136

A W a k e N R E M S le e p R E M S le e p 1 0 0 1 0 0 2 5 C o n tro l (n = 4)

W ith d ra w (n = 8)

8 0 8 0 2 0

)

/ /

+ + /

6 0 6 0 1 5

(

e e 4 0 4 0 e 1 0

+ +

T T 2 0 2 0 T 5

In a c tiv e P e rio d A c tiv e P e rio d In a c tiv e P e rio d A c tiv e P e rio d In a c tiv e P e rio d A c tiv e P e rio d 0 0 0 -4 0 4 -4 0 4 -4 0 4 -4 0 4 -4 0 4 -4 0 4 T im e (H o u r) T im e T im e (H o u r)

B W a k e N R E M S le e p R E M S le e p 6 9 6 1 2 1 .5 2 .4

C o n

) )

W ith d r a w )

r r

u ** ** u * u

o 4 o

6 4 8 * o 1 .0 1 .6

( (

( *

e e *** e 2

m 3 2 4 0 .5 0 .8

T T

0 0 0 0 0 .0 0 .0 In a c tiv e A c tiv e T o ta l In a c tiv e A c tiv e T o ta l In a c tiv e A c tiv e T o ta l

Figure 4-7. Sleep in D:D during Acute Withdrawal from Alcohol.

(A). Wake, NREM and REM Sleep Time during the first couple days after acute removal of the alcohol treatment. Data under D:D were aligned to the acrophase (Time 0 of active period) and trough (Time 0 of inactive period) of the temperature rhythms estimated by sine-wave fitting.

While/Grey shadings: light/dark schedule. Crosses (+): significantly different from controls by

Bonferroni post-hoc tests. (B). Mean Wake, NREM and REM Sleep Time during the Light /

Dark period, and the Total 24-hr Period. Asterisks: significantly different from controls by

Bonferroni post-hoc tests, * p < 0.05, ** p < 0.01, *** p < 0.001.

137

A B o d y T e m p e r a t u r e 1 0 1 C o n tr o l (n = 9 )

N o A lc o h o l (n = 8 )

r u

) +

n a

a 1 0 0

l m

e +

9 9

d (

o + + + + B

9 8 0 6 1 2 1 8 2 4

Z e it g e b e r T im e (H o u r)

B B o d y T e m p e r a t u r e 1 0 1 1 0 1 C o n

N o A lc

)

a e

u 1 0 0 1 0 0

s a

m 9 9 9 9

T (

9 8 9 8 L ig h t D a r k T o t a l

Figure 4-8. Temperature in L:D after Prolonged Abstinence.

(A). Daily Rhythms of Body Temperature. X-axis: Zeitgeber time. White/Grey shadings: light/dark schedule. Two weeks of data were normalized to baseline to generate the daily profiles

(1-hr bin). Crosses (+): significantly different from controls by Bonferroni post-hoc tests. (B).

Mean Body Temperature during the Light/Dark Period, and the Total 24 hrs. Asterisks: significantly different from controls by Bonferroni post-hoc tests, * p < 0.05, ** p < 0.01, *** p

< 0.001.

138

Table 4. Rhythm Parameters for Body Temperature

Light Treatment Parameters Control (n = 9) Alcohol (n = 8) p Value Condition Period (hour) 24.11 ± 0.09 24.03 ± 0.06 ns Amplitude (% Baseline) - - - 12h:12h Baseline Robustness (%) 46.2 ± 3.7 54.4 ± 3.3 ns L:D Circadian Energy (%) 50 ± 4 58 ± 4 ns Circadian Variability (hour) 0.79 ± 0.09 0.66 ± 0.07 ns

Period (hour) 24.03 ± 0.03 23.95 ± 0.03 0.074 Amplitude (% Baseline) 103.2 ± 10.1 61.0 ± 3.7 < 0.01 12h:12h Robustness (%) 40.8 ± 3.0 21.2 ± 2.7 < 0.001 L:D Circadian Energy (%) 48 ± 3 28 ± 3 < 0.001 Chronic Circadian Variability (hour) 0.81 ± 0.06 1.3 ± 0.18 < 0.05 Alcohol Treatment Period (hour) 24.23 ± 0.05 24.38 ± 0.06 0.078 Amplitude (% Baseline) 95.99 ± 7.6 76.41 ± 6.1 0.068 D:D Robustness (%) 37.7 ± 4.1 32.8 ± 4.1 ns Circadian Energy (%) 46 ± 4 41 ± 4 ns Circadian Variability (hour) 0.92 ± 0.12 0.91 ± 0.11 ns

Period (hour) 24.31 ± 0.07 24.38 ± 0.07 ns Amplitude (% Baseline) 95.1 ± 9.0 66.0 ± 3.0 < 0.05 D:D Robustness (%) 37.9 ± 2.9 28.2 ± 2.5 < 0.05 Circadian Energy (%) 45 ± 3 35 ± 2 < 0.05 No Circadian Variability (hour) 0.78 ± 0.07 0.93 ± 0.10 ns Alcohol Period (hour) 24.02 ± 0.04 24.08 ± 0.02 ns Amplitude (% Baseline) 85.7 ± 7.0 81.8 ± 6.9 ns 12h:12h Robustness (%) 32.9 ± 3.6 38.5 ± 3.6 ns L:D Circadian Energy (%) 42 ± 3 46 ± 4 ns Circadian Variability (hour) 0.60 ± 0.07 0.66 ± 0.06 ns Column of p values: significant difference between groups by unpaired two-tailed student t-tests;

NS: not significantly different.

139

Reference

Aalto J (1986) Circadian drinking rhythms and blood alcohol levels in two rat lines developed for their alcohol consumption. Alcohol 3 (1): 73-75.

Balsalobre A (2002) Clock genes in mammalian peripheral tissues. Cell and Tissue Research 309 (1): 193-199.

Colrain IM, Nicholas CL and Baker FC (2014) Alcohol and the sleeping brain, ELSEVIER.

Dwyer SM and Rosenwasser AM (1998) Neonatal clomipramine treatment, alcohol intake and circadian rhythms in rats. Psychopharmacology (Berl) 138 (2): 176-183.

Guo R, Simasko SM and Jansen HT (2016) Chronic alcohol consumption in rats leads to desynchrony in diurnal rhythms and molecular clocks. Alcoholism: Clinical and Experimental Research 40 (2): 291-300.

Huang MC, Ho CW, Chen CH, Liu SC, Chen CC and Leu SJ (2010) Reduced expression of circadian clock genes in male alcoholic patients. Alcoholism: Clinical and Experimental Research 34 (11): 1899-1904.

Imatoh N, Nakazawa Y, Ohshima H, Ishibashi M and Yokoyama T (1986) Circadian rhythm of REM sleep of chronic alcoholics during alcohol withdrawal. Drug and alcohol dependence 18 (1): 77-85.

King DP and Takahashi JS (2000) Molecular genetics of circadian rhythms in mammals. Annual review of neuroscience 23 (1): 713-742.

Knowles JB, Laverty SG and Kuechler HA (1968) Effects on REM sleep. Quarterly Journal of Studies on Alcohol 29 (2): 342.

Ko CH and Takahashi JS (2006) Molecular components of the mammalian circadian clock. Human molecular genetics 15 (Suppl 2): R271-R277.

140

Kumagai Y, Shiga T, Sunaga K, Fukushima C, Cornélissen G, Ebihara A and Halberg F (1992) Repeated alcohol intake changes circadian rhythm of ambulatory blood pressure. Chronobiologia 20 (1-2): 77-85.

Majumdar SK and Miles A (1987) Disturbed melatonin secretion in chronic alcoholism and withdrawal. Clinical Chemistry 33 (7): 1291.

Moore RY (2013) The suprachiasmatic nucleus and the circadian timing system. Chronobiology: Biological timing in health and disease.

Rosenwasser AM, Fecteau ME and Logan RW (2005a) Effects of ethanol intake and ethanol withdrawal on free-running circadian activity rhythms in rats. Physiology & Behavior 84 (4): 537-542.

Rosenwasser AM, Logan RW and Fecteau ME (2005b) Chronic ethanol intake alters circadian period-responses to brief light pulses in rats. Chronobiology International 22 (2): 227- 236.

141

Stokes PE (1973) Adrenocortical activation in alcoholics during chronic drinking. Annals of the New York Academy of Sciences 215 (1): 77-83.

Van Dongen HP, Olofsen E, Dinges DF and Maislin G (2004) Mixed-model regression analysis and dealing with interindividual differences. Methods in Enzymology 384 139-171.

Wasielewski JA and Holloway FA (2001) Alcohol's interactions with circadian rhythms. Alcohol Research & Health 25 94-100.

142

CHAPTER FIVE

GENERAL DISCUSSION

Summary of Findings

We first subjected chronic alcohol-treated and control rats to 6 hours of acute sleep deprivation and examined the quantity and quality of the recovery sleep to determine directly whether the homeostatic regulation of sleep was impaired by our chronic alcohol treatment. We found that both control and alcohol-treated rats showed robust compensatory increase in sleep time and sleep intensity (as measured by slow wave amplitude) within 24 hours after the sleep deprivation challenge. However, the recovery sleep of alcohol-treated rats was delayed and fragmented. Results from this study suggest that chronic alcohol exposure may weaken the stability of sleep states leading to sleep fragmentation, but the homeostatic regulatory mechanisms were most likely not dramatically disrupted.

We next determined how chronic alcohol exposure affected the integrity of circadian systems by examining its effects on diurnal rhythms of body temperature, locomotor activity, plasma corticosterone concentrations, and ex vivo Per1 expression in the master pacemaker

(suprachiasmatic nucleus, SCN) and in peripheral clocks within the HPA axis (pituitary and adrenal glands). We found that after chronic alcohol treatment these rhythmic processes became less robust and more variable. Most importantly, the phase relationships among diurnal physiological rhythms, and between the central and peripheral molecular clocks, were significantly altered leading to a state of internal desynchrony after chronic alcohol exposure.

143

We lastly determined the contribution of circadian disruptions to sleep disturbances observed after chronic alcohol treatment by recording sleep when the control and alcohol-treated rats were placed under constant darkness to remove the external light Zeitgeber. In this study we found that constant darkness stabilized the dampened rhythms of body temperature and locomotor activity and normalized the blunted distribution of sleep/wake time caused by chronic alcohol exposure, suggesting that chronic alcohol might have led to a desynchrony between sleep timing and the external light Zeitgeber.

In conclusion, the findings reported in this dissertation suggest that chronic alcohol treatment may disrupt sleep through the circadian regulation process instead of the homeostatic regulation process. Chronic alcohol exposure compromises the ability of the central pacemaker – the SCN- to maintain a tight synchronization among internal clocks and to integrate sleep timing with the external light Zeitgeber. This desynchrony is at least partially responsible for the observed sleep disturbances after chronic alcohol use.

144

Possible Candidate Mechanisms

A. Body Temperature

Chronic alcohol use can cause significant changes in body temperature which may contribute to the system-wide circadian desynchrony and sleep disturbances we have observed in our studies.

Body temperature has a very strong circadian pattern and reflects the integrated influence of locomotor activity, sleep/wake cycles, and metabolism (Refinetti and Menaker, 1992). The daily rhythm of body temperature is closely coupled to the sleep/wake cycle, although the 24- hour rhythmicity of heat production remains during total sleep deprivation (van Someren, 2006).

Core body temperature is the reflection of a balance between heat production and loss. The heat production and loss during sleep are intrinsically different from those during wakefulness. The basal metabolic rate during sleep is lower than that during resting wakefulness. In humans, the core body temperature generally declines in sleep during the night. When body temperature reaches its trough, wake onset is normally initiated and body temperature rises again in the morning. Rather than a passive result of energy metabolism, body temperature is proposed to serve as a universal resetting cue for oscillators downstream of the SCN, which helps synchronize circadian clocks of various peripheral systems (Buhr et al., 2010). Notably, the molecular clock in the SCN is temperature-compensated and therefore is resistant to the normal daily fluctuations of body temperature (Ruby et al., 1999). In addition, body temperature also plays an active modulatory role in sleep regulation (van Someren, 2006). In SCN-lesioned rats, the coupling between the ultradian rhythm of brain temperature and the slow-wave activity

145 persists, although the normal daily rhythm of brain temperature has been abolished (Baker et al.,

2005).

Acute alcohol consumption by its own has a profound effect on lowering body temperature of healthy subjects. Acute consumption of alcohol in the early afternoon reduces body temperature immediately while evening consumption had little acute effect (Devaney et al.,

2003). Yet, both day and night drinking increases body temperature during sleep and reduces the amplitude of the temperature rhythm in healthy men (Danel et al., 2001; Eastman et al., 1994). In rats, acute alcohol in rats can shorten the periodicity and reduce the amplitude of body temperature rhythms; whether alcohol causes phase advance or phase delay depends on the time of administration (Baird et al., 1998).

Chronic alcohol consumption also disrupts temperature. Moderate habitual wine drinking in the night changes the periodicity of the oral and body temperature, but not the acrophase of sleep-wake cycles (Reinberg et al., 2010). AUD patients who experience significant tremor and sweating in acute withdrawal show blunted rhythm of skin temperature with an abnormal late night rising (White et al., 1994). Three weeks after the acute withdrawal, the body temperature rhythm of AUD patients, especially depressive AUD patients, still remains phase-advanced

(Kodama et al., 1988). In mice during withdrawal from 1 week of alcohol exposure in liquid diet, the extent of the hypothermia correlates with the severity of withdrawal symptoms (Ritzmann and Tabakoff, 1976).

In the context of the current studies, we find that chronic alcohol treatment in rats decreases body temperature, especially in the dark/active period, and phase-advances the diurnal temperature rhythm. Whether the lowered set-point of thermoregulation and the shifted timing

146 of the daily rhythm contribute to chronic alcohol-induced sleep and circadian disruptions should be further explored.

B. The Corticotrophin-Releasing Factor (CRF) System, HPA Axis, and Glucocorticoids

(GCs)

Another potential contributor to the sleep and circadian disruptions we observed is the

CRF system and activities of the HPA axis, particularly the CORT rhythm.

The Corticotrophin-Releasing Factor/Hormone (CRF/CRH) is a 41-amino-acid peptide produced mainly by the paraventricular nucleus (PVN). CRF receptors are G-protein coupled receptors, and CRF binding will activate adenylate cyclase and initiate the cAMP cascade. There are two subtypes of CRF receptors with different coding genes, distributions, and functions:

CRF-R1 and CRF-R2. CRF has a high affinity for CRF-R1 only (Vale, 1997). The CRF system has both hormonal and neuronal actions. As a hormone, CRF drives the hypothalamic- pituitary- adrenal (HPA) axis, resulting in the systemic release of glucocorticoids, such as cortisol in humans or corticosterone in rodents. The hormonal action of CRF is mediated mainly through

CRFR1 in the anterior pituitary (Chen, 1993; Van Pett, 2000). As a neurotransmitter, CRF is involved in various endocrine, autonomic, behavioral and immune responses. Neurons with

CRF-like immunoreactivity (CRF-LI) and the expression of CRF-receptor mRNAs are broadly distributed in brain areas outside of the PVN. CRF is critical for behavioral responses to stress

(Smagin, 2001). The CRF system is also involved in addiction and drugs of abuse. Activation of the HPA axis is proposed to be associated with dependence and withdrawal. Microinjections of

CRF antagonists into the central nucleus of amygdala (CeA) can reverse anxiogenic-like effects during withdrawal after chronic administration of drugs such as cocaine (Koob, 1996). CRF can

147 also affect body temperature, food intake, micturition and metabolic functions, which may be collateral to the stress-like effects of CRF or independent of the releasing of glucocorticoids.

CRF may also increase brain excitability and cause seizure-like activity (Ehlers, 1983).

The CRF system has a very strong circadian component. Daily profiles of cortisol and

CORT have been well documented: in humans, plasma cortisol peaks in the late afternoon towards the onset of darkness while in rats/mice plasma CORT peaks before entering the light period. The central CRF activity also exhibits a daily rhythm. CRF in the monkey CSF peaks in the early evening and reaches the nadir in the morning, while cortisol in the CSF peaks in the late morning and reaches the nadir in the evening (Garrick, 1987). In rats, CRF-LI in the hypothalamus peaks right before and after the plasma CORT peak, and has a trough consistent with a massive CRF release leading to subsequent ACTH and CORT production (Moldow,

1984). Circadian regulatory inputs from the suprachiasmatic nucleus (SCN) are necessary to maintain the daily rhythm of the CRF system. SCN-lesion in rats abolishes the daily rhythms and causes a constantly elevated CORT level (Buijs, 1993). The rhythm of hypothalamic CRF persists in adrenalectomized rats with decreased plasma CORT and increased ACTH (Hiroshige,

1971). Hypophysectomized rats have decreased CORT and ACTH and first a small decrease then a significant increase of hypothalamic CRF-IL content (Moldow, 1982).

The CRF system plays an important role in regulating sleep-wake cycles. The HPA axis and sleep have a bidirectional interaction: HPA hormones affect sleep while changes of sleep affect the production of HPA hormones (Steiger, 2002). Plasma cortisol relates to the intensity of transitions among wake and sleep stages on a history-and-time-dependent manner (Yassouridis,

1999). Sleep deepening relates to descending cortisol levels, while sleep lightening relates to ascending cortisol levels (Gronfier, 1999). Healthy humans with higher cortisol levels have

148 longer total sleep time (Späth-Schwalbe, 1992). Administration of ACTH or cortisol affects sleep structure in both humans and rats. Changes in sleep-wake cycles also affect HPA hormone secretion. Partial or total sleep deprivation (SD) usually causes elevated cortisol levels (Leproult,

1997). Acute diurnal sleep only changes the cortisol rhythm subtly (Pietrowsky, 1994), while after long-term shifts, daily cortisol rhythm of is usually blunted, as in shift workers (Weibel,

1998). As a neurotransmitter, endogenous CRF has an arousal effect and promotes wakefulness

(Chang, 1998; Steiger, 2003; Uchida, 2007). CRF is important in sleep disturbances under stresses. Administration of CRF, CRF antagonists or CRF agonists also has various effects on sleep, depending on the kind of drug and the time, method and dosage of drug administration.

Alcohol affects various aspects of the CRF system. Acute alcohol induces a stress response and activates the HPA axis, which causes increases in plasma ACTH (Zakhari, 1993) and CORT levels (Tabakoff, 1978) in rats, while another study fails to see alcohol-induced changes in plasma cortisol levels in healthy males (Davis, 2008). In rats, acute alcohol activates the noradrenergic neurons in the locus coeruleus and the (nor)adrenergic neurons in the medulla, and lesions of these (nor)adrenergic circuits or blockade of α1 adrenergic receptors attenuates the alcohol-induced ACTH release, indicating the involvement of catecholamine system in the effect of alcohol (Lee, 2011). IP injection of alcohol increases the mRNA level of CRF-R1 in rat hypothalamus (Lee, 1997). On the other hand, chronic alcohol consumption usually causes a flattening of the normal daily rhythm of plasma CORT levels in rats and mice (Kakihana, 1976;

Sipp, 1993). In humans, chronic alcohol can increase salivary cortisol levels (Adinoff, 2003), while one study reports hospitalized alcoholics with regular drinking have a normal cortisol rhythm (Bertello, 1982). During abstinence, the acrophase and amplitude of the cortisol rhythm in former alcoholics are different v.s. non-alcoholics (Iranmanesh, 1989; Mukai M., 1998;

149

Valimaki, 1984). In rats during alcohol abstinence, the CRF-IL in the amygdala and in the bed nucleus of the stria terminalis is elevated (Olive, 2002; Pich, 1995).

The CRF system is important in alcohol dependence and alcohol withdrawal (Hellig,

2007). During alcohol withdrawal, rats exhibit various anxiogenic-like responses such as decrease of exploration time in the elevated plus maze test, decrease of social interaction and increase of performance in alcohol self-administration, and these responses can be blocked by injection of CRF antagonists, especially CRF-R1 selective antagonists (Baldwin, 1991;

Menzaghi, 1994; Overstreet, 2004; Rassnick, 1993; Valdez, 2002). The CRF system also relates to alcohol-preferring. One of the CRF-related peptides - urocortin (Ucn) is highly expressed in the Edinger-Westphal (EW) nucleus, and has been shown to be associated with alcohol consumption level and alcohol-preferring. Also, alcohol-preferring rats have lower endogenous

CRF levels in the hypothalamus, amygdala, prefrontal cortex, cingulate cortex and PVN, suggesting that CRF receptors are down-regulated in the alcohol-preferring line (Ehlers, 1992;

Hwang, 2004).

It is essential for survival that the hypothalamic-pituitary-adrenal (HPA) axis produces neuroendocrine, immune, and behavioral adaptations in response to stress. Hypophysiotropic neurons at the medial parvocellular paraventricular nucleus (mpPVN) secret arginine vasopressin (AVP) and corticotropin-releasing factor (CRF) to the anterior pituitary. CRF acts mainly through corticotropin-releasing factor type I receptors (CRF-R1) as the central HPA regulator. Binding to CRF-R1 activates the adenylate cyclase and following cAMP pathways, leading to pro-opiomelanocortin (POMC) gene transcription and subsequent production of adrenocorticotropic hormone (ACTH) in the pituitary. AVP modifies the pituitary CRF responsiveness via vasopressin V1b (V3) receptors (AVP-V1b). ACTH released into circulation

150 then binds to melanocortin type II receptors (MC2-R) on adrenocortical cells and activates cAMP pathways leading to steroidogenesis and secretion of glucocorticoids (primates: cortisol; rodents: corticosterone (CORT)). For a review of HPA organization: (Abel and Majzoub, 2005;

Herman and Cullinan, 1997; Herman et al., 2003; Smith and Vale, 2006; Ziegler and Herman,

2002). Glucocorticoids (GCs) are main downstream effectors in producing homeostatic stress responses (Munck et al., 1984; Sapolsky et al., 2000), and also directly feedback control the HPA axis (Laryea et al., 2015). GCs have heterogeneous effects on multiple systems, such as metabolism and immune functions (Munck et al., 1984; Reddy et al., 2009; Sapolsky et al.,

2000). GCs bind to glucocorticoid receptors (GRs), which can act as transcription factors targeting glucocorticoid response elements (GREs) in the promoter region or directly interact with other transcription factors to regulate gene expression (Reddy et al., 2009). It’s critical for organisms to effectively cope with stress and to restore homeostasis after a disruption.

Dysfunctional HPA axis can cause Cushing's syndrome (hypercortisolism) and Addison's disease

(hypocortisolism) (Liddle, 1972). Abnormal HPA activities have also been reported in many mental disorders and pathological processes (de Kloet et al., 2005), such as schizophrenia

(Bradley and Dinan, 2010), depression (Pariante and Lightman, 2008), autoimmune disorder

(Tsigos and Chrousos, 1994), metabolic syndrome (Pasquali et al., 2006) and neurodegenerative diseases (Landfield, 1994). Disrupted HPA axis may also be responsible for the transition from occasional alcohol use to alcohol dependence/abuse by modulating the brain reward and anti- reward (stress) systems (Koob, 2008) in the allostasis model of alcoholism (Koob, 2003).

Acute alcohol could act as a stressor and stimulate the HPA axis in a dose-dependent manner, despite that low dosages of alcohol (especially with BAC < 100 mg/dl (Jenkins and

Connolly, 1968)) usually failed to be effective (Davis and Jeffcoate, 1983; Gianoulakis et al.,

151

1997; Ida et al., 1992; Leppäluoto et al., 1975) or produced inconsistent results (Ekman et al.,

1994; Rasmussen et al., 1998; Waltman et al., 1993). Alcohol can readily diffuse among tissues and through the brain, potentially affecting all levels of the HPA axis. Acute alcohol can promote adrenal ascorbic acid and cholesterol depletion (Forbes and Duncan, 1951), increase secretions of GCs, ACTH, AVP and CRF (Colbern et al., 1985; Inder et al., 1995; Rivier et al., 1984), dampen the ACTH response to CRF (Waltman et al., 1994) and stress responses to other stressors (Pohorecky et al., 1980), up-regulate CRF and CRF-R1 gene expressions in the PVN

(Lee and Rivier, 1997; Rivier and Lee, 1996; Zoeller and Rudeen, 1992), elevate pituitary

POMC hnRNA levels, and activate neural activities in the PVN (Rivier et al., 1996) and other

HPA-regulating brain areas such as the prefrontal cortex, the central nuclei of amygdala (CeA), and the locus coerulesu (Chang et al., 1995; Knapp et al., 2001; Lee et al., 2011). Human studies were limited by possible measurements of HPA activity and confounding factors, and thus provided few insights into mechanisms underlying alcohol-induced HPA activation. Animal research from Rivier, Lee and others revealed that acute alcohol stimulated the HPA axis via central mechanisms in the PVN, as well as CRF (CRF-R1) and AVP (AVP-V1 b) pathways, independent of peripheral blood alcohol content (László et al., 2001; Lee and Rivier, 1997; Lee et al., 2004; Lee et al., 2001; Lolait et al., 2007; Ogilvie et al., 1997a; Ogilvie et al., 1997b;

Rivest and Rivier, 1994; Rivier and Lee, 1996; Rivier et al., 1996). Early in vitro studies showed that alcohol could directly stimulate the adrenal CORT and the pituitary ACTH productions

(Cobb et al., 1981; Kalant et al., 1963; Redei et al., 1986), reduce the adrenal responsiveness to

ACTH (Guaza and Borrell, 1984), and increase CRF mRNA expression and CRF secretion in the

PVN through cAMP-PKA pathways (Cannizzaro et al., 2010; Li et al., 2005). Acetaldehyde

(ACD), as the intermediate metabolite of alcohol, may partially mediate the central and

152 peripheral HPA-stimulating effects (Cannizzaro et al., 2010; Kinoshita et al., 2001; Pastor et al.,

2004).

Chronic alcohol exposure produces long-lasting HPA disturbances. Human studies showed unaffected (Bertello et al., 1982) or overall elevated (Stokes, 1973) cortisol rhythms and increased rhythm variations (Margraf et al., 1967) in drinking alcoholics, while studies on abstinent patients yielded various results depending on the length of abstinence (Risher-Flowers et al., 1988). Often, abstinent alcoholics would show elevated circulating cortisol and ACTH levels (Boschloo et al., 2011; Wand and Dobs, 1991; Willenbring et al., 1984), and some exhibited hypercortisolism to the extent that resembled Cushing’s syndrome (Besemer et al.,

2011). Dexamethasone suppression and metyrapone blockage failed in some patients, and their responses to ACTH, CRF, and insulin-induced hypoglycemia were compromised (Berman et al.,

1990; Margraf et al., 1967; Wand and Dobs, 1991; Willenbring et al., 1984). In animals, the tolerance to the HPA-simulating effect of acute alcohol developed after prolonged or repeated alcohol exposure (Kakihana et al., 1971; Richardson et al., 2008). Stress responses were blunted

(Mohn et al., 2011; Ratcliffe, 1972; Silva and Madeira, 2012) or potentiated (Macho et al., 2003;

Patterson-Buckendahl et al., 2005), depending on the alcohol exposure procedure and the nature of the stressor. In animal studies, continuous exposure to alcohol for a prolonged period of time equivalent to decades of excessive drinking in human alcoholics was not always guaranteed, and thus opposite or negative results have been reported (Aragon et al., 1986; Richardson et al.,

2008). Previous research using rodent models reported disrupted (Tabakoff et al., 1978), shifted

(Helms et al., 2012), or flattened/dampened (Kakihana and Moore, 1976; Sipp et al., 1993) diurnal GC rhythms and dampened ACTH (Helms et al., 2013; Helms et al., 2012) rhythm.

153

Chronic alcohol exposure interferes with HPA functions on multiple levels. Centrally, chronic alcohol hindered HPA activities by decreasing the number of CRF and AVP neurons and their mRNA and peptide levels, potentially leading to blunted responses to CRF and AVP and reduced HPA responsiveness to stressors (Ishizawa et al., 1990; Lee, 1995; Lee et al., 2000;

Richardson et al., 2008; Silva et al., 2002b; Silva et al., 2002a; Silva et al., 2009). Chronic alcohol also impaired adrenal ACTH sensitivity (Wand and Dobs, 1991) and pituitary responsiveness to CRF (Berman et al., 1990; Dave et al., 1986; Rivier et al., 1990). Adrenal hypertrophy and thymus involution were common after chronic alcohol exposure (Rasmussen et al., 2000; Spencer and McEwen, 1990). GCs played a permissive role in mediating effects of chronic alcohol, through GR-depended pathways (Sze, 1977). Chronic alcohol decreased GRs in many brain regions including the PVN (Roy et al., 2002), while daily administration of a GR blocker reduced the development of tolerance to acute alcohol (Tabakoff and Yanai, 1979).

Acute withdrawal from alcohol is stressful and actives the HPA axis. CRF, ACTH and

GC levels usually peaked within the first couple days of alcohol discontinuation and gradually returned to normal following abstinence, along with the alleviation of withdrawal symptoms

(Adinoff et al., 1991; Burov et al., 1986; Holsboer et al., 1986; Keedwell et al., 2001). Many alcohol-induced HPA abnormalities, including the hyperactivity and abnormal HPA responses, resolved by themselves after protracted abstinence (Ozsoy and Esel, 2008). In fact, stress hormone levels might be lower in dry alcoholics after extended abstinence than in controls (Esel et al., 2001). However, the impaired HPA function might persist for months or even years after abstinence (Bailly et al., 1989; Silva et al., 2009), contributing to alcohol craving/drinking and relapsing (Junghanns et al., 2003; Sinha, 2001; Stephens and Wand, 2012). Alcohol-induced

HPA dysfunction was proposed to cause irreversible neuro-adaptations in the prefrontal cortex

154 through GC-dependent pathways, leading to increased propensity to relapse (Lu and Richardson,

2014).

The HPA axis receives numerous circadian regulations and exhibits prominent rhythms.

Stress responses of the HPA axis to stimuli varied diurnally (Atkinson et al., 2006; Gibbs, 1970;

Kalsbeek et al., 2003; Sage et al., 2001). CRF transcript and peptide concentrations in the PVN and CRF levels in the cerebrospinal fluid (CSF) presented circadian variations (Cai and Wise,

1996; Garrick et al., 1987; Kalin et al., 1987; Kwak et al., 1993; Kwak et al., 1992; Owens et al.,

1990; Watts and Swanson, 1989; Watts et al., 2004). ACTH secretion was moderately rhythmic

(Carnes et al., 1989; Girotti et al., 2009), while adrenal sensitivity and responsiveness to ACTH showed significant diurnal differences (Kaneko et al., 1981; Kaneko et al., 1980; Sage et al.,

2002; Ungar and Halberg, 1962). Moreover, MC2-Rs and downstream components in steroidogenesis, such as steroidogenic acute regulatory (StAR) protein, were rhythmically expressed in the adrenal glands (Dickmeis, 2009; Oster et al., 2006). GC secretion exhibit the most robust diurnal rhythm in the HPA axis in addition to its ultradian pulsatile release pattern

(Waite et al., 2012): rising prior to awakening or the activity onset; dropping to nadir at the beginning of sleep or during the inactive period (Clow et al., 2010; Girotti et al., 2009; van

Cauter et al., 1996). Circadian inputs to the HPA axis is governed centrally by the master pacemaker - the suprachiasmatic nucleus (SCN), which synchronizes and coordinates various physiological processes and entrain animals to the external light/dark cycle (Menaker et al.,

2013). The SCN uses a dual-mechanism to refine HPA activities: SCN neurons regulate mpPVN

CRF activity through projections to the subparaventricular zone (subPVZ) and the dorsomedial nucleus of the hypothalamus (DMH), and via very few direct connections (Buijs et al., 1993); more importantly, the SCN regulates adrenal sensitivity and GC production by communicating

155 with adrenal glands through multi-synaptic autonomic pathways via the splanchnic nerve, independent of ACTH (Buijs et al., 1999; Engeland and Arnhold, 2005; Jasper and Engeland,

1994). Clock genes, whose transcriptional-translational feedback loops constitute the core mechanism of molecular clocks, are expressed not only in the SCN, but also in mostly all tissues, including the PVN, pituitary (Bur et al., 2010; von Gall et al., 2002) and adrenal glands (Bittman et al., 2003; Girotti et al., 2009). The robust GC rhythm is a combined result of SCN integration and the control of peripheral adrenal clocks, which gates adrenal sensitivity to ACTH and regulates steroidogenesis (Oster et al., 2006). Perturbed circadian control of the HPA axis instead of dysfunctional HPA components per se may contribute to diseases. Thus, it is important to update the understanding of HPA dysregulation after chronic alcohol exposure from a circadian perspective.

In our study, we found that chronic alcohol exposure in rats phase-advanced the diurnal rhythm of plasma CORT levels and the molecular clock (Per1) in peripheral HPA sites (adrenal and pituitary) but not at the central pacemaker (SCN). Glucocorticoids such as CORT can act as potent resetting cues to entrain peripheral clocks via glucocorticoid response elements found in many genes, including several clock genes (Balsalobre et al., 2000). Therefore, chronic alcohol exposure may have led to alterations in the CORT rhythm via peripheral effects at which point the changes could have been propagated to other organs via an endocrine action. The exact role played by the CRF system, the HPA axis, and GCs should be tested in future studies.

156

Implications

Results from our studies suggest that chronic alcohol exposure 1) weakens the stability of sleep states leading to unstable and fragments sleep, 2) compromises the ability of SCN to synchronize internal clocks and to integrate sleep timing with the external light Zeitgeber, and 3) does not prevent the build-up of sleep pressure during a homeostatic sleep challenge. Our study supports the idea that chronic alcohol exposure disrupts sleep possibly through the circadian regulation process rather than the homeostatic regulation process. This interpretation can be very helpful in directing future researches to determine he underlying mechanisms by which chronic alcohol exposure disrupts sleep and circadian rhythms.

Instead of focusing on a particular sleep-regulating circuit or sleep promoting substance, future studies should especially pay attention to the SCN. Although in our study we did not see changes in Per1 expression from the ex vivo culture of the SCN slices after chronic alcohol treatment and our results and others’ also suggest that chronic alcohol treatment does not stop the

SCN from entraining to the external L:D cycles, it is possible that chronic alcohol exposure could interfere with normal functions of the SCN. To address this, future experiments using various cellular, molecular, electrorheological, and imaging techniques are needed. It will be important to understand whether chronic alcohol consumption 1) changes the anatomical structures within the SCN, 2) modulates synaptic connections and couplings between SCN neurons, and 3) affects the connection and communication between the SCN and its downstream oscillators. In addition, one of the most interesting findings in our study is that constant darkness alleviated some of the effects of chronic alcohol exposure on sleep and on the body temperature

157 rhythms, suggesting that the sleep and circadian disturbances we observed may be due to alcohol-induced disruptions in the ability of the SCN to integrate sleep timing with the external light Zeitgeber. To determine whether chronic alcohol exposure alters the response of SCN to light signals, an experiment is needed to measure the phase response curve of control and alcohol-treated animals after they have been exposed to light pulses at various circadian times.

Moreover, attentions should be given to examine the pathways and structures associated with the entrainment and the integration of external light Zeitgeber by the SCN.

Finally, more and more studies in the literature are addressing the contribution of circadian disruptions, especially of circadian desynchrony, in other AUD-related health problems, such as leaky gut and alcoholic liver. Clock genes are involved with molecular pathways critical for addiction and also with exerting effects of chronic drug use in the peripheral systems. The circadian system not only may contribute to the general well-being of patients and affect their vulnerability to drugs of abuse but also may directly mediate multiple negative effects of chronic alcohol use on many organs and tissues.

158

References

Abel KB and Majzoub JA (2005) Molecular biology of the HPA axis. In: STECKLER T, KALIN NH and REUL J (eds.) Handbook of Stress and the Brain, Part 1: The Neurobiology of Stress. Elsevier.

Adinoff B, Risher-Flowers D, De Jong J, Ravitz B, Bone GH, Nutt DJ, Roehrich L, Martin PR and Linnoila M (1991) Disturbances of hypothalamic-pituitary-adrenal axis functioning during ethanol withdrawal in six men. American Journal of Psychiatry 148 (8): 1023- 1025.

Aragon CM, Nicoletti S, Rogan F and Amit Z (1986) Corticosterone response to an acute dose of ethanol in naive and ethanol experienced rats. Alcohol and Alcoholism (Supplement1): 345-349.

Atkinson HC, Wood SA, Kershaw YM, Bate E and Lightman SL (2006) Diurnal variation in the responsiveness of the hypothalamic-pituitary-adrenal axis of the male rat to noise stress. Journal of Neuroendocrinology 18 (7): 526-533.

Bailly D, Dewailiy D, Beuscart R, Couplet G, Dumont P, Racadot A, Fossati P and Parquet PJ (1989) Adrenocorticotropin and cortisol responses to ovine corticotropin-releasing factor in alcohol dependence disorder. Hormone Research in Paediatrics 31 (1-2): 72-75.

Baird TJ, Briscoe RJ, Vallett M, Vanecek SA, Holloway FA and Gauvin DV (1998) Phase- response curve for ethanol: alterations in circadian rhythms of temperature and activity in rats. Pharmacology Biochemistry and Behavior 61 (3): 303-315.

Baker FC, Angaraj C, Szymusiak R and McGinty D (2005) Persistence of sleep-temperature coupling after suprachiasmatic nuclei lesions in rats. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology (289): R827-838.

Baldwin HA, Rassnick S, Rivier J, Koob GF and Britton KT (1991) CRF antagonist reverses the “anxiogenic” response to ethanol withdrawal in the rat. Psychopharmacology 103 (2): 227-232.

159

Berman JD, Cook DM, Buchman M and Keith LD (1990) Diminished adrenocorticotropin response to insulin-induced hypoglycemia in nondepressed, actively drinking male alcoholics. Journal of Clinical Endocrinology and Metabolism 71 (3): 712-717.

Besemer F, Pereira AM and Smit JW (2011) Alcohol-induced Cushing syndrome. Hypercortisolism caused by alcohol abuse. Netherlands Journal of Medicine 69 (7/8): 318-323.

Bittman EL, Doherty L, Huang L and Paroskie A (2003) Period gene expression in mouse endocrine tissues. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 285 (3): R561-R569.

Boschloo L, Vogelzangs N, Licht CM, Vreeburg SA, Smit JH, van den Brink W, Veltman DJ, de Geus EJ, Beekman AT and Penninx BW (2011) Heavy alcohol use, rather than alcohol dependence, is associated with dysregulation of the hypothalamic-pituitary-adrenal axis and the autonomic nervous system. Drug and Alcohol Dependence 116 (1-3): 170-6.

Bradley AJ and Dinan TG (2010) A systematic review of hypothalamic-pituitary-adrenal axis function in schizophrenia: implications for mortality. Journal of Psychopharmacology 24 (Suppl 4): 91-118.

Buhr ED, Yoo SH and Takahashi JS (2010) Temperature as a universal resetting cue for mammalian circadian oscillators. Science 330 (6002): 379-385.

Buijs RM, Kalsbeek A, van der Woude TP, van Heerikhuize JJ and Shinn S (1993) Suprachiasmatic nucleus lesion increases corticosterone secretion. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 246 (6): R1186- R1192.

Buijs RM, Markman M, Nunes‐Cardoso B, Hou YX and Shinn S (1993) Projections of the suprachiasmatic nucleus to stress‐related areas in the rat hypothalamus: A light and electron microscopic study. Journal of Comparative Neurology 335 (1): 42-54.

Buijs RM, Wortel J, Van Heerikhuize JJ, Feenstra MG, Ter Horst GJ, Romijn HJ and Kalsbeek A (1999) Anatomical and functional demonstration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway. European Journal of Neuroscience 11 (5): 1535-1544.

Bur IM, Zouaoui S, Fontanaud P, Coutry N, Molino F, Martin AO, Mollard P and Bonnefont X (2010) The comparison between circadian oscillators in mouse liver and pituitary gland reveals different integration of feeding and light schedules. PLOS ONE 5 (12): e15316.

160

Burov YV, Treskov VG, Vedernikova NN and Shevelyova OS (1986) Types of alcohol withdrawal syndrome and dexamethasone suppression test. Drug and Alcohol Dependence 17 (1): 81-88.

Cai A and Wise PM (1996) Age-related changes in the diurnal rhythm of CRH gene expression in the paraventricular nuclei. American Journal of Physiology-Endocrinology And Metabolism 33 (2): E238.

Cannizzaro C, La Barbera M, Plescia F, Cacace S and Tringali G (2010) Ethanol modulates corticotropin releasing hormone release from the rat hypothalamus: does acetaldehyde play a role? Alcoholism: Clinical and Experimental Research 34 (4): 588-593.

Carnes M, Lent S, Feyzi J and Hazel D (1989) Plasma adrenocorticotropic hormone in the rat demonstrates three different rhythms within 24 h. Neuroendocrinology 50 (1): 17-25.

Chang FC and Opp MR (1998) Blockade of corticotropin-releasing hormone receptors reduces spontaneous waking in the rat. American Journal of Physiology (275): R793-R802.

Chang SL, Patel NA and Romero AA (1995) Activation and desensitization of Fos immunoreactivity in the rat brain following ethanol administration. Brain Research 679 (1): 89-98.

Chen R, Lewis KA, Perrin MH and Vale WW (1993) Expression cloning of a human corticotropin-releasing-factor receptor. Proceedings of the National Academy of Sciences 90 (19): 8967-8971.

Clow A, Hucklebridge F, Stalder T, Evans P and Thorn L (2010) The cortisol awakening response: more than a measure of HPA axis function. Neuroscience and Biobehavioral Reviews 35 (1): 97-103.

Cobb CF, van Thiel DH, Gavaler JS and Lester R (1981) Effects of ethanol and acetaldehyde on the rat adrenal. Metabolism 30 (6): 537-543.

Colbern DL, ten Haaf J, Tabakoff B and van Wimersma Greidanus TB (1985) Ethanol increases plasma vasopressin shortly after intraperitoneal injection in rats. Life Sciences 37 (1): 1029-1032.

Danel T, Libersa C and Touitou Y (2001) The effect of alcohol consumption on the circadian control of human core body temperature is time dependent. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 281 (1): R52-R55.

Dave JR, Eiden LE, Karanian JW and Eskay RL (1986) Ethanol exposure decreases pituitary corticotropin-releasing factor binding, adenylate cyclase activity, proopiomelanocortin biosynthesis, and plasma β-endorphin levels in the rat. Endocrinology 118 (1): 280-286.

161

Davis JRE and Jeffcoate WJ (1983) Lack of effect of ethanol on plasma cortisol in man. Clinical Endocrinology 19 (4): 461-466. de Kloet ER, Joels M and Holsboer F (2005) Stress and the brain: from adaptation to disease. Nature Reviews Neuroscience 6 (6): 463-75.

Devaney M, Graham D and Greeley J (2003) Circadian variation of the acute and delayed response to alcohol: investigation of core body temperature variations in humans. Pharmacology Biochemistry and Behavior 75 (4): 881-887.

Dickmeis T (2009) Glucocorticoids and the circadian clock. Journal of Endocrinology 200 (1): 3-22.

Eastman CI, Stewart KT and Weed MR (1994) Evening alcohol consumption alters the circadian rhythm of body temperature. Chronobiology International 11 (2): 141-142.

Ehlers CL, Chaplin RI, Wall TL, Lumeng L, Li TK, Owens MJ and Nemeroff CB (1992) Corticotropin releasing factor (CRF): studies in alcohol preferring and non-preferring rats. Psychopharma-cology 106 (3): 359-364.

Ehlers CL, Henriksen SJ, Wang M, Rivier J, Vale W and Bloom FE (1983) Corticotropin releasing factor produces increases in brain excitability and convulsive seizures in rats. Brain Research Bulletin 278 (1): 332-336.

Ekman AC, Vakkuri O, Vuolteenaho O and Leppäluoto J (1994) Delayed pro-opiomelanocortin activation after ethanol intake in man. Alcoholism: Clinical and Experimental Research 18 (5): 1226-1229.

Engeland WC and Arnhold MM (2005) Neural circuitry in the regulation of adrenal corticosterone rhythmicity. Endocrine 28 (3): 325-332.

Esel E, Sofuoglu S, Aslan SS, Kula M, Yabanoglu I and Turan MT (2001) Plasma levels of beta- endorphin, adrenocorticotropic hormone and cortisol during early and late alcohol withdrawal. Alcohol and Alcoholism 36 (6): 572-576.

Forbes JC and Duncan GM (1951) The effect of acute alcohol intoxication on the adrenal glands of rats and guinea pigs. Quarterly Journal of Studies on Alcohol 12 (3): 355-359.

Garrick NA, Hill JL, Szele FG, Tomai TP, Gold PW and Murphy DL (1987) Corticotropin- releasing factor: a marked circadian rhythm in primate cerebrospinal fluid peaks in the evening and is inversely related to the cortisol circadian rhythm. Endocrinology 121 (4): 1329-34.

Gianoulakis C, Guillaume P, Thavundayil J and Gutkowska J (1997) Increased plasma atrial natriuretic peptide after ingestion of low doses of ethanol in humans. Alcoholism: Clinical and Experimental Research 21 (1): 162-170.

162

Gibbs FP (1970) Circadian variation of ether-induced corticosterone secretion in the rat. American Journal of Physiology - Legacy Content 219 (2): 288-292.

Girotti M, Weinberg MS and Spencer RL (2009) Diurnal expression of functional and clock- related genes throughout the rat HPA axis: system-wide shifts in response to a restricted feeding schedule. American Journal of Physiology - Endocrinology and Metabolism 296 (4): E888-97.

Gronfier C, Simon C, Piquard F, Ehrhart J and Brandenberger G (1999) Neuroendocrine processes underlying ultradian sleep regulation in man. Journal of Clinical Endocrinology and Metabolism 84 (4): 2686-2690.

Guaza C and Borrell J (1984) Effect of ethanol on corticosterone production by dispersed adrenal cells of the rat. Life Sciences 35 (11): 1191-1196.

Hellig M, Koob GF (2007) A key role for corticotropin-releasing factor in alcohol dependence. Trends in Neurosciences (30): 399-406.

Helms CM, Gonzales SW, Green HL, Szeliga KT, Rogers LS and Grant KA (2013) Diurnal pituitary-adrenal activity during schedule-induced polydipsia of water and ethanol in cynomolgus monkeys (Macaca fascicularis). Psychopharmacology (Berl) 228 (4): 541- 549.

Helms CM, McClintick MN and Grant KA (2012) Social rank, chronic ethanol self- administration, and diurnal pituitary-adrenal activity in cynomolgus monkeys. Psychopharmacology (Berl) 224 (1): 133-143.

Herman JP and Cullinan WE (1997) Neurocircuitry of stress: central control of the hypothalamo- pituitary-adrenocortical axis. Trends in Neurosciences 20 (2): 78-84.

Herman JP, Figueiredo H, Mueller NK, Ulrich-Lai Y, Ostrander MM, Choi DC and Cullinan WE (2003) Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Frontiers in Neuroendocrinology 24 (3): 151-180.

Hiroshige T and Sakakura M (1971) Circadian rhythm of corticotropin-releasing activity in the hypothalamus of normal and adrenalectomized rats. Neuroendocrinology 7 (1): 25-36.

Holsboer F, Von Bardeleben U, Buller R, Heuser I and Steiger A (1986) Stimulation response to corticotropin-releasing hormone (CRH) in patients with depression, alcoholism and panic disorder. Hormone and Metabolic Research Supplement (16): 80-88.

Hwang BH, Stewart R, Zhang JK, Lumeng L and Li TK (2004) Corticotropin-releasing factor gene expression is down-regulated in the central nucleus of the amygdala of alcohol-

163

preferring rats which exhibit high anxiety: a comparison between rat lines selectively bred for high and low alcohol preference. Brain Research 1026 (1): 143-150.

Ida Y, Tsujimaru S, Nakamaura K, Shirao I, Mukasa H, Egami H and Nakazawa Y (1992) Effects of acute and repeated alcohol ingestion on hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal functioning in normal males. Drug and Alcohol Dependence 31 (1): 57-64.

Inder WJ, Joyce PR, Wells JE, Evans MJ, Ellis MJ, Mattioll L and Donald RA (1995) The acute effects of oral ethanol on the hypothalamic-pituitary-adrenal axis in normal human subjects. Clinical Endocrinology 42 (1): 65-71.

Iranmanesh A, Veldhuis JD, Johnson ML and Lizarralde G (1989) 24-hour pulsatile and circadian patterns of cortisol secretion in alcoholic men. Journal of Andrology 10 (1): 54- 63.

Ishizawa H, Dave JR, Liu LI, Tabakoff B and Hoffman PL (1990) Hypothalamic vasopressin mRNA levels in mice are decreased after chronic ethanol ingestion. European Journal of Pharmacology: Molecular Pharmacology 189 (2): 119-127.

Jasper MS and Engeland WC (1994) Splanchnic neural activity modulates ultradian and circadian rhythms in adrenocortical secretion in awake rats. Neuroendocrinology 59 (2): 97-109.

Jenkins JS and Connolly J (1968) Adrenocortical response to ethanol in man. British Medical Journal 2 (5608): 804.

Junghanns K, Backhaus J, Tietz U, Lange W, Bernzen J, Wetterling T, Rink L and Driessen M (2003) Impaired serum cortisol stress response is a predictor of early relapse. Alcohol and Alcoholism 38 (2): 189-193.

Kakihana R and Moore JA (1976) Circadian rhythm of corticosterone in mice: the effect of chronic consumption of alcohol. Psychopharmacologia 46 (3): 301-305.

Kakihana R, Butte JC, Hathaway A and Noble EP (1971) Adrenocortical response to ethanol in mice: modification by chronic ethanol consumption. Acta Endocrinologica 67 (4): 653- 664.

Kalant H, Hawkins RD and Czaja C (1963) Effect of acute alcohol intoxication on steroid output of rat adrenals in vitro. American Journal of Physiology - Legacy Content 204 (5): 849- 855.

Kalin NH, Shelton SE, Barksdale CM and Brownfield MS (1987) A diurnal rhythm in cerebrospinal fluid corticotrophin-releasing hormone different from the rhythm of pituitary-adrenal activity. Brain Research 426 (2): 385-391.

164

Kalsbeek A, Ruiter M, La Fleur SE, Van Heijningen C and Buijs RM (2003) The diurnal modulation of hormonal responses in the rat varies with different stimuli. Journal of Neuroendocrinology 15 (12): 1144-1155.

Kaneko M, Hiroshige T, Shinsako J and Dallman MF (1980) Diurnal changes in amplification of hormone rhythms in the adrenocortical system. American Journal of Physiology 239: R309-R316.

Kaneko M, Kaneko K, Shinsako J and Dallman MF (1981) Adrenal sensitivity to adrenocorticotropin varies diurnally. Endocrinology 109 (1): 70-75.

Keedwell PA, Poon L, Papadopoulos AS, Marshall E and Checkley SA (2001) Salivary cortisol measurements during a medically assisted alcohol withdrawal. Addiction Biology 6 (3): 247-257.

Kinoshita H, Jessop DS, Finn DP, Coventry TL, Roberts DJ, Ameno K, Jiri I and Harbuz MS (2001) Acetaldehyde, a metabolite of ethanol, activates the hypothalamic-pituitary- adrenal axis in the rat. Alcohol and Alcoholism 36 (1): 59-64.

Knapp DJ, Braun CJ, Duncan GE, Qian Y, Fernandes A, Crews FT and Breese GR (2001) Regional specificity of ethanol and NMDA action in brain revealed with fos-like immunohistochemistry and differential routes of drug administration. Alcoholism: Clinical and Experimental Research 25 (11): 1662-1672.

Kodama H, Nakazawa Y, Kotorii T, Nonaka K, Inanaga K, Ohshima M and Yokoyama T (1988) Biorhythm of core temperature in depressive and non-depressive alcoholics. Drug and Alcohol Dependence 21 (1): 1-6.

Koob GF (1996) Drug addiction: the yin and yang of hedonic homeostasis. Neuron 16 (5): 893- 6.

Koob GF (2003) Alcoholism: allostasis and beyond. Alcoholism: Clinical and Experimental Research 27 (2): 232-43.

Koob GF (2008) A role for brain stress systems in addiction. Neuron 59 (1): 11-34.

Kwak SP, Morano MI, Young EA, Watson SJ and Akil H (1993) Diurnal CRH mRNA rhythm in the hypothalamus: decreased expression in the evening is not dependent on endogenous glucocorticoids. Neuroendocrinology 57 (1): 96-105.

Kwak SP, Young EA, Morano I, Watson SJ and Akil H (1992) Diurnal corticotropin-releasing hormone mRNA variation in the hypothalamus exhibits a rhythm distinct from that of plasma corticosterone. Neuroendocrinology 55 (1): 74-83.

Landfield PW (1994) The role of glucocorticoids in brain aging and Alzheimer's disease: An integrative physiological hypothesis. Experimental Gerontology 29 (1): 3-11.

165

Laryea G, Muglia L, Arnett M and Muglia LJ (2015) Dissection of glucocorticoid receptor- mediated inhibition of the hypothalamic-pituitary-adrenal axis by gene targeting in mice. Frontiers in Neuroendocrinology 36 150–164.

László FA, Varga C, Pávó I, Gardi J, Vecsernyés M, Gálfi M, Morschl E, László F and Makara GB (2001) Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 280 (2): R458-R465.

Lee S and Rivier C (1995) Altered ACTH and corticosterone responses to Interleukin-1β in male rats exposed to an alcohol diet: possible role of vasopressin and testosterone. Alcoholism: Clinical and Experimental Research 19 (1): 200-208.

Lee S, Craddock Z and Rivier C (2011) Brain stem catecholamines circuitry: activation by alcohol and role in the hypothalamic-pituitary-adrenal response to this drug. Journal of Neuroendocrinology 23 (6): 531-41.

Lee S and Rivier C (1997) Alcohol increases the expression of type 1, but not type 2α corticotropin-releasing factor (CRF) receptor messenger ribonucleic acid in the rat hypothalamus. Molecular Brain Research 52 (1): 78-89.

Lee S, Schmidt D, Tilders F, Cole M, Smith A and Rivier C (2000) Prolonged exposure to intermittent alcohol vapors blunts hypothalamic responsiveness to immune and non- immune signals. Alcoholism: Clinical and Experimental Research 24 (1): 110-122.

Lee S, Selvage D, Hansen K and Rivier C (2004) Site of action of acute alcohol administration in stimulating the rat hypothalamic-pituitary-adrenal axis: comparison between the effect of systemic and intracerebroventricular injection of this drug on pituitary and hypothalamic responses. Endocrinology 145 (10): 4470-9.

Lee S, Smith GW, Vale W, Lee KF and Rivier C (2001) Mice that lack Corticotrophin-Releasing Factor (CRF) receptors type 1 show a blunted acth response to acute alcohol despite up- regulated constitutive hypothalamic CRF gene expression. Alcoholism: Clinical and Experimental Research 25 (3): 427-433.

Leppäluoto J, Rapeli M, Varis R and Ranta T (1975) Secretion of anterior pituitary hormones in man: effects of ethyl alcohol. Acta physiologica Scandinavica 95 (4): 400-406.

Leproult R, Copinschi G, Buxton O and Van Cauter E (1997) Sleep loss results in an elevation of cortisol levels the next evening. Sleep 20 (10): 865-870.

Li Z, Kang SS, Lee S and Rivier C (2005) Effect of ethanol on the regulation of corticotropin- releasing factor (CRF) gene expression. Molecular and Cellular Neuroscience 29 (3): 345-354.

166

Liddle GW (1972) Pathogenesis of glucocorticoid disorders. The American Journal of Medicine 53 (5): 638-648.

Lolait SJ, Stewart LQ, Roper JA, Harrison G, Jessop DS, Young W and O'Carroll AM (2007) Attenuated stress response to acute lipopolysaccharide challenge and ethanol administration in vasopressin V1b receptor knockout mice. Journal of Neuroendocrinology 19 (7): 543-551.

Lu YL and Richardson HN (2014) Alcohol, stress hormones, and the prefrontal cortex: A proposed pathway to the dark side of addiction. Neuroscience 277 139-151.

Macho L, Zorad S, Radikova Z, Patterson-Buckedahl P and Kvetnansky R (2003) Ethanol consumption affects stress response and insulin binding in tissues of rats. Endocrine Regulations 37 (4): 195-202.

Margraf HW, Moyer CA, Ashford LE and Lavalle LW (1967) Adrenocortical function in alcoholics. Journal of Surgical Research 7 (2): 55-62.

Menaker M, Murphy ZC and Sellix MT (2013) Central control of peripheral circadian oscillators. Current Opinion in Neurobiology 23 (5): 741-746.

Menzaghi F, Rassnick S, Heinrichs S, Baldwin H, Pich EM, Weiss F and Koob GF (1994) The Role of Corticotropin-Releasing Factor in the Anxiogenic Effects of Ethanol Withdrawal. Annals of the New York Academy of Sciences 739 (1): 176-184.

Mohn CE, Fernandez-Solari J, De Laurentiis A, Bornstein SR, Ehrhart-Bornstein M and Rettori V (2011) Adrenal gland responses to lipopolysaccharide after stress and ethanol administration in male rats. Stress 14 (2): 216-226.

Moldow RL and Fischman AJ (1982) Hypothalamic CRF-like immunoreactivity in the rat after hypophysectomy or adrenalectomy. Peptides 3 (2): 143-147.

Moldow RL and Fischman AJ (1984) Circadian rhythm of corticotropin releasing factor-like immunoreactivity in rat hypothalamus. Peptides 5 (6): 1213-1215.

Mukai M, Uchimura N, Hirano T, Ohshima H, Ohshima M and Nakamura J (1998) Circadian rhythms of hormone concentrations in alcohol withdrawal. Psychiatry and Clinical Neurosciences 52 (2): 238-240.

Munck A, Guyre PM and Holbrook NJ (1984) Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocrine Reviews 5 (1): 25-44.

Ogilvie KM, Lee S and Rivier C (1997a) Effect of three different modes of alcohol administration on the activity of the rat Hypothalamic-Pituitary-Adrenal Axis. Alcoholism: Clinical and Experimental Research 21 (3): 467-476.

167

Ogilvie KM, Lee S and Rivier C (1997b) Role of arginine vasopressin and corticotropin- releasing factor in mediating alcohol-induced adrenocorticotropin and vasopressin secretion in male rats bearing lesions of the paraventricular nuclei. Brain Research 744 (1): 83-95.

Olive MF, Koenig HN, Nannini MA and Hodge CW (2002) Elevated extracellular CRF levels in the bed nucleus of the stria terminalis during ethanol withdrawal and reduction by subsequent ethanol intake. Pharmacology Biochemistry and Behavior 72 213-220.

Oster H, Damerow S, Kiessling S, Jakubcakova V and Abraham D (2006) The circadian rhythm of glucocorticoids is regulated by a gating mechanism residing in the adrenal cortical clock. Cell Metabolism 4 (2): 163-173.

Overstreet DH, Knapp DJ and Breese GR (2004) Modulation of multiple ethanol withdrawal- induced anxiety-like behavior by CRF and CRF1 receptors. Pharmacology, Biochemistry, And Behavior 77 (2): 405.

Owens MJ, Bartolome J, Schanberg SM and Nemeroff CB (1990) Corticotropin-releasing factor concentrations exhibit an apparent diurnal rhythm in hypothalamic and extrahypothalamic brain regions: differential sensitivity to corticosterone. Neuroendocrinology 52 (6): 626-631.

Ozsoy S and Esel E (2008) Hypothalamic-pituitary-adrenal axis activity, dehydroepiandrosterone sulphate and their relationships with aggression in early and late alcohol withdrawal. Progress in Neuro-Psychopharmacology and Biological Psychiatry 32 (2): 340-7.

Pariante CM and Lightman SL (2008) The HPA axis in major depression: classical theories and new developments. Trends in Neurosciences 31 (9): 464-468.

Pasquali R, Vicennati V, Cacciari M and Pagotto U (2006) The Hypothalamic-Pituitary-Adrenal axis activity in obesity and the metabolic syndrome. Annals of the New York Academy of Sciences 1083 (1): 111-128.

Pastor R, Sanchis-Segura C and Aragon CM (2004) Brain catalase activity inhibition as well as opioid receptor antagonism increases ethanol-induced hpa axis activation. Alcoholism: Clinical and Experimental Research 28 (12): 1898-1906.

Patterson-Buckendahl P, Kubovcakova L, Krizanova O, Pohorecky LA and Kvetnansky R (2005) Ethanol consumption increases rat stress hormones and adrenomedullary gene expression. Alcohol 37 (3): 157-166.

Pich EM, Lorang M, Yeganeh M, Rodriguez de Fonseca F, Raber J, Koob GF and Weiss F (1995) Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis. The Journal of Neuroscience 15 (8): 5439-5447.

168

Pietrowsky R, Meyrer R, Kern W, Born J and Fehm HL (1994) Effects of diurnal sleep on secretion of cortisol, luteinizing hormone, and growth hormone in man. Journal of Clinical Endocrinology and Metabolism 78 (683-683).

Pohorecky LA, Rassi E, Weiss JM and Michalak V (1980) Biochemical evidence for an interaction of ethanol and stress: Preliminary studies. Alcoholism: Clinical and Experimental Research 4 (4): 423-426.

Rasmussen DD, Boldt BM, Bryant CA, Mitton DR, Larsen SA and Wilkinson CW (2000) Chronic daily ethanol and withdrawal: 1. Long‐term changes in the Hypothalamo‐ Pituitary‐Adrenal axis. Alcoholism: Clinical and Experimental Research 24 (12): 1836- 1849.

Rasmussen DD, Bryant CA, Boldt BM, Colasurdo EA, Levin N and Wilkinson CW (1998) Acute alcohol effects on opiomelanocortinergic regulation. Alcoholism: Clinical and Experimental Research 22 (4): 789-801.

Rassnick S, Heinrichs SC and Britton KT (1993) Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal. Brain Research 605 (25-32).

Ratcliffe F (1972) Hypothalamic sensitivity in rats following prolonged consumption of ethanol. Archives Internationales de Pharmacodynamie et de Thérapie 197 (2): 305.

Reddy TE, Pauli F, Sprouse RO, Neff NF, Newberry KM, Garabedian MJ and Myers RM (2009) Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation. Genome Research 19 (12): 2163-71.

Redei E, Branch BJ and Taylor AN (1986) Direct effect of ethanol on adrenocorticotropin (ACTH) release in vitro. Journal of Pharmacology and Experimental Therapeutics 237 (1): 59-64.

Refinetti R and Menaker M (1992) The circadian rhythm of body temperature. Physiology and Behavior 51 (3): 613-637.

Reinberg A, Touitou Y, Lewy H and Mechkouri M (2010) Habitual moderate alcohol consumption desynchronizes circadian physiologic rhythms and affects reaction-time performance. Chronobiology International 27 (9-10): 1930-42.

Richardson HN, Lee SY, O’Dell LE, Koob GF and Rivier CL (2008) Alcohol self-administration acutely stimulates the hypothalamic-pituitary-adrenal axis, but alcohol dependence leads to a dampened neuroendocrine state. European Journal of Neuroscience 28 (8): 1641- 1653.

169

Risher-Flowers D, Adinoff B, Ravitz B, Bone GH, Martin PR, Nutt D and Linnoila M (1988) Circadian rhythms of cortisol during alcohol withdrawal. Advances in Alcohol and Substance Abuse 7 (3-4): 37-41.

Ritzmann RF and Tabakoff B (1976) Body temperature in mice: a quantitative measure of alcohol tolerance and physical dependence. Journal of Pharmacology and Experimental Therapeutics 199 (1): 158-170.

Rivest S and Rivier C (1994) Lesions of hypothalamic PVN partially attenuate stimulatory action of alcohol on ACTH secretion in rats. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 266 (2): R553-R558.

Rivier C, Imaki T and Vale W (1990) Prolonged exposure to alcohol: effect on CRF mRNA levels, and CRF-and stress-induced ACTH secretion in the rat. Brain Research 520 (1): 1-5.

Rivier C, Bruhn T and Vale W (1984) Effect of ethanol on the hypothalamic-pituitary-adrenal axis in the rat: role of corticotropin-releasing factor (CRF). Journal of Pharmacology and Experimental Therapeutics 229 (1): 127-131.

Rivier C and Lee S (1996) Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin. Brain Research 726 (1): 1-10.

Rivier C, Rivier J and Lee S (1996) Importance of pituitary and brain receptors for corticotrophin-releasing factor in modulating alcohol-induced ACTH secretion in the rat. Brain Research 721 (1): 83-90.

Sage D, Maurel D and Bosler O (2001) Involvement of the suprachiasmatic nucleus in diurnal ACTH and corticosterone responsiveness to stress. American Journal of Physiology - Endocrinology and Metabolism 280 (2): E260-E269.

Sage D, Maurel D and Bosler O (2002) Corticosterone-dependent driving influence of the suprachiasmatic nucleus on adrenal sensitivity to ACTH. American Journal of Physiology -Endocrinology and Metabolism 28 (2): E458-E465.

170

Sanna PP, Folsom DP, Barizo MJ, Hirsch MD, Melia KR, Maciejewski-Lenoir D and Bloom FE (1993) Chronic ethanol intake decreases vasopressin mRNA content in the rat hypothalamus: a PCR study. Molecular Brain Research 19 (3): 241-245.

Sapolsky RM, Romero LM and Munck AU (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions 1. Endocrine Reviews 21 (1): 55-89.

Silva SM and Madeira MD (2012) Effects of chronic alcohol consumption and withdrawal on the response of the male and female hypothalamic-pituitary-adrenal axis to acute immune stress. Brain Research 1444 27-37.

Silva SM, Madeira MD, Ruela C and Paula-Barbosa MM (2002a) Prolonged alcohol intake leads to irreversible loss of vasopressin and oxytocin neurons in the paraventricular nucleus of the hypothalamus. Brain Research 925 (1): 76-88.

Silva SM, Paula-Barbosa MM and Madeira MD (2002b) Prolonged alcohol intake leads to reversible depression of corticotropin-releasing hormone and vasopressin immunoreactivity and mRNA levels in the parvocellular neurons of the paraventricular nucleus. Brain Research 954 (1): 82-93.

Silva SM, Santos-Marques MJ and Madeira MD (2009) Sexually dimorphic response of the hypothalamo-pituitary-adrenal axis to chronic alcohol consumption and withdrawal. Brain Research 1303 61-73.

Sinha R (2001) How does stress increase risk of drug abuse and relapse? Psychopharmacology 158 (4): 343-359.

Sipp TL, Blank SE, Lee EG and Meadows GG (1993) Plasma corticosterone response to chronic ethanol consumption and exercise stress. Experimental Biology and Medicine 204 (2): 184-190.

Smagin GN, Heinrichs SC and Dunn AJ (2001) The role of CRH in behavioral responses to stress. Peptides 22 (5): 713-724.

Smith SM and Vale WW (2006) The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress. Dialogues in Clinical Neuroscience 8 (4): 383-395.

Späth-Schwalbe E, Schöller T, Kern W, Fehm HL and Born J (1992) Nocturnal adrenocorticotropin and cortisol secretion depends on sleep duration and decreases in association with spontaneous awakening in the morning. Journal of Clinical Endocrinology and Metabolism 75 (6): 1431-1435.

Spencer RL and McEwen BS (1990) Adaptation of the hypothalamic-pituitary-adrenal axis to chronic ethanol stress. Neuroendocrinology 52 (5): 481-489.

171

Steiger A (2002) Sleep and the hypothalamo-pituitary-adrenocortical system. Sleep Medicine Reviews 6 (2): 125-138.

Steiger A (2003) Sleep and endocrinology. Journal of Internal Medicine (254): 13-22.

Stephens M and Wand G (2012) Stress and the HPA axis: role of glucocorticoids in alcohol dependence. Alcohol Research: Current Reviews 34 (4): 468.

Stokes PE (1973) Adrenocortical activation in alcoholics during chronic drinking. Annals of the New York Academy of Sciences 215 (1): 77-83.

Sze PY (1977) The permissive role of glucocorticoids in the development of ethanol dependence and tolerance. Drug and Alcohol Dependence 2 (5): 381-396.

Tabakoff B, Jaffe RC and Ritzmann RF (1978) Corticosterone concentrations in mice during ethanol drinking and withdrawal. Journal of Pharmacy and Pharmacology 30 (1): 371- 374.

Tabakoff B and Yanai J (1979) Cortexolone antagonizes development of alcohol tolerance in mice. Psychopharmacology 64 (1): 123-124.

Tsigos C and Chrousos GP (1994) Physiology of the hypothalamic-pituitary-adrenal axis in health and dysregulation in psychiatric and autoimmune disorders. Endocrinology and Metabolism Clinics of North America 23 (3): 451-466.

Uchida M (2007) Effects of Urocortin, Corticotropin-Releasing Factor (CRF) Receptor Agonist, and Astressin, CRF Receptor Antagonist, on the Sleep-Wake Pattern Analysis by Radiotelemetry in Conscious Rats. Biological and Pharmaceutical Bulletin 30 (10): 1895-1897.

Ungar F and Halberg F (1962) Circadian rhythm in the in vitro response of mouse adrenal to adrenocorticotropic hormone. Science 137 (3535): 1058-1060.

Valdez GR, Roberts AJ, Chan K, Davis H, Brennan M, Zorrilla EP and Koob GF (2002) Increased Ethanol Sel-Administration and Anxiet-Like Behavior During Acute Ethanol Withdrawal and Protracted Abstinence: Regulation by Corticotropin-Releasing Factor. Alcoholism: Clinical and Experimental Research 26 (10): 1494-1501.

Vale W, Vaughan J and Perrin M (1997) Corticotropin-releasing factor (CRF) family of ligands and their receptors. The Endocrinologist 7 (1): 3S.

Valimaki M, Pelkonen R, Harkonen M and Ylikahri R (1984) Hormonal changes in noncirrhotic male alcoholics during ethanol withdrawal. Alcohol and Alcoholism 19 (3): 235-242.

172 van Cauter E, Leproult R and Kupfer DJ (1996) Effects of gender and age on the levels and circadian rhythmicity of plasma cortisol. Journal of Clinical Endocrinology and Metabolism 81 (7): 2468-2473. van Pett K, Viau V, Bittencourt JC, Chan RK, Li HY, Arias C and Sawchenko PE (2000) Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. Journal of Comparative Neurology 428 (2): 191-212. van Someren EJ. (2006) Mechanisms and functions of coupling between sleep and temperature rhythms. Progress in Brain Research 153: 309-324. von Gall C, Garabette ML, Kell CA, Frenzel S, Dehghani F, Schumm-Draeger PM, Weaver DR, Korf HW, Hastings MH and Stehle JH (2002) Rhythmic gene expression in pituitary depends on heterologous sensitization by the neurohormone melatonin. Nature Neuroscience 5 (3): 234-8.

Waite EJ, McKenna M, Kershaw Y, Walker JJ, Cho K, Piggins HD and Lightman SL (2012) Ultradian corticosterone secretion is maintained in the absence of circadian cues. European Journal of Neuroscience 36 (8): 3142-3150.

Waltman C, Blevins Jr LS, Boyd G and Wand GS (1993) The effects of mild ethanol intoxication on the hypothalamic-pituitary-adrenal axis in nonalcoholic men. Journal of Clinical Endocrinology and Metabolism 77 (2): 518-522.

Waltman C, McCaul ME and Wand GS (1994) Adrenocorticotropin responses following administration of ethanol and ovine Corticotropin-Releasing Hormone in the sons of alcoholics and control subjects. Alcoholism: Clinical and Experimental Research 18 (4): 826-830.

Wand GS and Dobs AS (1991) Alterations in the hypothalamic-pituitary-adrenal axis in actively drinking alcoholics. Journal of Clinical Endocrinology and Metabolism 72 (6): 1290- 1295.

Watts AG and Swanson LW (1989) Diurnal Variations in the Content of Preprocorticotropin- Releasing Hormone Messenger Ribonucleic Acids in the Hypothalamic Paraventricular Nucleus of Rats of Both Sexes as Measured by in Situ Hybridization. Endocrinology 125 (3): 1734-1738.

Watts AG, Tanimura S and Sanchez-Watts G (2004) Corticotropin-releasing hormone and arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of unstressed rats: daily rhythms and their interactions with corticosterone. Endocrinology 145 (2): 529-40.

Weibel L and Brandenberger G (1998) Disturbances in hormonal profiles of night workers during their usual sleep and work times. Journal of Biological Rhythms 13 (3): 202-208.

173

White JM, Frewin CR, Kaur M, Flavel S and McGregor C (1994) Twenty-four hour ambulatory monitoring of tremor, sweating, skin temperature and locomotor activity in the alcohol withdrawal syndrome. Clinical Autonomic Research 4 (1-2): 15-18.

Willenbring ML, Morley JE, Niewoehner CB, Heilman RO, Carlson CH and Shafer RB (1984) Adrenocortical hyperactivity in newly admitted alcoholics: prevalence, course and associated variables. Psychoneuroendocrinology 9 (4): 415-422.

Yassouridis A, Steiger A, Klinger A and Fahrmeir L (1999) Modelling and exploring human sleep with event history analysis. Journal of Sleep Research 8 (1): 25-36.

Zakhari S (1993) Alcohol and the endocrine system. (No. 93). National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism.

Ziegler DR and Herman JP (2002) Neurocircuitry of stress integration: anatomical pathways regulating the hypothalamo-pituitary-adrenocortical axis of the rat. Integrative and Comparative Biology 42 (3): 541-551.

Zoeller RT and Rudeen PK (1992) Ethanol blocks the cold-induced increase in thyrotropin- releasing hormone mRNA in paraventricular nuclei but not the cold-induced increase in thyrotropin. Molecular Brain Research 13 (4): 321-330.

174