Dr. Youssef Sari, Committee Chair 19 20 21 ______22 Dr

Home , Norbinaltorphimine

1 A Dissertation 2 3 Entitled 4 5 Role of Modulating Glutamate Transporters on Hydrocodone and Alcohol Co-Abuse in 6 Alcohol-Preferring Rats 7 8 9 By 10 11 Fahad Alshehri 12 13

Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the 14

Doctor of Philosophy Degree in Experimental Therapeutics 15

16 17 ______18 Dr. Youssef Sari, Committee Chair 19 20 21 ______22 Dr. F. Scott Hall, Committee Member 23 24 25 ______26 Dr. Zahoor Shah, Committee Member 27 28 29 ______30 Dr. Amit K. Tiwari, Committee Member 31 32 33 ______34 Dr. Amanda Bryant-Friedrich, Dean 35 College of Graduate Studies 36

38 39 The University of Toledo 40 August-2018 41 1

This document is copyrighted material. Under copyright law, no parts of this document 24 may be reproduced without the express written permission of the author. 25 An Abstract of

Role of Modulating Glutamate Transporters on Hydrocodone and Alcohol Co-Abuse in Alcohol-Preferring Rats

Fahad Alshehri

Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the Doctor of Philosophy Degree in Experimental Therapeutics

The University of Toledo

August-2018

Alcoholism and opioid addiction are a significant issue worldwide. According to the

National Institute on Alcohol Abuse and Alcoholism, the lifetime incidence of alcohol use to the point of intoxication for is 86.4% for US adults (>17 years of age). Concurrent ethanol use is very common among illicit opioid users. Evidence shows a strong association between opioid and alcohol use and changes in glutamate homeostasis.

Therefore, in this project, we investigated the effects of hydrocodone (HYD), which is one of the most prescribed opioid drugs in the United States, on the expression of key astroglial glutamate transporters. Primary astrocyte cell cultures were used to examine the expression of astroglial glutamate transporter 1 (GLT-1), cystine-glutamate transporter

(xCT), and glutamate/aspartate transporter (GLAST). We found that treatment of primary astrocytes for five days with HYD caused downregulation of GLT-1 and xCT expression, but no effect was observed on GLAST expression. Relapse to opioids is one of the most challenging aspects of opioid addiction. Accordingly, we investigated HYD reinstatement of drug-seeking behavior using the conditioned place preference (CPP) in alcohol-

iii preferring (P) rats. We found that HYD reinstatement is associated with reduction in xCT expression in the nucleus accumbens (NAc) and hippocampus (HIP). We then examined the effects of ceftriaxone (CEF), a β-lactam compound known to upregulate GLT-1 and xCT, on HYD reinstatement. We found that CEF treatment attenuated the reinstatement to HYD and restored xCT expression in the NAc and HIP. Finally, as ethanol co-abuse commonly occurs with prescription opioids, we investigated the effects of chronic ethanol drinking and HYD reinstatement on astroglial glutamate transporters in P rats. HYD and ethanol exposure caused downregulation of both GLT-1 and xCT expression in the NAc, dorsomedial prefrontal cortex (dmPFC) and HIP. However, CEF attenuated HYD- reinstatement and reduced ethanol drinking. CEF treatment restored the expression of

GLT-1 and xCT in the NAc, dmPFC and HIP, but had no effect in the amygdala (AMY).

Thus, restoring the levels of these glutamate transporters using CEF could have therapeutic potential for treating relapse to opioid and/or alcohol dependence.

This dissertation is dedicated to the memory of father, Sultan Alshehri. A man whom I still miss every day.

Acknowledgments

First, I would like to express my deepest appreciation to my advisor

Dr. Youssef Sari, who helped and guided me throughout this project. His guidance helped me during my research and writing this dissertation. I also would like to thank my committee members Dr. Zahoor Shah, and Dr. F. Scott Hall, and Dr. Amit K. Tiwari, I am sincerely thankful for their assistance and guidance throughout my dissertation. I am thankful for Dr. Jeffrey Sarver for being my graduate representative.

I would also like to express my sincere gratitude to all my friends and current lab members,

Alqasem Hakami, Fawaz Alasmari, and Hasan Alhadad their support and assistance during this project. Also, I would like to thank all the former lab members for their help, Atiah

Almalki, Yusuf Althobaiti, Alaa Hammad, and Sujan Das for their help.

I would like to thank my family for their unconditional support during my project; I would never have been able to complete my dissertation without their support.

I also thank Umm Al-Qura University for giving me and my family the financial support to continue my graduate studies. I also thank The University of Toledo and the Department of Pharmacology and Experimental Therapeutics for their assistance and support since the beginning of my study.

Table of Contents

Abstract ……………………………………………………...... iii

Acknowledgements...... vi

Table of contents...... vii

List of Tables...... xiii

List of Figures...... xiv

List of Abbreviations...... xix

List of Symbols...... xxi

1. Opioid Drugs and Alcohol: Role of Glutamate and Nitric Oxide………...... 12

Overall Introduction...... 1

Reference...... 6

Introduction Part I...... 12

1.1. Glutamatergic system...... 17

1.2. Opioids and glutamate...... 20

1.2.1. Opioids and glutamate transporters...... 22

1.2.2. Opioids and glutamate receptors...... 24

1.3. Alcohol and glutamate...... 30

vii

1.3.1. Alcohol and glutamate transporters...... 30

1.3.2. Alcohol and glutamate receptors...... 32

1.4. Opioids and alcohol...... 33

1.5. Opioids and NO...... 34

1.5.1. Opioids and nitric oxide synthase inhibitors...... 37

1.5.2. Opioids, NO and glutamate receptors antagonists...... 39

1.5.3. Opioids, NO and opioids receptors antagonists...... 40

1.5.4. Opioids rewards and NO...... 40

1.6. Opioids and body temperature...... 43

1.6.1. Effects of opioids receptors blocker on body temperature...... 44

1.6.2. Effects of modulating the glutamate receptors and transporters in

opioids-induced changes in body temperature...... 45

1.6.3. Effects of modulating NO transmission in opioids-induced changes

in body temperature...... 46

References...... 50

2. Effect of Hydrocodone on Astroglial Glutamate Transporters in Primary

Astrocyte Cell Culture...... 88

Introduction...... 90

viii

2.1. Materials and Methods...... 91

2.1.1. Drugs...... 91

2.1.2. Experimental design...... 91

2.1.3. Western blot for GLT-1, xCT and GLAST expression...... 93

2.1.4. Statistical analyses...... 93

2.2. Results...... 94

2.2.1. Immunocytochemical detection of astrocyte, microglia and

neurons...... 94

2.2.2. The effect of HYD treatment on astroglial glutamate transporters

expression in primary astrocyte cell culture...... 95

2.3. Discussion...... 98

References...... 106

3. Eﬀects of Ceftriaxone on Hydrocodone Seeking Behavior and Glial Glutamate

Transporters in P Rats...... 110

Introduction...... 112

3.1. Materials and Methods...... 115

3.1.1. Drugs...... 115

3.1.2. Animals and Drug Dosing...... 115

3.1.3. Apparatus...... 116

3.1.4. Experimental Procedure...... 116

3.1.5. Brain Tissue Extraction...... 119

3.1.6. Immunoblots Procedure...... 119

3.1.7. Statistical Analyses...... 120

3.2. Results...... 120

3.2.1. Eﬀect of SAL administration on animal preference using CPP....120

3.2.2. Eﬀect of CEF on HYD-induced reinstatement using CPP...... 121

3.2.3. Eﬀect of CEF on the expression of GLT-1, xCT and GLAST in the

NAc and dmPFC in HYD-induced reinstatement...... 123

3.2.4. Eﬀect of CEF on the expression of GLT-1, xCT and GLAST in the

HIP and AMY in HYD-induced reinstatement...... 126

3.3. Discussion...... 129

References...... 139

4. Ceftriaxone Attenuate Alcohol Drinking and Hydrocodone Reinstatement: Role of Modulating Astroglial Glutamate Transporters in Alcohol-Preferring P Rats...... 153

Introduction...... 155

4.1. Materials and Methods...... 157

4.1.1. Drugs...... 157

4.1.2. Animals and drug dosing...... 157

4.1.3. CPP Apparatus...... 158

4.1.4. Animal groups and experimental procedure...... 159

4.1.5. Brain Tissue Extraction...... 161

4.1.6. Immunoblots Procedure...... 162

4.1.7. Statistical Analyses...... 163

4.2. Results...... 163

4.2.1. Effect of SAL exposure on animal preference using CPP...... 163

4.2.2. Effect of HYD administration on animal preference using CPP...164

4.2.3. Effect of CEF administration on HYD reinstatement...... 165

4.2.4. Effect of HYD exposure on ethanol and water drinking during

conditioning phase...... 166

4.2.5. Effect of CEF treatment on ethanol and water drinking during

extinction phase...... 167

4.2.6. Effect of HYD and CEF exposure on weight changes during

conditioning and extinction...... 172

4.2.7. Effect of CEF on HYD reinstatement on the expression of GLT-1,

xCT, and GLAST in NAc and dmPFC...... 173

4.2.8. Effect of CEF on HYD-induced reinstatement on the expression of

GLT-1, xCT, and GLAST in HIP and AMY...... 177

4.3. Discussion...... 179

References...... 188

5. Summary...... 199

5.1. Experimental designs...... 200

5.2. Outcomes...... 202

References...... 205

A List of Articles Published based on this Dissertation...... 275

xii

List of Tables

3-1 Animal groups and treatment during the conditioning, extinction and reinstatement phases...... 118

4-1 Classification of animal groups based on treatments during conditioning, extinction and reinstatement...... 160

xiii

List of Figures

1-1 Illustration of the glutamatergic synapse showing the localization of the glutamatergic receptors and transporters...... 18

1-2 Summary of potential targets involving the glutamatergic system that might attenuate chronic opioid effects...... 29

1-3 Schematic representation of the effects of chronic exposure to opioids on the glutamatergic system as well as NO production...... 42

1-4 Summary of the potential targets that modulate body temperature associated with exposure to opioids...... 48

2-1 Experimental timeline with HYD 3 doses assessed at 3 time points (Day 1, Day 3, and Day 5) ...... 92

2-2 Immunocytochemistry staining of primary astrocyte cell culture...... 94

2-3 The effect of HYD (0.5 µM, 1 µM, and 2 µM) on the GLT-1 expression on primary astrocytes cell culture at Day 1, Day 3 and Day 5 ...... 96

2-4 The effect of HYD (0.5 µM, 1 µM, and 2 µM) on the xCT expression on primary astrocytes cell culture on Day 1, Day 3, and Day 5...... 97

xiv

2-5 The effect of HYD (0.5 µM, 1 µM, and 2 µM) on the GLAST expression on primary astrocytes cell culture on Day 1, Day 3, and Day 5...... 98

3-1 Timeline of the experimental procedure during the conditioning, extinction and reinstatement phases...... 118

3-2 Effect of SAL (i.p.) administration alone on CPP...... 121

3-3 Time spent in the conditioning chamber during pre-conditioning, post- conditioning, extinction, and reinstatement tests...... 123

3-4 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the expression of GLT-1, xCT and GLAST in the NAc...... 125

3-5 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the expression of GLT-1, xCT and GLAST in the dmPFC...... 126

3-6 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the expression of GLT-1, xCT and GLAST in the HIP...... 128

3-7 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the expression of GLT-1, xCT and GLAST in the AMY...... 129

3-8 Proposed mechanistic events associated with changes in the xCT expression in the

NAc and HIP for the attenuation of HYD-seeking behavior with CEF treatment...... 137

4-1 Timeline for the chronic ethanol drinking and exposure to HYD using CPP paradigm...... 161

4-2 Effect of SAL exposure on animals using CPP...... 164

4-3 The time spent in pre-conditioning (PRE), post-conditioning (POST), extinction

(EXT), and reinstatement (RE)...... 166

4-4 Effect of HYD exposure on chronic ethanol drinking during the conditioning phase

(A)...... 169

4-4 Effect of CEF treatment on chronic ethanol drinking during the extinction phase

(B)...... 169

4-5 Effect of HYD exposure on water drinking during the conditioning phase (A)...170

4-5 Effect of CEF treatment on water drinking during the extinction phase (B)...... 170

4-6 Effect of HYD exposure on total fluid intake during the conditioning phase

(A)...... 171

4-6 Effect of CEF treatment on total fluid intake during the extinction phase (B).....171

4-7 Effect of HYD exposure on average body weight during the conditioning phase

(A)...... 173

xvi

4-7 Effect of CEF treatment on average body weight during the extinction phase

(B)...... 173

4-8 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and CEF

(200 mg/kg, i.p.) in GLT-1, xCT and GLAST expression in NAc...... 175

4-9 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and CEF

(200 mg/kg, i.p.) in GLT-1, xCT, and GLAST expression in dmPFC...... 176

4-10 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and CEF

(200mg/kg) in GLT-1, xCT, and GLAST expression in HIP...... 178

4-11 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and CEF

(200 mg/kg, i.p.) in GLT-1, xCT, and GLAST expression in AMY...... 179

5-1 Summary of all experimental designs for the studies used in this project...... 201

5-2 Schematic representation showing the effects of repeated exposure to HYD on primary astrocytes culture (A)...... 202

5-2 Schematic representation showing the effects of repeated exposure to HYD on primary astrocytes culture (B)...... 202

5-3 Proposed mechanistic events associated with changes in the xCT expression in the

NAc and HIP underlying the attenuation of HYD-seeking behavior with CEF treatment...... 203

xvii

5-4 Proposed mechanistic events associated with changes in the xCT and GLT-1 expression in the NAc, dmPFC and HIP underlying the attenuation of HYD-seeking behavior and ethanol drinking with CEF treatment...... 204

xviii

List of Abbreviations

ADMA...... Asymmetric dimethylarginine ALS...... Amyotrophic lateral sclerosis. AMPAR...... α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. AMY...... Amygdala ANOVA...... Analysis of variance ATP/ADP...... Adenosine tri/diphosphate

CaMKII...... Ca2+/calmodulin-dependent protein kinase II, cGMP...... Cyclic guanosine monophosphate CNS...... Central nervous system CPP...... Conditioned place preference CSF...... Cerebrospinal fluid dmPFC...... Dorsomedial prefrontal cortex DST...... Dorsal striatum

EAAT...... Excitatory amino acid transporter ERK...... Extracellular-signal-regulated kinase

GLAST...... Glutamate aspartate transporter GLT-1...... Glutamate Transporter 1 GluR...... Glutamate receptor HIP...... Hippocampus HYD...... Hydrocodone iGluR...... Ionotropic glutamate receptor IL-1β...... Interleukins 1 beta I.P...... Intraperitoneal IT...... Intrathecal

KAR...... Kainate receptor. L-NA...... NG-nitro-L-arginine o

xix

L-NAME...... L-NG-nitro arginine methyl ester L‐NIO...... N‐iminoethyl‐l‐ornithine L-NPLA...... L-N-propylarginine

MND...... Motor Neuron Disease. MAPK...... Mitogen‐activated protein kinase mGluR...... Metabotropic glutamate receptor MPEP...... 2-methyl-6-phenylethynyl-pyridine mRNA...... Messenger ribonucleic acid

NAc...... Nucleus accumbens NMDAR...... N-methyl-D-aspartate receptor. NO...... Nitric oxide NOS...... Nitric oxide synthase NR……………………...... N-methyl-D-aspartate receptor subtype

PFC...... Prefrontal cortex PKC...... Protein Kinases P rat...... Alcohol-preferring rat

S.C...... Subcutaneous sGS...... Soluble guanylyl cyclase SNAP...... Donor S-nitroso-N-acetylpenicillamine

TNFα...... Tumor necrosis factor alpha vGluT...... Vesicular glutamate transporter VTA...... Ventral tegmental area xCT...... Cystine/glutamate transporter.

List of Symbols

α...... Alfa ……………………………. Betta

μl………………………….....Microliter μM ...... Micro molar

°C ...... Degree Celsius

Mg ...... Milligram G……………………………..Gram Kg...... Kilogram

M……………………………..Molar μmol………………………….Micromole mmol…………………………Millimole

Ca+2………………………...... Calcium Na+…………………………...Sodium K+...... Potassium

xxi

Chapter 1 4

Overall Introduction 5

Drug addiction is a worldwide problem with devastating economic, societal and public 7 health effects. Addiction is a brain disorder associated with repetitive uncontrolled 8 responses to certain rewarding stimuli despite the negative consequences. Repeated use of 9 drugs of abuse can lead to permanent changes in the neurocircuitry underlying brain reward 10 and reinforcement, decision-making, and impulse control. Drugs of abuse, including 11 opioids, are associated with a high degree of liability to develop drug use and dependence 12 disorders. The opioids morphine, hydrocodone (HYD) and oxycodone are only the most 13 recent types of opioids used by humans and is the product of modern medicine and science. 14

The human use of opium for pain management, and recreationally, dates to at least the 4th 15 century BCE [ for review see (Brownstein, 1993)]. While opioids have beneficial effects 16 in pain management (Ballantyne and LaForge, 2007), their non-medical uses have grown 17 rapidly in the last few years. According to the Substance Abuse and Mental Health 18

Services Administration (SAMHSA), non-medically prescribed opioids are considered the 19

1 second most commonly used illicit drug after marijuana. Prescription opioid overuse is 1 considered one of the major current public health problems in the United States (White et 2 al., 2009, Birnbaum et al., 2011). HYD was one of the most common causes of emergency 3 medical visits between 2004 and 2008 in nonmedical use of prescribed drugs (Control and 4

Prevention, 2010). In 2013, HYD was one of the most prescribed opioids with about 124 5 million prescriptions (National Prescription Audit Plus). A recent report found that HYD 6 is one of the top 10 drugs that frequently cause death in the United States (Warner et al., 7

2016). 8

HYD is a semi-synthetic opioid used in pain management. HYD was reported to have 9 equal potency for analgesic properties compared to morphine (Meert and Vermeirsch, 10

2005). HYD is known to activate the mu opioid receptor to produce analgesia, cough 11 suppression, and euphoria. Three main opioid receptors have been identified: mu opioid 12 receptor, delta opioid receptor, and the kappa opioid receptor (Reisine and Bell, 1993, 13

Minami and Satoh, 1995). Opioid receptors are found in the brain, spinal cord, 14 cardiopulmonary system, gastrointestinal tract, and various peripheral tissues. Activation 15 of individual receptors can produce different responses depending on the type of receptor 16 and its location [for review, see ref (Waldhoer et al., 2004)]. Generally, opioids can induce 17 all characteristics of addiction, including tolerance, withdrawal, dependence, and relapse. 18

Chronic use of opioids can lead to the development of tolerance, which requires increasing 19 the dose to achieve the same effect (Christie, 2008). Studies have found that using opioids 20 in chronic pain management is associated with a higher chances to abuse other illicit drugs 21

(Manchikanti et al., 2001, Atluri and Sudarshan, 2003). In addition, patients using opioids 22 for a long time have high chances to develop addiction (Fields, 2007). 23

Several studies have examined the rewarding effects of opioids, focusing on their effects 1 on GABAergic and dopaminergic systems (Johnson and North, 1992, Bonci and Williams, 2

1997). However, little is known about the involvement of glutamate, especially for HYD. 3

Alternatively, others have reported that repeated exposure to morphine can increase 4 glutamate and aspartate concentrations in the spinal cord (Wen et al., 2004). It has been 5 found that morphine-sensitized rats show an increase in extracellular glutamate when 6 challenged with morphine in the hippocampus (HIP), and naloxone prevents this increase, 7 which suggests an association between opioid exposure and glutamate homeostasis 8

(Farahmandfar et al., 2011). Astrocytes are one of the most abundant types of glial cells 9 in the brain; they provide support to the neurons and perform different metabolic functions 10 in the brain. The astroglial glutamate transporters, glutamate transporter 1(GLT-1), 11 cystine-glutamate transporter (xCT) and glutamate-aspartate transporter (GLAST), have 12 been suggested to play a major role in maintaining the glutamate homeostasis (Danbolt, 13

2001). Thus, GLT-1, in particular, is responsible for clearing the majority of glutamate 14 from the extracellular space and maintaining glutamate homeostasis (Danbolt, 2001). 15

Also, xCT exchanges glutamate for cystine in astrocytes. This transporter is believed to 16 be responsible for regulating the release of glutamate from astrocytes into the extracellular 17 compartment (Danbolt, 2001, Baker et al., 2002). GLAST is believed to be responsible for 18 clearing both glutamate and aspartate, but is more expressed in the cerebellum (Storck et 19 al., 1992). However, less is known about the effect of HYD treatment on astroglial 20 glutamate transporters. Thus, the first study reported here investigated the effect of HYD 21 treatment for five days using on the astroglial glutamate transporters using the primary 22 astrocytes. 23

Relapse after a long period of abstinence is a major problem in the treatment of drug 1 dependence (O’Brien, 1996). The high rate of relapse associated with opioids remains as 2 one of the most challenging clinical problems in opioid dependence. Recently, studies 3 have shed light on the possible role of glutamate in drug seeking and reinstatement for 4 many drugs of abuse (Cornish and Kalivas, 2000, Zhao et al., 2006, Dravolina et al., 2007, 5

LaLumiere and Kalivas, 2008). In addition, high concentrations of extracellular glutamate 6 in the brain are involved in relapse for most drugs of abuse (Knackstedt and Kalivas, 2009). 7

Studies have found an increase in extracellular glutamate concentrations during drug 8 seeking and reinstatement for many drugs of abuse, including heroin (Shen et al., 2014), 9 alcohol (Das et al., 2015), nicotine (Gipson et al., 2013), and cocaine (Baker et al., 2003). 10

Reinstatement in animals can be examined by different paradigms such as self- 11 administration and conditioned place preference (CPP) (Shaham et al., 2003). CPP is less 12 invasive than intravenous self-administration (Carr et al., 1989). Since little is known 13 about the effect of HYD reinstatement on the astroglial glutamate transporters, the second 14 study investigated the reinstatement of HYD on the astroglial glutamate transporters as 15 well as the effect of ceftriaxone (CEF) on HYD reinstatement in alcohol-preferring (P) rats. 16

The concomitant use of ethanol and opioids is a substantial problem (Ottomanelli, 1999, 17

Gossop et al., 2000). It has been found that adolescents frequently combine non-medical 18 prescription opioids with other drugs of abuse, including ethanol (McCabe et al., 2012). 19

18.5% of opioid-related emergency visits and 22.1% of drug-related deaths are linked to 20 ethanol use (Jones et al., 2014). Both opioids and ethanol may have an overlapping 21 endogenous activity. Ethanol is proposed to produce its rewarding and reinforcing effects, 22 in part, via the endogenous opioid system (Herz, 1997, Oswald and Wand, 2004). Studies 23

4 have shown that low doses of ethanol increase β-endorphin release from the pituitary gland 1 and hypothalamus (De Waele and Gianoulakis, 1993) as well as the NAc and ventral 2 tegmental area (VTA) (Rasmussen et al., 1998, Olive et al., 2001). In addition, mu opioid 3 receptor knockout mice have been shown to consume less ethanol compared to wild-type 4 mice (Hall et al., 2001). Equally important, chronic ethanol drinking has been shown to 5 increase glutamate concentrations and reduce its clearance in P rats (Das et al., 2015). It 6 has also been shown that chronic ethanol drinking is associated with reduction in GLT-1 7 and xCT expression in P rats (Alhaddad et al., 2014, Goodwani et al., 2015, Hakami et al., 8

2016). However, less is known about the effect of combining ethanol and HYD. 9

Therefore, the last study reported here investigated the effect of exposing P rats to chronic 10 ethanol drinking and then repeated HYD injections in a CPP reinstatement procedure. The 11 effects of combining ethanol and HYD was investigated from different perspectives: 1) the 12 effects of HYD on chronic ethanol drinking; 2) the effect of chronic ethanol drinking on 13

HYD preference and reinstatement using the CPP paradigm; 3) the effect of combining 14

HYD and ethanol on the expression of astroglial glutamate transporters; and 4) the ability 15 of CEF to attenuate HYD reinstatement and ethanol drinking via modulating the astroglial 16 glutamate transporters. 17

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Introduction: Part I 2

Opioid Drugs and Alcohol: Role of Glutamate and Nitric Oxide 4

Fahad S. Alshehri 1, Katelyn Berry1, Youssef Sari 1* 5

1 Department of Pharmacology & Experimental Therapeutics, College of Pharmacy and 7 Pharmaceutical Sciences, University of Toledo, Health Science Campus, 3000 Arlington 8 Avenue, Toledo, OH 43614, USA 9 10

* Corresponding author: 13 Dr. Youssef Sari 14 University of Toledo, College of Pharmacy & Pharmaceutical Sciences 15 Department of Pharmacology & Experimental Therapeutics 16 Health Science Campus, 3000 Arlington Avenue 17 Toledo, OH 43614, USA 18 E-mail: [email protected] 19 Tel: 419-383-1507 20 21

23 24 25 26 27 28

Abstract 1

Recent studies revealed that exposure to opioids alters glutamate homeostasis. Different 2 mechanisms have been suggested to be involved, including alterations in the expression of 3 glutamate transporters and receptors in the brain. Modulation of these transporters and 4 receptors was shown to improve effects associated with chronic opioid use. Similarly, 5 chronic exposure to alcohol has been shown to reduce the expression of astroglial 6 glutamate transporters such as glutamate transporter -1 and the cystine-glutamate 7 transporter. Upregulation of these transporters has been shown to reduce alcohol 8 consumption and reinstatement in animal models. Evidence has also found that nitric oxide 9

(NO) levels are affected by chronic opioid exposure. Changes in NO levels are associated 10 with the changes in glutamate neurotransmission through the activation of glutamate 11 receptors and modulation of glutamate release. Blocking NO synthesis can modulate 12 opioid tolerance, withdrawal, dependence, and relapse. Similarly, opioid use is associated 13 with changes in body temperature, which was also found to be associated, in part, with NO 14 and glutamate as well as opioid receptors. This review discusses in detail the changes 15 associated with opioid use on responses to alcohol, as well as glutamate and NO function. 16

Introduction 1

Substantial evidence suggests that excessive extracellular glutamate is associated with 2 neurotoxicity (Ankarcrona et al., 1995) and neurodegenerative diseases (Rothstein et al., 3

1992, Zumkehr et al., 2015). Several drugs of abuse have been shown to increase 4 extracellular glutamate concentrations in several brain regions (Nash and Yamamoto, 5

1992, Gioanni et al., 1999, Cornish and Kalivas, 2000, Lambe et al., 2003, Abulseoud et 6 al., 2012, Das et al., 2015). Furthermore, the glutamatergic system was found to be 7 involved in opioid dependence, withdrawal, and reinstatement in animal models 8

(Tokuyama et al., 1996, Ozawa et al., 2004, Tahsili-Fahadan et al., 2010). For instance, 9 heroin reinstatement was found to be mediated in animals, in part, through changes in the 10 extracellular glutamate (Shen et al., 2014). It has been found that glutamate and aspartate 11 levels were elevated in the cerebrospinal fluid (CSF) with long-term infusion of morphine 12 in rats (Tai et al., 2007). In addition, chronic morphine exposure has been reported to 13 increase the release of glutamate and reduces its uptake in the hippocampus (HIP) (Wang 14 et al., 2016). Studies have suggested that long-term use of opioids (e.g. morphine) can lead 15 to reduction in the expression of different glutamate transporters, which can consequently 16 reduce glutamate clearance (Marek et al., 1991, Dunbar and Pulai, 1998, Mao et al., 2002a). 17

The elevation in glutamate levels and reduction of its uptake after chronic exposure to 18 opioids (e.g. morphine) was suggested to be due to the reduction in the expression of the 19 glutamate transporter 1 (GLT-1) (Ozawa et al., 2001), as well as the glutamate-aspartate 20 transporter (GLAST) (Tai et al., 2007). The deficiency in glutamate clearance by the 21 glutamate transporters was also reported to be associated with opioid tolerance (decrease 22 in response to the same giving dose), hyperalgesia (increased sensitivity to pain) and 23

14 allodynia (pain due to stimuli that usually do not produce pain) (Marek et al., 1991, Dunbar 1 and Pulai, 1998, Mao et al., 2002a). Similarly, opioid use was found to produce 2 neuroadaptive changes in several ionotropic glutamate receptors (iGluRs). In fact, long- 3 term use of opioids was reported to be associated with an increase in the expression and 4 activity of several iGluRs (Fitzgerald et al., 1996, Inoue et al., 2003, Bajo et al., 2006, 5

Murray et al., 2007). 6

Several studies have found that the glutamatergic system is also associated with nitric oxide 7

(NO) production (Garthwaite et al., 1989, Knowles et al., 1989, Heinzel et al., 1992, Grima 8 et al., 1998). In fact, activation of the N-methyl-D-aspartate receptor (NMDAR), was 9 found to enhance Ca2+ influx, which facilitates the catalytic process involving L-arginine 10 and nitric oxide synthase (NOS) in the production of NO (Grima et al., 1998). Therefore, 11 studies have examined the connection between NO production and opioid exposure on the 12 glutamatergic system. Several studies have found that opioid exposure is associated with 13 an increase in NO production in the nervous system (Elliott et al., 1994, Bhargava, 1995, 14

Fricchione and Stefano, 2005) through the activation of NMDAR. Therefore, several 15 approaches have been implemented to block the production of NO production, including 16 the administration of NOS inhibitors and glutamate receptors blockers, as well as opioid 17 receptor blockers, to prevent the negative consequences of excess NO production (Kivastik 18 et al., Elliott et al., 1994, Herman et al., 1995). Blocking the production of NO may result 19 in reduction in opioid-induced hyperalgesia/allodynia effects and attenuation of the effects 20 of chronic opioid exposure, including tolerance, withdrawal and dependence (Kolesnikov 21 et al., 1992, Kolesnikov et al., 1993, Bhargava et al., 1998, Karami et al., 2002). 22

Opioids have also been reported to affect body temperature. Different studies examined 1 these effects in animals (Handler et al., 1992, Morrison and Nakamura, 2011). It has been 2 found that activation of the opioid receptors can induce increase or decrease body 3 temperature depending on which receptor is activated (Handler et al., 1992). The activation 4 of the mu-opioid receptor produces hyperthermia, while activation of the kappa-opioid 5 receptor and delta-opioid receptor produce hypothermia. Therefore, different studies have 6 investigated different possible methods to attenuate the hyperthermic effects of the mu 7 opioid receptor agonists such morphine (Rawls et al., 2007) and fentanyl (Cao and 8

Morrison, 2005). Pretreatment with opioid receptors blockers was found to have beneficial 9 effects in blocking hyperthermia (Cao and Morrison, 2005). Glutamate and NO were 10 shown to be involved in temperature changes associated with opioid use. Several studies 11 have investigated the effect of modulating the glutamate receptor antagonist and NOS 12 inhibitors on the hyperthermic effects of opioids (Benamar et al., 2001, Benamar et al., 13

2003, Rawls et al., 2003, Cao and Morrison, 2005). The use of iGluRs blocker and/or 14 neuronal (nNOS) inhibitors were shown to attenuate the temperature elevation associated 15 with opioids (Benamar et al., 2001, Benamar et al., 2003, Rawls et al., 2003). Therefore, 16 blocking these receptors could be a potential target for modulating hyperthermia associated 17 with opioids. Together, this review discusses the effect of opioids and alcohol on the 18 glutamatergic system. The effects of chronic opioid use on NO transmission are also 19 discussed. Finally, this review discusses the effect of acute opioids on body temperature 20 and its association with NO and glutamate transmission. 21

1.1. Glutamatergic system 1

Glutamate is a major excitatory neurotransmitter in the nervous system. The glutamatergic 2 system constitutes of several types of glutamate transporters and receptors (Figure 1-1). 3

There are five glutamate transporters, which called the excitatory amino acid transporters 4

(EAAT 1-5) (Goodwani et al., 2017). The EAATs co-transport Na+ and K+, so are also 5 called Na+ and K+ coupled glutamate transporters (Danbolt, 2001). EAAT1 is also called 6

GLAST, which transports both glutamate and aspartate in the nervous system. It is 7 expressed mostly in the spinal cord and cerebellum (Storck et al., 1992). EAAT2 is also 8 called GLT-1, which is responsible for the majority of glutamate uptake (Danbolt, 2001, 9

Jensen et al., 2015). GLT-1 is expressed more in astroglial cells than in neurons (Danbolt, 10

2001, Jensen et al., 2015). EAAT 3 (EAATC1) and EAAT4 are expressed more in 11 neuronal terminals (Kanai and Hediger, 1992, Fairman et al., 1995). EAAT5 is expressed 12 mainly in the retina (Arriza et al., 1997). The cystine/ glutamate exchanger (xCT) is 13 believed to be responsible for regulating the release of glutamate from astrocytes into the 14 extracellular space (Baker et al., 2002). xCT is involved in neural protection via regulation 15 of glutathione biosynthesis (Griffith, 1999). Changes in glutamate levels have been shown 16 to be associated with neurodegenerative diseases (Blandini et al., 1996, Butterfield and 17

Pocernich, 2003, Frigo et al., 2012), drug seeking and relapse (Self and Nestler, 1998, 18

Cornish and Kalivas, 2000), ischemia (Choi and Rothman, 1990, Arundine and Tymianski, 19

2004), mood disorders (Sanacora et al., 2008), and lack of response in pain management 20 with opioids (Wong et al., 2002). Therefore, glutamate transporters have been suggested 21 to be a critical target in modulating glutamate clearance in the nervous system that may 22 have therapeutic potential for these conditions. 23

Figure 1-1 Illustration of the glutamatergic synapse showing the localization of the 3 glutamatergic receptors and transporters. After the glutamate is released from the 4 presynaptic neurons, it activates several iGluRs and mGluRs. Then, the majority of 5 glutamate is cleared by the astroglial glutamate transporter GLT-1; GLAST has also a role 6 in glutamate uptake. In astrocytes the glutamate is converted into glutamine via glutamine 7 synthase. Glutamine defuses back into the presynaptic neurons, where it is converted back 8 into glutamate by glutaminase. This glutamate is transported and stored in the vesicles via 9 the vesicular glutamate transporter (vGluT). 10

In regard to glutamate receptors, there are iGluRs, the NMDAR has an essential role in 11 neural plasticity and neural excitability. NMDAR has been suggested as a potential target 12

18 in neurodegenerative disease, drug addiction, and neural excitotoxicity (Mody and 1

MacDonald, 1995, Shankar et al., 2007, Mao et al., 2009). AMPAR is composed of 2 tetrameric/heteromeric complexes. AMPAR is activated by glutamate, allowing Na+ and 3

Ca2+ influx into the postsynaptic neurons. AMPARs are found in both neurons and 4 astrocytes; but, it is less expressed in astrocytes. The AMPARs are known to mediate 5 glutamate excitotoxicity in several neurodegeneration diseases as well as ischemia (Noh et 6 al., 2005, Lai et al., 2006, Araujo et al., 2007, Chen et al., 2007). 7

Kainate acid receptors (KARs) are asimilar to NMDAR and AMPAR. The Kainate acid 8

(KA) is a potent excitatory amino acid that activates KAR and AMPAR (Fritsch et al., 9

2014). In fact, the activation of the glutamate receptors with KA was reported to cause 10 regional damages in several brain areas, including the thalamic, amygdala (AMY), 11 hypothalamus, and HIP (Fuller and Olney, 1979). Similar to other iGluRs, the permeability 12 of KAR ion channels (Na+ and Ca2+) varies according to the family and the subunit 13 composition of the receptor. For example, pre-synaptic activation of KARs in HIP may 14 inhibit the glutamate transmission (Vignes et al., 1998). However, other studies have 15 shown that KARs are also expressed in the postsynaptic terminals, which participate in the 16 excitatory postsynaptic currents of glutamate in the spinal cord (Li et al., 1999), AMY (Li 17 and Rogawski, 1998) and retina (DeVries and Schwartz, 1999). 18

The metabotropic glutamate receptors (mGluRs) are membrane-bound G protein-coupled 19 resceptors. These receptors are slower in their actions as compared to the iGluRs. The 20 activation of these receptors can affect glutamate release and transmission. Three groups 21 of the mGluRs have been identified. Group 1 receptors are expressed more in postsynaptic 22

19 neurons. These receptors have been found to have an essential role in many brain 1 functions, including memory, drug dependence, and motor function (Aiba et al., 1994, 2

Chiamulera et al., 2001, Niswender and Conn, 2010). Group 2 receptors (mGluR2/3) are 3 expressed both in pre- and post-synaptic neurons. Activation of presynaptic receptors may 4 result in the inhibition of glutamate release (Swanson et al., 2005). Group 3 receptors are 5 expressed mostly in the presynaptic neuron and have similar functions to Group 2 receptors 6

(Swanson et al., 2005). mGluRs have been shown to be involved in neurodegenerative 7 diseases such as Alzheimer’s disease (Haas et al., 2016) and Amyotrophic lateral sclerosis 8

(ALS) (Brownell et al., 2015). In addition, several studies have shed light on the 9 involvement of mGluRs in addiction and dependence to several drugs of abuse, including 10 cocaine (Justinova et al., 2016), methamphetamine (Caprioli et al., 2015) and heroin (Lou 11 et al., 2014). However, studies are still needed to explore more about their involvement 12 and possible ways of modulating their function in opioid addiction and dependence. 13

1.2. Opioids and glutamate 14

Opioid drugs (exogenous opioids) are used in pain management, including morphine, 15 hydrocodone, oxycodone, and hydromorphone. Endogenous opioids (endorphins, 16 enkephalins, and dynorphins) also bind and activate the opioid receptors (mu opioid 17 receptor, kappa opioid receptor, and delta opioid receptor) (Raynor et al., 1994). Opioid 18 receptors are G-coupled protein receptors that negatively control the transmission of the 19 glutamatergic and GABAergic neurons (Cherubini and North, 1985). Opioids are highly 20 efficacious analgesics; however, prolonged use is associated with a high potential for 21 tolerance, addiction and abuse. Typically, acute administration of opioid analgesics 22

20 activates primarily mu opioid receptor and consequently triggers a signaling cascade and 1 regulatory events that include activation of the inhibitory proteins Go/Gi (Duman et al., 2

1988), and phosphorylation of GRK, protein kinases (PKC) (Zheng et al., 2008). However, 3 repeated exposure to opioids can lead to mu opioid receptor desensitization and 4 endocytosis (Borgland et al., 2003) through different mechanisms, including activation of 5 the extracellular-signal-regulated kinase (ERK) (Rozenfeld and Devi, 2007), p38 mitogen‐ 6 activated protein kinase (MAPK) (Macé et al., 2005) and reduction of mu opioid receptor 7 expression (Horner and Zadina, 2004). These mechanisms have been suggested to lead to 8 opioid tolerance and loss of opioid analgesic effects. Other mechanisms have also been 9 thought to participate in chronic opioids effects. 10

Evidence has shown a strong relationship between opioid use and the glutamatergic system. 11

In general, administration of opioids (e.g. morphine) has shown to produce biphasic effects 12 with an initial reduction in locomotor activity followed by enhanced locomotor activity 13

(Babbini and Davis, 1972). Furthermore, it has been shown that opioid exposure reduces 14 the basal glutamate level and evoked the release of glutamate in different brain regions: 15 nucleus accumbens (NAc) (Sepulveda et al., 1998, Martin et al., 1999), cerebral cortex 16

(Coutinho-Neito et al., 1980, Coutinho-Netto et al., 1982, Nicol et al., 1996) and dorsal 17 striatum (DST) (Desole et al., 1996, Enrico et al., 1998). It has been suggested that opioids 18 can mediate their action through interacting with opioid receptors presynaptically on 19 glutamatergic neurons to reduce glutamate transmission (Satoh et al., 1975, Gass and 20

Olive, 2008). The reduction in glutamate transmission is a result of decreases in the pre- 21 synaptic Ca2+ influx (Yang et al., 2004). However, repeated administration of morphine 22 has shown to sensitize the neuronal response, when administered again after a period of 23

21 time (Satoh et al., 1976, Fry et al., 1980). Therefore, we discuss here the effects of repeated 1 administration of opioids, which are associated with tolerance, dependence, and 2 withdrawal. These opioid effects have been shown to produce adaptive changes in iGluRs, 3 mGluRs and glutamate transporters, discussed below in detail. A summary of the potential 4 targets in the glutamatergic system to attenuate chronic opioid effects is shown in Figure 5

1-2. 6

1.2.1. Opioids and glutamate transporters 7

The glutamate transporters regulate glutamate transmission and play a critical role in the 8 development of morphine tolerance and abnormal pain sensitivity. GLT-1 is considered 9 the primary transporter, which is responsible for clearing about 90% of extracellular 10 glutamate (Danbolt, 2001). The mRNA expression of GLT-1 is downregulated with 11 repeated morphine treatment in different brain regions such as NAc, ST, and thalamus 12

(Ozawa et al., 2001). However, withdrawal of morphine is associated with an increase of 13

GLT-1 expression in the striatum (Ozawa et al., 2001) and HIP (Xu et al., 2003). 14

Alternatively, heroin relapse was shown to be associated with a reduction in GLT-1 15 expression in the NAc core (Shen et al., 2014). Therefore, activating or upregulating GLT- 16

1 is considered a potential approach for attenuating opioid dependence and relapse. 17

Different studies have examined drug reward using the conditioned place preference (CPP) 18 procedure (Prus et al., 2009). The effect of MS-153, a glutamate transporter activator, was 19 investigated in mice on morphine CPP (Nakagawa et al., 2005). Co-administration of MS- 20

153 (10 mg/kg) with morphine (5 mg/kg) blocked morphine-induced CPP. In the same 21 study, MS-153 was able to block methamphetamine and cocaine preference as well. Thus, 22

22 maintaining glutamate levels by modulating GLT-1 expression could be a possible 1 approach in preventing opioid dependence and relapse. 2

The xCT system has an essential role in maintaining the glutamate levels in the 3 extracellular space (Bannai et al., 1989, Bassi et al., 2001, Danbolt, 2001). Only a limited 4 number of studies have investigated the xCT transporter and opioids. However, restoring 5 the function of xCT with N-Acetylcysteine prevents heroin reinstatement in an animal 6 model (Zhou and Kalivas, 2008). Recently, it was reported that ceftriaxone attenuated 7 hydrocodone seeking through modulating the expression of xCT in alcohol-preferring (P) 8 rats (Alshehri et al., 2018). The reduction in xCT expression may affect the inhibitory 9 mechanism of presynaptic mGluR2/3, which could facilitate glutamate release upon 10 reinstatement of hydrocodone (Alshehri et al., 2018). Therefore, N-Acetylcysteine and 11 ceftriaxone could be beneficial compounds with to prevent reinstatement in animals 12 through restoring the xCT function. GLAST can transport both glutamate and aspartate, 13 which is more expressed in the cerebellum and spinal cord (Storck et al., 1992). Repeated 14 morphine treatment was shown to increase the levels of glutamate and aspartate in the CSF 15

(Wong et al., 2002). Moreover, morphine infusion into the spinal cord was found to reduce 16

GLAST expression (Tai et al., 2006, Tai et al., 2007). In addition, it has been reported that 17 intrathecal injections of morphine were associated with a reduction in GLAST and EAAC1 18 expression (Mao et al., 2002a, Tai et al., 2006, Tai et al., 2007). Other glutamate 19 transporters have been reported to be affected by repeated morphine administration. For 20 instant, EAAT3 expression was found to be reduced in the prefrontal cortex (PFC) after 21 repeated administration of morphine (Wu et al., 2013). Thus, the repeated use of opioids 22 can be associated with a deficit in glutamate clearance due to the impairment of several 23

23 glutamate transporters. Therefore, targeting these transporters can provide potential 1 benefits in attenuating opioid-induced dysregulation of glutamate clearance. 2

1.2.2. Opioids and glutamate receptors 3

It has been suggested that glutamatergic systems mediate, in part, changes in neural 4 plasticity associated with morphine tolerance and dependence (Nakagawa et al., 2001). 5

Glutamate neurotransmission is critically involved in chronic opioid-induced neuronal 6 adaptation, including opioid tolerance, dependence, withdrawal and chronic pain- 7 associated hyperalgesia (Ben-Eliyahu et al., 1992, Herman et al., 1995, Bilsky et al., 1996, 8

Ta et al., 2000, Inoue et al., 2003, Do Couto et al., 2005). Acute administration of morphine 9 activates MOR on corticostriatal glutamatergic terminals leading to a reduction in 10 glutamate release (Fricchione and Stefano, 2005). However, upon repeated exposure, 11 glutamate clearance is dramatically reduced, increasing glutamate levels, which may 12 facilitate the development of tolerance. In fact, it has been reported that prolonged 13 exposure to morphine facilitates apoptotic cell death in spinal cord through overstimulation 14 of NMDAR, which can lead to a reduction in opioid analgesia, and the behavioral 15 manifestation of morphine tolerance (Mao et al., 2002b). In addition, it has been found 16 that morphine induces an increase in glutamate and aspartate concentrations in rat spinal 17

CSF dialysates, which is associated with the loss of morphine antinociceptive effects (Tai 18 et al., 2006). Equally important, it has been reported that opioid withdrawal is also 19 associated with an increase in glutamate and aspartate release in the NAc (Sepulveda et al., 20

1998) and locus coeruleus (Aghajanian et al., 1994). 21

Dependence and relapse are still among the most challenging aspect of opioid treatment 1 management. Studies have investigated glutamate receptor expression to identify new 2 insights into their influence in opioid dependence. Morphine seeking behavior is 3 associated with high levels of NMDAR subtype NR2B in NAc and HIP, but not in PFC 4

(Ma et al., 2007). NMDAR antagonists attenuated morphine reinstatement (Do Couto et 5 al., 2005, Popik et al., 2006). Alternatively, morphine extinction behavior was associated 6 with elevation in AMPAR subtype GluR1 phosphorylation in the HIP (Billa et al., 2009). 7

Similar to NMDAR antagonists, AMPAR antagonists attenuated morphine dependence 8

(McLemore et al., 1997). The inhibition of Ca2+/calmodulin-dependent protein kinase II 9

(CaMKII), primary NMDAR and AMPAR signaling pathways, was found to prevent 10 morphine reinstatement (Liu et al., 2012). Repeated morphine administration increases 11

NMDAR protein expression in different brain regions, including periaqueductal gray 12 matter, ventral tegmental area (VTA), and NAc, but not AMY or HIP (Inoue et al., 2003, 13

Bajo et al., 2006, Murray et al., 2007). However, repeated morphine exposure also was 14 associated with downregulation in NMDAR subtype NR2B in the HIP (Wang et al., 2016). 15

Although repeated morphine induces changes in NMDAR protein expression in different 16 brain regions, no changes were observed in NR1, NR2A, or NR2B protein expression in 17 the PFC (Scheggi et al., 2002). Repeated morphine administration increases the long-term 18 potentiation (LTP) at mossy fiber synapses in the HIP (Harrison et al., 2002), which could 19 be due to an increase in GluRA1 and GluRA2/3 (Zhong et al., 2006). Furthermore, 20

AMPAR subunit-GluRA1 knockout mice showed reduction in morphine sensitization to 21 context (Vekovischeva et al., 2001). Moreover, repeated morphine administrations leads 22 to an elevation in the expression of GluRA1in the VTA (Fitzgerald et al., 1996). However, 23

GluRA1 expression was found to be reduced in the PFC, but not in the NAc or ventral 1 pallidum (Mickiewicz and Napier, 2011). 2

NMDAR and non-NMDAR antagonists reduce the response to different noxious stimuli 3

(thermal, chemical, mechanical, and electric) (Dougherty et al., 1992, Neugebauer et al., 4

1993). The inhibition of tolerance and dependence by MK-801 (NMDAR blocker) 5 suggests that NMDARs are involved in the behavioral changes and therefore presumably 6 in the neural adaptations produced by repeated morphine administration. This suggested a 7 potential role of NMDARs in the development of opiate tolerance and dependence. MK- 8

801 can interfere with the development of sensitization to several stimulant drugs, 9 suggesting that the excitatory amino acid systems may be involved in experience- 10 dependent changes produced by repeated exposure to a variety of drugs (Trujillo and Akil, 11

1991). In fact, a study examined different doses of MK-801(0.1, 0.2 and 0.3 mg/kg) and 12 memantine (2.5, 5, 10, 20 and 40 mg/kg), NMDAR blockers, on morphine (40 mg/kg) CPP 13

(Do Couto et al., 2004). This study found that co-administration of these blockers can 14 attenuate morphine-induced CPP in mice (Do Couto et al., 2004). Another study tested the 15 effect of riluzole, a possible NMDAR blocker, to attenuate morphine-induced CPP. Co- 16 administration of riluzole (4 mg/kg) with morphine (10 mg/kg) blocked morphine-induced 17

CPP in rats (Tzschentke and Schmidt, 1998). This effect was suggested to be through 18 blocking the NMDAR, but others have suggested that riluzole could reduce glutamate 19 release through inactivation of glutamatergic nerve terminals (voltage-dependent Na+ 20 channels) as well as activation of the G-protein-dependent signal transduction process 21

(Doble, 1996). Interestingly, riluzole produced preference in CPP when administrated 22 alone; however, others found that riluzole at the same dose did not produce any preference 23

26 in rats (Jin et al., 2006). Of note, studies have shown that MK-801 alone could produce 1 preference in CPP paradigm (Layer and Kaddis, 1993, Steinpreis et al., 1995). 2

The activation of non-NMDAR (KAR, AMPAR) within the spinal cord is implicated in 3 the development of hyperalgesia in rats with experimental neuropathic pain and peripheral 4 inflammation. The role of KAR is more complicated compared to NMDAR or AMPAR. 5

KAR is expressed both in pre- and post-synaptic glutamatergic neurons, where they have 6 facilitatory or inhibitory effects on glutamate transmission (Ruscheweyh and Sandkühler, 7

2002). In general, KAR antagonists attenuate allodynia and hyperalgesia associated with 8 nerve injury in rat models (Ta et al., 2000); however, these antagonists have minimal 9 effects on acute spinal nociceptive responses (Procter et al., 1998). More studies are still 10 needed to investigate the role of KAR and AMPAR in morphine tolerance. Together, 11

NMDAR and AMPAR antagonists may attenuate opioid withdrawal (Herman et al., 1995, 12

Rasmussen et al., 1996), development of tolerance (Trujillo and Akil, 1991, Bilsky et al., 13

1996, Dunbar and Yaksh, 1996) and antinociceptive effects (Ben-Eliyahu et al., 1992, 14

Grass et al., 1996). 15

Several reports have indicated that modulation of mGluRs can alter pain and nociception 16

(Fundytus, 2001, Neugebauer, 2002). It has been proposed that MOR and mGluRs share 17 some common intracellular signaling pathways, which could explain, in part, the effects of 18 mGluR antagonist in preventing morphine withdrawal (Fundytus and Coderre, 1997, 19

Fundytus et al., 1997). Since the mGluRs are distributed in both pre- and postsynaptic 20 neurons, these receptors can affect different behaviors according to their location 21

(Niswender and Conn, 2010). Rats lacking mGluR1 have a better response to opioids in a 22

27 nerve injury model of neuropathic pain (Fundytus et al., 2001). mGluR1 also modulates 1 opioid antinociceptive effects by activating protein kinase C (PKC) and reducing the 2

NMDAR activity (Cerne and Randic, 1992, Kelso et al., 1992, Skeberdis et al., 2001). 3

Pretreatment with mGluR1 antagonists enhances buprenorphine and dezocine (mu opioid 4 receptor agonists) antinociceptive effects in the hot plate test in C57BL/6 mice (Fischer et 5 al., 2008). Moreover, pretreatment with mGluR2/3 agonists attenuates heroin cue-induce 6 reinstatement (Bossert et al., 2005). However, blocking mGluR2/3 was shown to facilitate 7 relapse to morphine (Tahsili-Fahadan et al., 2010). Regarding the mGluR5 receptors, 8 systemic administration of mGluR5 antagonist 2-methyl-6-phenylethynyl-pyridine 9

(MPEP) has been shown to attenuate the inflammatory hyperalgesia; however, failed to 10 attenuate mechanical hyperalgesia in neuropathic pain model in rats (Walker et al., 2001). 11

Also, inhibiting mGluR5 has been shown to attenuate morphine withdrawal induced by 12 naloxone (Palucha et al., 2004, Rasmussen et al., 2004). Thus, blocking the mGluR5 with 13

MPEP with different doses (5, 15 and 50 nmol) was found to attenuate heroin-seeking 14 behavior (Lou et al., 2014). These studies suggest that mGluR5 could be a potential target 15 in preventing opioid reinstatement in animals 16

Figure 1-2 Summary of potential targets involving the glutamatergic system that might 3 attenuate chronic opioid effects. Chronic opioid exposure is associated with reduction of 4 glutamate clearance and accumulation in the synapse. This could lead to overstimulation 5 of the iGluRs. Blocking iGluRs with NMDAR or non-NMDAR antagonists would 6 attenuate the overstimulation of iGluRs. Activating mGluR2/3 with LY379268 and 7 blocking mGluR1/5 with MPEP are also potential targets to attenuate chronic opioids 8 effects. β-lactam compounds, NAC and MS-153 have been shown to increase the 9 expression of GLT-1 and xCT, which can lead to reduction in glutamate accumulation and 10 improve its clearance. 11

1.3. Alcohol and glutamate 1

Recently, reports have focused on the link between alcohol and the glutamatergic system 2

(Gonzales and Jaworski, 1997, Dodd et al., 2000, Allgaier, 2002). Acute alcohol can 3 inhibit the glutamatergic transmission through decreasing NMDAR conductance (Lovinger 4 et al., 1989). However, upon chronic use, tolerance can develop, which is associated with 5 adaptive changes in glutamate transporters and receptors (Trevisan et al., 1994, Rao and 6

Sari, 2012, Rao and Sari, 2014, Das et al., 2015, Hakami et al., 2017). In addition, 7 withdrawal from alcohol is associated with hyperactivity of the glutamatergic system 8

(Rossetti and Carboni, 1995, Tsai et al., 1998). Thus, the effect of alcohol use, dependence, 9 and withdrawal on the glutamatergic transporters and receptors are discussed below in 10 details. 11

1.3.1. Alcohol and glutamate transporters 12

It has been suggested that acute and chronic alcohol exposure is associated with changes 13 in glutamatergic transmission (Gass et al., 2011). Acute alcohol (6 g/kg) administration 14 for seven days downregulates the expression of GLT-1 in NAc (Althobaiti et al., 2016), 15 striatum, and HIP (Alshehri et al., 2017). In addition, chronic alcohol use can increase 16 glutamate in several brain areas including NAc (Das et al., 2015), VTA (Ding et al., 2013) 17 and other areas in the mesocortical system. Thus, the elevation in glutamate levels in 18 chronic alcohol drinking is possibly due to a downregulation in GLT-1 expression in the 19

NAc (Goodwani et al., 2015), HIP, and AMY (Aal‐Aaboda et al., 2015). Also, the 20 expression of xCT was downregulated with chronic alcohol drinking in NAc (Goodwani 21 et al., 2015, Hakami et al., 2016), HIP, and AMY (Aal‐Aaboda et al., 2015). Therefore, 22

30 targeting glutamate homeostasis attenuates chronic alcohol drinking in animals. Beta- 1 lactam compounds upregulate GLT-1 and xCT expression in in vivo (Goodwani et al., 2

2015, Alasmari et al., 2016, Hakami et al., 2016, Hakami et al., 2017) and GLT-1 in vitro 3

(Rothstein et al., 2005). In fact, ceftriaxone was shown to attenuate alcohol consumption, 4 which was associated with upregulation of GLT-1 and xCT expression (Rao and Sari, 5

2014). In addition, MS-153, a GLT-1 activator, attenuated alcohol drinking and 6 normalized both GLT-1 and xCT levels (Alhaddad et al., 2014b, Aal‐Aaboda et al., 2015). 7

However, further studies are still needed to explore the changes in other glutamate 8 transporters expression in chronic alcohol use. 9

In alcohol withdrawal, glutamate is elevated in several brain areas including NAc, HIP, 10 and striatum (Rossetti and Carboni, 1995, Dahchour and Witte, 1999, Das et al., 2015). 11

Also, it was found that repeated cycles of alcohol withdrawal can elevate glutamate levels 12 in rats (Dahchour and De Witte, 2003). Regarding the expression of the glutamate 13 transporters in alcohol withdrawal, it has been found that binge alcohol withdrawal in 14 alcohol-preferring (P) rats is associated with a downregulation in GLT-1expression in NAc 15 and mPFC (Das et al., 2016). Ceftriaxone upregulates GLT-1 expression and attenuates 16 alcohol withdrawal symptoms (Das et al., 2015, Das et al., 2016). Alcohol relapse is still 17 one of most challenging aspects of alcohol addiction management. Chronic alcohol can 18 lead to neuroadaptive changes in the glutamatergic system, which is important in alcohol 19 craving. Several studies have investigated the effect of beta-lactam compounds like 20 ceftriaxone during alcohol abstinence to attenuate alcohol reinstatement. In fact, it has 21 been shown that ceftriaxone attenuates alcohol relapse via upregulating GLT-1 and xCT 22 expression in NAc and PFC in P rats (Qrunfleh et al., 2013, Alhaddad et al., 2014a). 23

Together, these data confirm the strong association between acute and chronic alcohol use 1 and changes in glutamate transporters, where modulating these transporters could have a 2 potential benefit in managing alcohol dependence, withdrawal and relapse. 3

1.3.2. Alcohol and glutamate receptors 4

Several studies have investigated the effects of alcohol on iGluRs, including NMDAR, 5

AMPAR and KAR. Alcohol can reduce the function of NMDAR in a dose-dependent 6 manner in vitro (Lovinger et al., 1989, Hoffman et al., 1990). Also, it has been found that 7 chronic alcohol use can increase the expression of NR2 and NR2B, as a response to the 8 chronic depression by alcohol (Kalluri et al., 1998). In fact, the upregulation and changes 9 in NMDAR signaling could be, in part, the reason behind the development of alcohol 10 tolerance with chronic use. Alternatively, it has been reported that alcohol can reduce the 11 ion current in AMPAR/KAR receptors (Lovinger, 1993). AMPARs have neuroadaptation 12 changes similar to NMDA receptors. Repeated alcohol administration can increase the 13 expression of AMPARs (GluR1 and GluR2) in the dorsal striatum in rats (Wang et al., 14

2012). 15

The neuroadaptation of NMDAR with chronic alcohol use, is involved in the increased 16 neuronal excitability seen in alcohol withdrawal syndrome (Hoffman et al., 1990, Tsai et 17 al., 1998). The upregulation of NMDARs augments the action of glutamate and increases 18

Ca2+ influx, which in part facilitates the neurotoxicity associated with alcohol in 19 withdrawal (Choi, 1992, Hoffman, 1995). Therefore, it has been suggested that NMDAR 20 antagonists could be beneficial in attenuating alcohol withdrawal symptoms. In fact, an 21

NMDAR antagonist (MK-801) reduced seizure severity associated with alcohol 22

32 withdrawal in a dose-dependent manner, while administering an NMDAR agonist 1 intensified withdrawal symptom in mice (Grant et al., 1990). Alternatively, alcohol 2 withdrawal can increase the expression of AMPARs (GluRA1 and GluRA2/3) in the 3 basolateral AMY (Christian et al., 2012). Glutamate receptors play an essential role in 4 alcohol consumption and withdrawal. Thus, memantine (an NMDAR antagonist) 5 attenuates cue induced reinstatement-like behavior in animals (Vengeliene et al., 2015). In 6 humans, memantine reduces alcohol craving in patients in a dose-dependent manner 7

(Krupitsky et al., 2007). Similarly, an AMPA antagonist (GYKI 52466) reduces alcohol- 8 seeking behavior in a dose-dependent fashion in animals (Sanchis-Segura et al., 2006). 9

Together, the glutamate receptors could serves as an important potential therapeutic target 10 in alcohol dependence management. 11

1.4. Opioids and alcohol 12

It has been shown that alcohol can induce an elevation in beta-endorphin release (De Waele 13 and Gianoulakis, 1993) as well as the expression of endorphin and enkephalin genes in 14 animals (Boyadjieva and Sarkar, 1997). Opioid receptors have been suggested to be 15 involved in alcohol drinking (Méndez and Morales-Mulia, 2008). In fact, chronic alcohol 16 exposure was associated with downregulation of mu opioid receptor in the NAc and 17 striatum (Turchan et al., 1999). Also, it has been reported that mu opioid receptor knockout 18 mice drink less alcohol as compared to wild mice (Roberts et al., 2000). Alternatively, 19 other opioid receptors have been investigated such as kappa opioid receptor and delta 20 opioid receptor in alcohol drinking. The delta opioid receptor blocker ICI 174864 was 21 shown to attenuate alcohol consumption equally as well as naloxone (Froehlich et al., 22

1991). However, blocking delta opioid receptor with D-Pen2,D-Pen5 was not effective in 1 reducing alcohol consumption (Honkanen et al., 1996). In fact, a single administration of 2 the delta opioid receptor antagonist norbinaltorphimine showed an opposite result to ICI 3

174864 and caused an increase in alcohol consumption in rats (Mitchell et al., 2005). This 4 could be due the possibility of loss of selectivity of these blockers as well as blocking 5 multiple opioids receptors when these blocker compounds are administered. In regard to 6 kappa opioid receptor, mice lacking kappa opioid receptors have been shown to drink less 7 alcohol (Kovacs et al., 2005). Together, these data show a strong relationship between 8 opioid receptors and alcohol drinking behavior. 9

1.5. Opioids and NO 10

NO is a diffusible chemical messenger that modulates several neuronal functions. NO is 11 produced from L-arginine via a catalytic process by NOS (Knowles et al., 1989) (Figure 1- 12

3). Different NOS subtypes have been identified, including neuronal NOS (nNOS), 13 endothelial NOS (eNOS) and inducible NOS (iNOS) (Bredt and Snyder, 1990). The 14 catalytic process mediated by nNOS in glutamatergic neurons is activated by increased 15

Ca2+ influx and the CaM-dependent pathway, which is triggered by NMDAR activation 16

(Garthwaite et al., 1989). It has been suggested that released NO diffuses back to the 17 presynaptic neuron where it increases the activity of soluble guanylyl cyclase (sGS) and 18 cyclic guanosine monophosphate (cGMP), which enhances the glutamate release (Dawson 19 et al., 1991, Manzoni et al., 1992, Kiss and Vizi, 2001, Uzbay and Oglesby, 2001). The 20 majority of NO effects on neuronal excitability depend on cGMP synthesis as well as the 21 activation of the sGC. This leads to enhancement of cGMP formation and cGMP- 22 dependent PKC, which have been suggested to be essential in glutamate presynaptic release 23

(Consolo et al., 1999, Prast and Philippu, 2001). Several studies have reported that 1 blocking the glutamate-NO-cGMP pathway in neurons can reduce glutamate-induced 2 neurotoxicity (Choi, 1987, Dawson et al., 1991, Montoliu et al., 1999). In fact, low doses 3 of NO donor S-nitroso-N-acetylpenicillamine (SNAP) has been shown to reduce the 4 glutamate release; however, high doses have been shown to enhance glutamate release in 5 the HIP in rats (Segieth et al., 1995). Similarly, hydroxylamine, a NO donor, has been 6 shown to produce different effects depending on the administered dose (Sequeira et al., 7

1997). Low concentrations of NO decrease the exocytotic release of glutamate; this 8 reduction is associated with an increase in sGS and cGMP (Sequeira et al., 1997). The 9 enhancement of glutamate release by hydroxylamine is due to the increase in sGS and 10 cGMP as well as reduction of adenosine tri/diphosphate (ATP/ADP) ratios and inhibition 11 of mitochondrial respiration (Sequeira et al., 1997, McNaught and Brown, 1998). 12

Together, these data suggest that NO may have dual functionality, where it inhibits the 13 glutamate release at low concentrations and enhances the glutamate release at higher 14 concentrations. 15

Glutamate is cleared from the synapses mostly by the astroglial glutamate transporters 16

GLT-1 and xCT. Astrocytes play an important role in glutamate clearance as well as the 17 glutamate-glutamine cycle (Figure 1-3). In addition, astrocytes express all of the opioid 18 receptors (Belcheva et al., 2005). It has been suggested that activation of mu opioid 19 receptor in astrocytes involves phosphorylation of ERK and PKC (Belcheva et al., 2005). 20

Moreover, the activation of mu opioid receptor increases Interleukin 1 beta (IL-1β) 21 expression in HIP astrocytes (Liu et al., 2011). The increase of IL-1β stimulates other 22 neuroactive substances, including iNOS, tumor necrosis factor alpha (TNFα), and IL-6, as 23

35 shown in human fetal astrocytes (Liu et al., 2000). iNOS has been shown to be expressed 1 in the astrocytes, and is responsible for formation of NO (Agulló and García, 1991, Galea 2 et al., 1992, Simmons and Murphy, 1992, Wallace and Fredens, 1992, Gabbott and Bacon, 3

1996, Loihl et al., 1999). On the other hand, glutathione reduces NO production (Chen et 4 al., 2001). This protective mechanism was suggested to involve a direct interaction 5 between glutathione and NO, producing an inactive compound called nitrosoglutathione 6

(GSNO) (Clancy et al., 1994, Hogg et al., 1996). However, with high levels of NO, 7 glutathione levels could reduce dramatically, which contributes to the toxic effects 8 associated with high levels of NO (Clancy et al., 1994, Padgett and Whorton, 1998). In 9 astrocytes, NO can also inhibit glutamine synthesis by the inhibition of glutamine 10 synthetase, which can be reversed by blocking the NO production with NOS inhibitors 11

(Kosenko et al., 2003). A list of NOS inhibitors and their selectivity are shown in Table 12

1-1. Glutamine synthetase is responsible for converting glutamate in the astrocytes into 13 glutamine (Meister, 1974). Then, glutamine is released into the extracellular space, where 14 it can be taken up by the neurons to be converted again into glutamate (Hertz et al., 1978). 15

Alternatively, ammonia can diffuse from the astrocytes due to its high permeability 16

(Brookes, 2000). Hyperammonemia has been suggested to reduce glutamate uptake in both 17 astrocytes and neurons (Butterworth, 1998). Acute increases in ammonia was shown to 18 increase glutamine synthetase activity and induced depletion of ATP, which was associated 19 with an increase in cGMP levels in rats (Kosenko et al., 2003). In fact, NO was found to 20 deplete neuronal ATP, but not astrocytic ATP, which contributes to mitochondrial 21 inhibition (Bal-Price and Brown, 2001). The inhibition of the neuronal mitochondrial 22 respiration was suggested to enhance glutamate release as mentioned earlier. The effect of 23

NO was blocked by using NMDAR antagonists, which supports the notion that high levels 1 of ammonia could produce a neurotoxic effect, in part, through activation of NMDARs 2

(Hermenegildo et al., 1996, Kosenko et al., 2003). Also, others have tested the effect of 3 the NOS inhibitor nitroarginine as a pretreatment, which reduced the production of NO and 4 attenuated ammonia-induced neurotoxic effects in rat brain (Kosenko et al., 1995). It is 5 important to note that activation of astrocytes can lead to production of IL-1β and TNFα 6 inflammatory mediators as well as NO, which participate in neuronal death through 7 different mechanisms, including increased glutamate release and excitotoxicity (Bal-Price 8 and Brown, 2001, Brown and Bal-Price, 2003). It has been found that IL-1β and TNFα 9 can reduce glutamate uptake as well as glutamine synthetase activity (Chao et al., 1995) 10

(Figure 1-3). Therefore, based on this evidence, we suggest here that opioid exposure may 11 be associated with an increase in NO production and glutamate release. Also, glutamate 12 uptake may be compromised, which can further increase excitotoxicity associated with 13 opioid exposure. 14

1.5.1. Opioids and nitric oxide synthetase inhibitors 16

As mentioned earlier, NO has an essential role in the development of opioid tolerance and 17 dependence (Majeed et al., 1994). Therefore, it has been found that inhibiting the NO 18 formation can decrease morphine tolerance and dependence (Kolesnikov et al., 1993). In 19 addition, pretreatment with a nNOS blocker, NG-nitro-L-arginine (L-NA) (1 mg/kg i.p), 20

15 min before and 1 hr after morphine injections (5 mg/kg) attenuated morphine tolerance 21

(Elliott et al., 1994). Furthermore, co-administration of L-NA (4 mg/kg) with morphine 22

(75 mg pellets) attenuated the development of tolerance (Kolesnikov et al., 1993). 1

Furthermore, co-administration of L-NA (8 mg/kg) with morphine (5 mg/kg) for 5 days 2 also prevented morphine-induced tolerance (Kolesnikov et al., 1992). In addition to the 3 effects of nNOS inhibitors on opioid tolerance, other studies have shown that pretreatment 4 with L-NA (7.5 mg/kg) and L-NG-nitro arginine methyl ester (L-NAME) (60 mg/kg) 5 attenuate naloxone- (0.5 mg/kg) induced withdrawal effects after morphine exposure (75 6 mg pellet s.c) for 8 days in rats (Kimes et al., 1993). It has been found that using L-NAME 7

(4 and 8 mg/kg, s.c) twice a day during morphine pellet implantation (75 mg, total 4 pellets 8 for 3 days) improved morphine analgesic effects and inhibited naltrexone (5 mg/kg) 9 induced withdrawal effects (Bhargava, 1995). Of note, a single high dose of L-NAME (20 10 mg/kg) after morphine pellet implantation (40 mg) enhanced morphine analgesia and 11 blocked withdrawal effects that were induced by naloxone (2 mg/kg) 72 hr after morphine 12 pellet implantation. However, exposure to L-Canavanine (200 mg/kg i.p), an iNOS 13 blocker, did not show any effect (Özek et al., 2003). Accordingly, a study reported that 14 administering a selective nNOS blocker, 7-Nitroindazol (7-NI) (25 ug), intrathecally (I.T) 15

15 min before morphine injections (15 ug IT) for 7 days significantly improved morphine 16 analgesia, and this effect was associated with reduction in p38 MAPK in spinal microglia 17 in rats (Liu et al., 2006). Similarly, the inhibition of NOS with L-NAME (400 ug, I.T) 18 improved morphine analgesic activity when examined in tail-flick tests in animals 19

(Przewłocki et al., 1993). Administration of L-NAME and morphine were used in a 20 behavioral test to determine if formalin evoked the release of nitrite/nitrate and glutamate 21 cord (Watanabe et al., 2003). This study found that co-administration of L-NAME (400 22

38 nmol, I.T) and morphine (1.25 nmol, I.T) improved morphine analgesic activity by 1 inhibiting NMDA receptors and NO formation in the spinal cord (Watanabe et al., 2003). 2

1.5.2. Opioids, nitric oxide and glutamate receptors antagonists 3

Studies have suggested that glutamate receptors play an essential role in the development 4 of morphine tolerance (Kest et al., 1997). AMPARs and NMDARs have similar, but not 5 identical, contributions to the development of opioid tolerance. Activation of these 6 receptors, particularly the NMDAR by glutamate, causes an influx in Ca2+ into neurons 7 leading to activation CaM-dependent NOS (Heinzel et al., 1992). This activation is 8 accompanied by an increase in the formation of NO (Garthwaite et al., 1989). It has been 9 shown that MK-801 exposure can improve morphine analgesic effects and prevent the loss 10 of this morphine analgesic effect that might be associated with repeated administration of 11

L-arginine (Bhargava et al., 1998). This shows that blocking the formation of NO through 12 attenuating the activation of glutamate receptors is crucial to improving opioids analgesic 13 effects. Alternatively, the non-NMDAR antagonist CNQX blocked KA-increased nitrite 14 excitotoxicity (Radenovic and Selakovic, 2005). Others have reported that CNQX may 15 lead to reduction of L-arginine, which is the primary precursor for NO formation (Grima 16 et al., 1998, Segieth et al., 2004). However, further studies are still needed to investigate 17 the role of non-NMDAR antagonists to evaluate if pretreatment with NMDAR or non- 18

NMDAR antagonists alters the NOS pathway and produce similar protection against the 19 negative effects associated with opioid use. 20

1.5.3. Opioids, nitric oxide and opioids receptors antagonists 1

It has been found that L-NA administration can attenuate tolerance to analgesia produced 2 by mu opioid receptor agonists, but not to kappa opioid receptor agonists (Kolesnikov et 3 al., 1993). Inhibition of nNOS potentiates mu opioid receptor, delta opioid receptor and to 4 a lesser extent kappa opioid receptor -mediated spinal antinociception in both acute and 5 prolonged pain (Machelska et al., 1997). Therefore, most studies have focused on the 6 effects of mu opioid receptor agonists because of their analgesic effects, including those 7 produced by morphine, hydrocodone, oxycodone, and other opioids. It has been found that 8 treating PC 12 cells with morphine in doses ranging from 10 to 1000 uM for 72 hr increases 9

NO production; however, pretreatment with naloxone (1 uM) reduces NO production 10

(Zarrindast et al., 2012). A single dose of morphine, a mu opioid receptor agonist, at dose 11 of 5 mg/kg (s.c) or chronic morphine (75 mg pellet, s.c implantation) increases the activity 12 of Ca+2 dependent NOS in the cerebellum, but not in frontal cortex or forebrain, which is 13 blocked with co-treatment with naloxone (1 mg/kg) in male CD1 mice (Leza et al., 1995). 14

Other studies found that chronic morphine can lead to an increase in the activity of nNOS 15 in different brain regions, including the olfactory bulb, olfactory nuclei, cerebellum, locus 16 coeruleus, and medulla oblongata (Cuéllar et al., 2000). Together, these data suggest that 17 blocking opioid receptors might attenuate the production of NO and chronic opioids 18 effects. 19

1.5.4. Opioids rewards and nitric oxide 20

Several studies have tested the effects of NOS inhibitors on opioid reward using CPP. 21

Intra-accumbal administration of L-NAME (0.3 - 3 ug/rat) attenuated morphine-induced 22

(5 mg/kg, i.p) CPP in sensitized rats (Sahraei et al., 2007). The morphine sensitization was 1 induced by exposure to morphine (5 mg/kg, i.p) for 3 sessions and re-exposure to lower 2 doses of morphine (0.25, 0.5 and 0.75 mg/kg, i.p) for 5 days in rats (Sahraei et al., 2007). 3

Furthermore, intra-accumbal administration of L-arginine, an NO precursor, at a dose of 4

0.03 and 0.05 ug/rat, facilitated the reinstatement of lower dose of morphine (0.5 mg/kg, 5 i.p) in Wistar rats using CPP (Gholami et al., 2002). However, similar doses of L-arginine 6 were infused in the HIP and did not facilitate morphine-induced preference. In the same 7 study, morphine-induced preference was blocked by administration of L-NAME (0.3 - 3.0 8 ug/rat) (Karami et al., 2002). Also, it has been shown that pretreatment with L-NA (20 9 mg/kg, i.p) blocked morphine-induced (3 mg/kg) CPP in rats (Kivastik et al.). 10

Furthermore, the NMDA-NO pathway in the HIP area was reported to modulate memory 11 processing in morphine-induced CPP. It was reported that morphine-induced CPP was 12 associated with an increase in the expression of nNOS, and sGC/cGMP-dependent PKC 13

(Shen et al., 2016). Blocking these proteins inhibited morphine-induced preference (Shen 14 et al., 2016). 15

Figure 1-3 Schematic representation of the effects of chronic exposure to opioids on the 3 glutamatergic system as well as NO production. Several compounds have been suggested 4 to attenuate the production of NO. NO is produced by NOS. Blocking the NOS enzyme 5 with NOS inhibitors can attenuate the synthesis of NO in both neurons and astrocytes. 6

Blocking MOR with naloxone can reduce the production of NO affecting opioid tolerance, 7 withdrawal and relapse. Chronic opioid exposure is associated with an accumulation of 8 glutamate and reduction in its clearance. Overstimulation of iGluRs can lead to dramatic 9 increases in NO production. Thus, blocking iGluRs with NMDAR and non-NMDAR 10 antagonists can attenuate NO production. β-lactam compounds and NAC can increase the 11 expression of GLT-1 and xCT, which reduces glutamate accumulation and improve its 12 clearance. Therefore, β-lactam compounds should also reduce NO production through 13 regulating glutamate homeostasis. 14

1.6. Opioids and body temperature 1

The opioid system plays an important role in many physiological functions in the body. 2

Evidence has shown that the opioid system regulates body temperature (Baker and Meert, 3

2002). Different factors can modulate the response of opioid effects on the body 4 temperature, including species, dose, ambient temperature, and route of administration 5

(Geller et al., 1986, Adler and Geller, 1993). It has been reported that low doses of 6 morphine induce hyperthermia (increased body temperature), while high doses can 7 produce hypothermia (decreased body temperature) (Adler and Geller, 1993). However, 8 intracerebroventricular (i.c.v) injections of morphine produce hyperthermia (Adler and 9

Geller, 1993). It has been reported that lower doses of morphine produce hyperthermia, 10 which reflect a possible physiologic action related to endogenous opioids (Clark, 1979). 11

However, it was found that rats showing tolerance to morphine may develop hypothermia 12 rather than hyperthermia (Cox et al., 1976). Of note, other study found that acute 13 administration of delta opioid receptor agonists like (+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5- 14 dimethyl-1-piperazinyl)-3methoxy-benzyl]-N,N-diethyl benzamide (SNC-80) produces 15 hypothermic effects (Rawls et al., 2006). Furthermore, a study examined the effect of i.c.v. 16 injections of morphine (0.5 - 65 ug) and heroin (10 - 100 ug) and found that both 17 compounds produced hyperthermia in rats (Geller et al., 1986). Another study has also 18 tested i.c.v. injections of PL-17 (mu opioid receptor agonist), which were associated with 19 hyperthermia, while dynorphin (a kappa opioid receptor agonist) reduced body 20 temperature, but DPDPE (a delta opioid receptor agonist) did not show any changes in 21 body temperature (Handler et al., 1992). The increase or decrease in metabolic rate can 22 affect the quantity of heat that is available to regulate body temperature through heat 23

43 exchange mechanisms (Handler et al., 1992). These alterations in metabolic rate and heat 1 exchange can be blocked with selective opioid receptor antagonist pretreatment (Handler 2 et al., 1992). Additionally, it appears that oxidative metabolism is extremely sensitive to 3 mu opioid receptor and kappa opioid receptor stimulation and is probably the primary cause 4 of body temperature alteration following kappa opioid receptor and kappa opioid receptor 5 activation (Handler et al., 1992). Central stimulation of delta opioid receptor by DPDPE 6 did not appear to have a direct effect on the metabolic functions that support the body 7 temperature (Handler et al., 1992). 8

1.6.1. Effects of opioid receptor blockers on body temperature 9

Evidence has shown that opioid receptors are somehow involved in the pathophysiology 10 of hyperthermic brain injury. A study examined the possible effects of naloxone (1, 5 and 11

10 mg/kg, i.p.) and naltrexone (1, 5 and 10 mg/kg, i.p.) as pretreatments to attenuate heat 12 stress in Wistar rats (Sharma et al., 1997). This study found that high doses of naloxone 13 or naltrexone (10 mg/kg, i.p.) were required to obtain a neuroprotective effect against heat 14 stress. The lower doses of these compounds were not effective. These observations 15 suggested that blocking kappa opioid receptor may be involved in these effects since high 16 doses of these drugs block all 3 receptors (Wang et al., 2007). High doses of these drugs 17 block kappa opioid receptors, but lower doses block mu opioid receptor and delta opioid 18 receptor (Black et al., 1991, Cruz and Granados-Soto, 2015). Another study examined the 19 effect of fentanyl, intravenously (100 ug/kg) as well as i.c.v (3.4 ug/rat), in rats on body 20 temperature (Cao and Morrison, 2005). This study revealed that fentanyl increased brown 21 adipose tissue thermogenesis and heart rate through possible activation of the sympathetic 22

44 nervous system; however, this effect was attenuated with pretreatment with naloxone (100 1 nmol in 10 ul, i.c.v). It is suggested that these effects could be mediated through the 2 activation of the dorsomedial hypothalamus, rostral raphe pallidus and rostral ventrolateral 3 medulla. 4

1.6.2. Effects of modulating glutamate receptors and transporters on opioid-induced 5 changes in body temperature. 6

The glutamatergic system is involved in thermoregulation (Singh and Gupta, 1997, 7

Morrison et al., 2008, Morrison and Nakamura, 2011). Intracisternal (within one of the 8 subarachnoid cisternae) injection of aspartate or glutamate at a dose of 20 ug/rat induced 9 hyperthermia in rats (Singh and Gupta, 1997). Several studies have examined the use of 10 glutamate receptor blockers to modulate the hyperthermic effects of opioids. Blocking 11

NMDARs with LY 235959 (0.1 - 1 mg/kg, s.c.) or dextromethorphan (5 - 15 mg/kg, s.c.) 12 attenuates i.c.v morphine-induced hyperthermia in rats (Rawls et al., 2003). Another study 13 showed that treatment with ceftriaxone, a β-lactam compound known to upregulate GLT- 14

1 and xCT, attenuated morphine-induced hyperthermia (Rawls et al., 2007). This study 15 found that pretreatment with ceftriaxone at a dose of 200 mg/kg (i.p) for 7 days prior to 16 morphine exposure (1, 4, 8 and 15 mg/kg, s.c.), significantly blocked morphine-induced 17 hyperthermia. This effect was suggested to be mediated through the ability of ceftriaxone 18 to enhance the glutamate clearance and homeostasis (Rawls et al., 2007). Other β-lactam 19 compounds were tested such as clavulanic acid. Similar to ceftriaxone, pretreatment with 20 clavulanic acid at a dose of 10 mg/kg (i.p.) for 7 days blocked morphine (4 mg/kg, s.c)- 21 induced hyperthermia (Schroeder et al., 2014). 22

1.6.3. Effects of modulating NO transmission in opioid-induced changes in body 1 temperature. 2

Studies have reported that the hyperthermic responses to a number of drugs, including 3 morphine, lipopolysaccharide, interleukin-1β, and prostaglandin E2, are linked to NO 4 production (Amir et al., 1991, Minano et al., 1997, Roth et al., 1998, Benamar et al., 2001). 5

Modulation of NO production with NOS inhibitors reduce hyperthermia. A study tested 6 the hyperthermic effects of morphine in rats at doses of 4 and 15 mg/kg, s.c., and examined 7 the co-administration of L-NAME (50 mg/kg, s.c.) with morphine (Benamar et al., 2001). 8

This study found that L-NAME attenuated morphine-induced hyperthermia at higher 9 doses, but failed in lower doses, which may inducate that NO may have a dual activity in 10 regulating the body temperature. Similarly, L-NAME (1 mg, i.c.v) blocked morphine- 11 induced hyperthermia at higher doses and failed at lower doses. It is important to note that 12

L-NAME attenuated morphine-induced hyperthermia for about 30 min, but then was 13 followed by hypothermia. However, L-NAME alone did not produce any changes in body 14 temperature (Benamar et al., 2001). Interestingly, L-NAME only blocked the effects of 15 higher doses of morphine but not lower doses; this effect was suggested to involve 16 antipyretic effects of adrenocorticotropic hormone (ACTH) and cortisone, which are 17 affected by morphine and NOS inhibitors. The production of NO was found to inhibit 18

ACTH and cortisone secretion (Bugajski et al., 1998), however, morphine and NOS 19 inhibitors stimulate their release (Nikolarakis et al., 1987, Nikolarakis et al., 1989). 20

Therefore, the additive effects of NOS with morphine could further increase the level of 21 these hormones. However, further studies are warranted to investigate these effects. 22

The effects of the different types of NOS inhibitors (nNOS, eNOS, and iNOS) were 1 examined to determine their role in morphine (15 mg/kg, s.c)-induced hyperthermia 2

(Benamar et al., 2003). This study showed that 7-NI, a selective nNOS inhibitor at a dose 3 of 5 and 10 mg/kg (s.c), was able to block morphine-induced hyperthermia, while both the 4 eNOS [N (5)-(-iminoethyl)-L-ornithine (L-NIO)] and iNOS [aminoguanidine (AG)] 5 inhibitors failed to block hyperthermia. This suggests that neither eNOS nor iNOS have a 6 role in opioid-induced hyperthermia in rats. Interestingly, the selectivity of 7-NI to nNOS 7 has many advantages over other non-selective NOS inhibitors. For instance, L-NAME was 8 associated with an increase in blood pressure, however, the selective nNOS inhibitor 7-NI 9 was not (MacKenzie et al., 1994). The finding with 7-NI provided promising results that 10 the neuronal production of NO could be a potential target for modulating opioid-induced 11 hyperthermia. The potential targets that might attenuate hyperthermia associated with 12 opioids are summarized in Figure 1-4. 13

Figure 1-4 Summary of potential targets that modulate body temperature associated with 12 exposure to opioids. Blocking the NOS enzyme with NOS inhibitors can attenuate the 13 synthesis of NO in both neurons and astrocytes, which was shown to normalize body 14 temperature. Blocking MOR could prevent MOR agonists like morphine from increasing 15 the body temperature, while blocking the kappa opioid receptor and delta opioid receptor 16 could lead to opposite effects. Overstimulation of iGluRs can lead to dramatic increase in 17

NO production. Thus, blocking the iGluRs with LY 235959 or dextromethorphan can 18 attenuate the hyperthermia associated with opioids. β-lactam compounds can increase the 19 expression of GLT-1 and xCT, which reduce glutamate accumulation and increase its 20 clearance. 21

Conclusion 1

This review discussed the strong relationship between exposure to opioids and glutamate 2 function. Several studies found that repeated exposure to opioids affects the expression of 3 glutamate receptors and glutamate transporters. Modulation of these receptors and 4 transporters were found to attenuate several effects of opioids, including aspects of 5 tolerance, dependence, withdrawal, and relapse. Also, chronic alcohol exposure has been 6 shown to produce several changes in the function and expression of glutamate receptors 7 and transporters. There are abundant connections between NO production and opioid 8 tolerance, withdrawal and dependence. Several studies investigated the modulatory effects 9 of NO using NOS inhibitors, opioid receptor blockers, and glutamate receptor blockers. 10

The effect of opioids on body temperature was found to occur via different mechanisms, 11 including effects of opioids. Together, findings showed that chronic opioid exposure 12 increases glutamate and NO transmission, so targeting these pathways could provide 13 potential benefits for opioid-induced tolerance, withdrawal, dependence, and relapse. 14

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Chapter 2 4

Effect of Hydrocodone on Astroglial Glutamate Transporters in Primary Astrocyte 6 Cell Cultures 7

Fahad S. Alshehri 1, Atiah H. Almalki2, Qasim Alhadidi2, Alqassem Y. Hakami1, Zahoor A. 9 Shah2, Youssef Sari1* 10 11 12 13 14 1 Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and 15 Pharmaceutical Sciences, University of Toledo, Health Science Campus, Toledo, OH, USA 16 17 2 Department of Medicinal and Biological Chemistry, College of Pharmacy and 18 Pharmaceutical Sciences, University of Toledo, Toledo, OH, USA 19

* Corresponding author: 22 Dr. Youssef Sari 23 University of Toledo, College of Pharmacy and Pharmaceutical Sciences 24 Department of Pharmacology and Experimental Therapeutics 25 Health Science Campus, 3000 Arlington Avenue 26 Toledo, OH 43614, USA 27 E-mail: [email protected] 28 Tel: 419-383-1507 29 30

Abstract 1

Glutamate is an excitatory neurotransmitter involved in several brain functions. Glutamate 2 homeostasis is regulated by several glutamate transporters. Among them, glutamate 3 transporter 1 (GLT-1) is responsible for the majority of glutamate clearance. In addition, 4 cystine-glutamate antiporter (xCT) and glutamate/aspartate transporter (GLAST) have 5 been shown to play roles in maintaining the glutamate homeostasis. Therefore, impairment 6 of these glutamate transporters can lead to dysregulation of glutamate clearance and 7 homeostasis. It has been shown that different drugs of abuse, including opioids, can alter 8 the functioning and expression of the glutamate transporters. Hydrocodone (HYD) is a 9 semisynthetic opioid that has been used for many years for pain management. HYD is one 10 of the most prescribed opioid analgesics. To examine the effect of HYD on astroglial 11 glutamate transporters, we investigated the effect of HYD on GLT-1, xCT, and GLAST 12 using primary astrocyte cell cultures. The primary astrocytes were isolated from brains of 13

3-4-day-old postnatal rat pups. HYD was exposed to astrocyte cultures at three different 14 doses (0.5 µM, 1 µM, and 2µM) and at three time periods (1, 3 or 5 days). We found that 15

HYD treatment for five days at a dose of 2 µM was associated with a reduction in the 16 expression of GLT-1 and xCT, but no effect was observed in GLAST expression. 17

Therefore, restoring GLT-1 and xCT expression could be a potential target to reverse the 18 effect of HYD on these glutamate transporters. 19

Introduction 1

Hydrocodone (HYD) is a semisynthetic opioid, derived chemically from codeine, used in 2 the management of mild to moderate pain (Armstrong and Cozza, 2003). HYD is one of 3 the most commonly prescribed opioids (Paulozzi et al., 2009). It has been suggested that 4

HYD has similar abuse liability compared to oxycodone and hydromorphone. Moreover, 5 dysregulation of glutamate clearance has been shown to decrease opioid efficacy in pain 6 management (Meller and Gebhart, 1993, Mao et al., 1995, Salvemini and Neumann, 2009). 7

Several studies have reported that opioid use is associated with changes in extracellular 8 glutamate levels in several brain regions, including the nucleus accumbens (NAc) 9

(LaLumiere and Kalivas, 2008), locus coeruleus (Aghajanian et al., 1994) and ventral 10 tegmental area (VTA). In fact, high extracellular concentrations of glutamate and aspartate 11 have been detected in the cerebrospinal fluid (CSF) of rats given long-term infusions of 12 morphine (Tai et al., 2007). Also, chronic use of opioids has been associated with 13 downregulation of spinal glial glutamate transporters (Mao et al., 2002). Several glutamate 14 transporters are involved in controlling extracellular glutamate levels: glutamate 15 transporter 1 (GLT-1), cystine-glutamate antiporter (xCT), and glutamate/aspartate 16 transporter (GLAST) (Danbolt, 2001). It is important to note that GLT-1 is responsible for 17 clearing the majority of glutamate in synapses (Danbolt, 2001). xCT exchanges glutamate 18 for cystine, playing a role in glutathione biosynthesis (Danbolt, 2001). GLAST transports 19 both glutamate and aspartate from the synapse into astrocytes (Danbolt, 2001); however it 20 is primarily expressed in the cerebellum (Storck et al., 1992). 21

Astrocytes are one of the most abundant types of glial cells in the brain (Kimelberg and 22

Norenberg, 1989). These cells are responsible for many functions in the brain, including 23

90 neurotransmission, electrolyte homeostasis, cell signaling, synapse modulation and 1 metabolic support to the endothelial cells in the blood-brain barrier (Kimelberg and 2

Norenberg, 1989, Abbott et al., 2006, Kimelberg and Ransom, 2012). Importantly, 3 astrocytes regulate the uptake of glutamate through astroglial glutamate transporters: GLT- 4

1, GLAST (Perego et al., 2000), and xCT (Seib et al., 2011). Thus, this study was 5 performed to investigate the effects of HYD on the expression of these glutamate 6 transporters GLT-1, xCT and GLAST in primary astrocyte cell cultures. 7

2.1. Materials and Methods 8

2.1.1. Drugs 9

HYD (+)- bitartrate salt and phosphate buffer solution (PBS) were purchased from 10

Sigma-Aldrich (St. Louis, MO). 11

2.1.2. Experimental design 12

Primary astrocytes culture was performed based on a previous study (Schildge et al., 2013). 13

Briefly, primary astrocytes were isolated from the brains of rat pups (postnatal, day 3 or 14

4). All animal procedures were approved by the University of Toledo Animal Care and 15

Use Committee and followed the guidelines of the National Institutes of Health. All 16 procedures were conducted under sterilized conditions to avoid possible contamination. 17

The pups were euthanized by CO2 inhalation and then rapidly decapitated. Collected 18 brains were kept in the cold EBSS/HEPES buffer. When the cortices were removed, tissues 19 were cut into small pieces in dissection solution (50 ml 10XHBSS, 5ml Pen-Strep, 5 ml 20

Na-Pyruvate, 10 ml HEPES, 5ml Glucose, and 425 ml ddH2O). Then, samples were 21

91 digested by using papain solution in EBSS/HEPES buffer for 30 min. DNase was then 1 added to the digested tissue and incubated for about 10 min to hydrolyze the DNA. The 2 cells were dissociated from the tissue by pipetting 5-10 times, and then collected, filtered 3 through 70 uM cell strainer, and counted using a hemocytometer. The cells were allowed 4 to grow on a pre-coated (poly-D-lysine) plate for seven days. Microglial cells were 5 removed by shaking the cells in an orbital shaker for 30 min at 180 rpm. Oligodendrocyte 6 cells were removed by shaking the cells for 6 hrs at 240 rpm at 37 °C. Astrocytes were 7 then plated and allowed to grow in 60 mm plates for 12 to 14 days. As shown in Figure 2- 8

1, astrocytes were divided into three main time point groups: Day 1, Day 3 and Day 5. In 9 each time point, each group was given three concentrations (0.5 uM, 1 uM, and 2 uM) of 10

HYD as a daily treatment. The HYD doses were selected based on the similarity between 11

HYD and morphine; morphine has been tested in astrocytes at similar doses (Hauser et al., 12

1996, Ikeda et al., 2010). HYD was dissolved in phosphate buffer saline (PBS) as a vehicle. 13

A control group was also tested with vehicle (PBS) for 1, 3, or 5 days (Day 1, Day 3 and 14

Day 5). 15

Figure 2-1 Experimental timeline with HYD 3 doses assessed at 3 time-points (Day 1, Day 19

3, and Day 5). 20

2.1.3. Western blot of GLT-1, xCT and GLAST protein expression 1

The Western blot procedure was performed as previously described (Sari et al., 2009, 2

Alshehri et al., 2017). All samples were lysed in lysis buffer (50 mM Tris–HCl, 150 mM 3

NaCl, 1 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS). Protein content was measured 4 in samples and loaded onto 10-20% Tris-glycine gels. After separation with 5 electrophoresis, the proteins were transferred onto PVDF membranes. Membranes were 6 then blocked using 3-5% fat-free milk in Tris-buffered saline-Tween 20 (10%) (TBST). 7

Rabbit-GLT-1 antibody (Abcam; 1:1000 dilution), Rabbit-xCT antibody (Abcam; 1:1000 8 dilution), and Rabbit-GAPDH antibody (Millipore 1:3000 dilution) were then added and 9 incubated overnight at 4°C. Membranes were then incubated with the Goat anti-rabbit as 10 a secondary antibody (Thermofisher 1:10000 dilution) . A Chemiluminescent Kit was used 11 to develop the blots using an SRX-101A machine. The blots were quantified digitally 12 using an MCID system. The data were calculated as ratios of GLT-1/GAPDH, 13 xCT/GAPDH and GLAST/GAPDH; and the control group was set as 100%. The changes 14 in the GLT-1, xCT and GLAST expression were compared to the control group. 15

2.1.4. Statistical analyses 16

The effects of HYD on GLT-1, xCT and GLAST expression were analyzed using one- 17 way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons. 18

GraphPad was used to analyze the data. P values of 0.05 or lower were considered to be 19 statistically significant. 20

2.2. Results 1

2.2.1 Immunocytochemical detection of astrocytes, microglia and neurons 2

As shown in Figures 2-2A and 2-2B, the primary astrocytes were tested by using 3 immunocytochemistry for the presence of microglia; goat anti-IBa1 was used as primary 4 antibody and then FITC labeled donkey anti-goat was used as secondary antibody. The 5 presence of neurons was also tested by using mouse anti-NeuroN antibody and then labeled 6 with FITC goat anti-mouse antibody. Astrocytes were also tested by using Anti-Glial 7

Fibrillary Acidic Protein (GFAP) and then labeled with Texas Red goat anti-rabbit 8 antibody. Cultures were found to consist of about 95% astrocytes, and about 5% microglia 9 with no presence of neurons, which is an acceptable ratio in astrocyte cultures (for review 10 see Saura, 2007). 11

A B 12 13 14 15 16 17 18 19 20 21 22 Figure 2-2 Immunocytochemistry staining of primary astrocyte cell cultures. (A) Anti- GFAP 23 straining shows the presence of astrocytes in red. Anti-NeuroN shows no presence of neurons; 24

Dapi-antibody-stained nuclei are stained in blue. (B) Anti-Glial Fibrillary Acidic Protein (GFAP) 25 staining shows the presence of astrocytes in red. Anti-IBa1shows the presence of only microglia 26 in green (about 5% of total cell counts). The overly is picture show the all pictures over each other. 27

2.2.2. The effect of HYD treatment on astroglial glutamate transporter expression in 1 primary astrocyte cell cultures 2

3 We investigated the effects of treatment with three different concentrations of HYD (0.5 4

µM, 1 µM and 2 µM) on GLT-1 expression in primary astrocytes. One-way ANOVA 5 showed no effects on GLT-1 expression on Day 1 [F (3, 4) = 0.2694, p=0.8451] or Day 3 6 of treatment [F (3, 8) = 0.2026, p=0.8917] (Figure 2-3A and 2-3B). Nonetheless, a 7 significant effect of HYD was observed on Day 5 [F (3, 8) = 7.712, p = 0.0096]. Dunnett's 8 multiple comparisons test showed a significant reduction in GLT-1 expression on Day 5 (p 9

< 0.05) in astrocytes treated with 2 µM HYD for 5 consecutive days in comparison to the 10 control group (Figure 2-3C). Furthermore, we examined the effects of HYD treatment (0.5 11

µM, 1 µM and 2 µM) on xCT expression in primary astrocyte cells. One-way ANOVA 12 revealed no significant effect on xCT expression on Day 1 [F (3, 4) = 0.003466, p = 0.9997] 13 or Day 3 [F (3, 8) = 0.1739, p=0.9111] (Figures 2-4A and 2-4B). However, five days of 14 treatment with 2 µM HYD revealed a significant decrease in xCT expression [F (3, 8) = 15

7.969, p = 0.0087] (Figure 2-4C). Dunnett's multiple comparisons tests found a significant 16 decrease in xCT expression on Day 5 (p < 0.05) when astrocytes were treated with 2 µM 17

HYD in comparison to the control group. Finally, we examined the effects of HYD (0.5 18

µM, 1 µM and 2 µM) on GLAST expression in primary astrocyte cells. One-way ANOVA 19 showed no significant effects on GLAST expression on Day 1 [F (3, 4) = 0.3098, p=0.8185] 20

(Figure 2-5A), Day 3 [F (3, 4) = 0.3098, p=0.8185] (Figure 2-5B) or Day 5 [F (3, 4) = 21

0.3098, p=0.8185] (Figure 2-5C). 22

1 2 A B C 3 4

5 6 7 Figure 2-3 The effect of HYD (0.5 µM, 1 µM and 2 µM) on GLT-1 expression in primary 8 astrocyte cell cultures on Day 1, Day 3 and Day 5 (A, B, and C, respectively). Upper panel: 9

Immunoblots represent the expression of GLT-1 and GAPDH. Lower panel: Quantitative 10 analysis showed no significant difference in GLT-1 expression on Day 1 and 3 between the 11 treatment groups (0.5 µM, 1 µM and 2 µM HYD) (A and B, respectively). Quantitative 12 analysis showed significant downregulation of GLT-1 expression with exposure of HYD (2 13

µM) for five days (C; Day 5). 14

15 16

A B C 1 2

3 4 5 6 Figure 2-4 The effect of HYD (0.5 µM, 1 µM and 2 µM) on xCT expression in primary 7 astrocyte cell cultures on Day 1, Day 3, and Day 5 (A, B, and C, respectively). Upper 8 panel: Immunoblots represent the expression of xCT and GAPDH. Lower panel: 9

Quantitative analysis found no significant difference in xCT expression on Day 1 or 3 10 between the treatment groups (0.5 µM, 1 µM and 2 µM HYD) (A and B, respectively). 11

Quantitative analysis showed significant downregulation of xCT expression with exposure 12 to HYD (2 µM) for five days as compared to the control group (C; Day 5). 13

14 15 16

A B C 1

2 3 4 Figure 2-5 The Effect of HYD (0.5 µM, 1 µM and 2 µM) on GLAST expression in primary 5 astrocyte cell cultures on Day 1, Day 3, and Day 5 (A, B, and C, respectively). Upper 6 panel: Immunoblots represent the expression of GLAST and GAPDH. Lower panel: 7

Quantitative analysis showed no significant differences in GLAST expression. 8

2.3. Discussion 9

Evidence has suggested a strong connection between the glutamatergic system and opioid 10 dependence (Nakagawa et al., 2001, Zhu et al., 2001). It has been reported that acute doses 11 of opioids (e.g. morphine) can decrease glutamate release from neurons (Satoh et al., 1975, 12

Coutinho-Neito et al., 1980, Gass and Olive, 2008). However, repeated exposure to opioids 13 was shown to increase levels of glutamate and aspartate in the brain and spinal cord (Wong 14 et al., 2002, Wang et al., 2016). Moreover, high levels of glutamate, perhaps due to deficits 15 in glutamate clearance, are associated with development of tolerance, hyperalgesia and 16 allodynia in repeated administration of opioids (Salvemini and Neumann, 2009, Yan et al., 17

2009, Rawls et al., 2010, Chen et al., 2012). Deficits in glutamate clearance could lead to 1 accumulation of glutamate in synapses (Yan et al., 2009, Rawls et al., 2010, Chen et al., 2

2012). Glutamate transporters are essential for glutamate uptake and homeostasis. Less is 3 known about the effects of HYD on glutamate transporters, including GLT-1, xCT, and 4

GLAST in astrocytes. Therefore, in this study, we investigated the impact of HYD on 5 these transporters using primary astrocyte cell cultures. We found that five days of daily 6 treatment of HYD (2 µM) downregulated the expression of GLT-1 and xCT, but, the 7 expression of GLAST was not changed. 8

Several studies have linked glutamate excitotoxicity to neurodegenerative diseases such as 9 multiple sclerosis (Frigo et al., 2012), Alexander disease (Tian et al., 2010), Alzheimer’s 10 disease (Shankar et al., 2007, Zumkehr et al., 2015) and Huntington’s disease (Miller et 11 al., 2008). Reductions in GLT-1 expression occurs with repeated exposure to several drugs 12 of abuse, including methamphetamine (Alshehri et al., 2017), cocaine (Hammad et al., 13

2017), alcohol (Hakami et al., 2016) and nicotine (Alasmari et al., 2017). GLT-1 is a 14 glutamate transporter that transports glutamate into astrocytes from extracellular synaptic 15 spaces; it accounts for about 90% of extracellular glutamate clearance (Danbolt, 2001). 16

Impairment of GLT-1 function can increase glutamate levels, which may lead to excessive 17 neuronal excitability (Schousboe and Frandsen, 1995). Several reports have revealed that 18 opioids, such as morphine, are associated with an increase in extracellular glutamate levels, 19 as well as disruption of glutamate homeostasis (Farahmandfar et al., 2011). Indeed, it has 20 been reported that implanting morphine pellets in rats for five days downregulates GLT-1 21 mRNA expression (Ozawa et al., 2001). Morphine dependence develops over a similar 22 period of five days (Nakagawa et al., 2001, Ozawa et al., 2001, Wen et al., 2005, Tai et al., 23

2007). In those studies, glutamate clearance was affected due to changes in the expression 1 of GLT-1 in several brain regions after five days of treatment. Accordingly, in this study, 2 we found that primary astrocytes treated with HYD (2 µM) for five days were associated 3 with a downregulation in the expression of GLT-1. However, other doses (0.5 µM and 1 4

µM) did not show any changes in GLT-1 expression on Day 1 or Day 3 of treatment. Thus, 5 it could be possible that it takes at least five days of opioid treatment to produce changes 6 in GLT-1 expression, which could explain the effect of HYD treatment on the expression 7 of GLT-1 after five days of treatment with 2 µM HYD. 8

We also investigated the expression of xCT in primary astrocyte cell cultures. We found 9 that xCT was also downregulated with HYD (2 µM) on Day 5. Studies have shown that 10 xCT exchanges glutamate for cystine in a 1:1 ratio, which is part of the glutathione 11 biosynthesis process (Bannai et al., 1989, Bassi et al., 2001, Danbolt, 2001). This exchange 12 has also been suggested to regulate presynaptic metabotropic glutamate receptor 13

(mGluR2/3) inhibitory effects on glutamate release (Bowers et al., 2004). It is important 14 to know that the role of xCT expression in opioid effects has not been extensively 15 investigated yet. Previous studies from our laboratory have reported a reduction in xCT 16 expression with different drugs of abuse, such as alcohol (Hakami et al., 2016), cocaine 17

(Hammad et al., 2017), and nicotine (Alasmari et al., 2017). Other studies have shown that 18 restoration of xCT function with N-acetylcysteine prevents drug-seeking behavior for 19 cocaine (Madayag et al., 2007) and heroin (Zhou and Kalivas, 2008) in animals. This is 20 the first study of the effects of HYD on the expression of xCT. It has been shown that 21 chronic exposure to morphine pellets for five days does not affect the mRNA expression 22 of GLAST in the cerebral cortex of rats or other brain areas (Ozawa et al., 2001). Similarly, 23

100 in this study, GLAST expression was not affected by HYD treatment. This may be because 1

GLAST is highly expressed in the cerebellum but has lower expression in other brain 2 regions (Storck et al., 1992), and in this study the astrocyte cell cultures were obtained 3 from the cerebrum. 4

In this study, we revealed HYD effects on astroglial glutamate transporters. We found that 5

GLT-1 and xCT expression were changed only after five days of treatments of HYD, with 6 only the highest dose (2uM). Additional studies are still needed to examine a wider range 7 of doses of HYD to examine if the downregulation of GLT-1 and xCT is dose-dependent. 8

Additionally, in this study we did not investigate the effect of HYD exposure on astrocyte 9 viability. In general, astrocytes are more resistant to morphine-induced apoptosis than 10 neurons or microglia (Hu et al., 2002). Treating astrocytes with morphine (1 uM) for 7 11 days did not induce apoptosis (Hu et al., 2002), and treating astrocytes for five days with 12 concentrations up to 100 uM did not induce apoptosis (Hu et al., 2002). In this study, there 13 was no effect on the expression of GLAST after treating astrocytes with HYD. Thus, we 14 suggest here that HYD did not induce astrocyte death. However, future studies are still 15 needed to examine the toxic effect of opioids on astrocytes. 16

In conclusion, this study examined for the first time the effect of HYD on glutamate 17 transporters (GLT-1, xCT, and GLAST) in primary astrocyte cell cultures. HYD treatment 18 for five days was associated with a downregulation in the expression of GLT-1 and xCT, 19 but no changes occurred in GLAST expression. Together, these findings demonstrate a 20 strong relationship between HYD and the glutamatergic system, which could be a potential 21 target to modulate the effects of repeated opioid exposure. 22

101

Disclosure Statements 1

The authors declare no conflicts of interest. 2

Acknowledgments 3

This work was supported in part by Award Number R01AA019458 (Y.S.) from the 4

National Institutes on Alcohol Abuse and Alcoholism and also by start-up funds from the 5

University of Toledo. Fahad S. Alshehri was supported by a scholarship from Umm Al- 6

Qura University, College of Pharmacy & Pharmaceutical Sciences, Makkah, Saudi Arabia 7

12 13 14 15 16 17 18 19 20 21 22 23 24

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14 15

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Chapter 3 4

Effects of Ceftriaxone on Hydrocodone Seeking Behavior and Glial Glutamate 6 Transporters in P Rats 7

Fahad S. Alshehri, Alqassem Y. Hakami, Yusuf S. Althobaiti, Youssef Sari* 9

Department of Pharmacology & Experimental Therapeutics, College of Pharmacy and 11 Pharmaceutical Sciences, University of Toledo, Health Science Campus, 3000 Arlington 12 Avenue, Toledo, OH 43614, USA 13

14 15 * Corresponding author: 16 Dr. Youssef Sari 17 University of Toledo, College of Pharmacy & Pharmaceutical Sciences 18 Department of Pharmacology & Experimental Therapeutics 19 Health Science Campus, 3000 Arlington Avenue 20 Toledo, OH 43614, USA 21 E-mail: [email protected] 22 Tel: 419-383-1507 23 Note: This chapter was published in Behavioural Brain Research: Volume 347, 16 July 24 2018, Pages 368-376 25

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Abstract 1

Hydrocodone (HYD) is one of the most widely prescribed opioid analgesic drugs. Several 2 neurotransmitters are involved in opioids relapse. Among these neurotransmitters, 3 glutamate is suggested to be involved in opioid dependence and relapse. Glutamate is 4 regulated by several glutamate transporters, including glutamate transporter 1 (GLT-1) and 5 cystine/glutamate transporter (xCT). In this study, we investigated the effects of 6 ceftriaxone (CEF) (200 mg/kg, i.p.), known to upregulate GLT-1 and xCT, on 7 reinstatement to HYD (5 mg/kg, i.p.) using the conditioned place preference (CPP) 8 paradigm in alcohol-preferring (P) rats. Animals were divided into three groups: 1) saline- 9 saline group (SAL-SAL); 2) HYD-SAL group; and 3) HYD-CEF group. The CPP was 10 conducted as follow: habituation phase, conditioning phase with HYD (i.p.) injections 11 every other day for four sessions, extinction phase with CEF (i.p.) injections every other 12 day for four sessions, and reinstatement phase with one priming dose of HYD. Time spent 13 in the HYD-paired chamber after conditioning training was increased as compared to pre- 14 conditioning. There was an increase in time spent in the HYD-paired chamber with one 15 priming dose of HYD in the reinstatement test. HYD exposure downregulated xCT 16 expression in the nucleus accumbens and hippocampus, but no effects were observed in 17 the dorsomedial prefrontal cortex and amygdala. Importantly, CEF treatment attenuated 18 the reinstatement effect of HYD and normalized xCT expression in the affected brain 19 regions. These findings demonstrate that the attenuating effect of HYD reinstatement with 20

CEF might be mediated through xCT. 21

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Introduction 1

Opioids have long been used in pain management (Ballantyne and LaForge, 2007). 2

However, their non-medical use has grown rapidly in the last few years. Hydrocodone 3

(HYD) is one of the most widely used short-acting opioids; with over 136.7 million 4 prescriptions in 2011 (Laxmaiah Manchikanti et al., 2012). HYD is a semi-synthetic opioid 5 used for analgesic and antitussive purposes. However, data shows that the nonmedical use 6 of HYD was one of the most common causes of emergency medical visits between 2004 7 and 2008 (Control and Prevention, 2010). HYD abuse liability and relative potency have 8 been shown to be similar to those of oxycodone and hydromorphone (Walsh et al., 2008). 9

In addition, a study suggested that HYD has similar effects to those of oxycodone and 10 morphine when administered intravenously (i.v) (Stoops et al., 2010). Relapse after a long 11 period of abstinence is a major problem in the treatment of drug dependence (O’Brien, 12

1996). The high rate of relapse associated with opioids remains as one of the most 13 challenging clinical problems in opioid dependence. It has been suggested that the 14 glutamatergic system has regulatory effects on opioid dependence (Ozawa et al., 2001), 15 withdrawal (Tokuyama et al., 1996) and reinstatement in animals (Tahsili-Fahadan et al., 16

2010). Indeed, memantine (N-methyl-D-aspartate receptor blocker) attenuated 17 reinstatement to morphine, while dopaminergic blockers failed (Do Couto et al., 2005). 18

Studies have found an increase in extracellular glutamate concentration in the nucleus 19 accumbens (NAc) with exposure to heroin (LaLumiere and Kalivas, 2008), nicotine (Reid 20 et al., 2000) and cocaine (Cornish and Kalivas, 2000). 21

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The extracellular glutamate is maintained via several glutamate transporters (also called 1 the excitatory amino acid transporters, EAATs), including glutamate transporter 1 (GLT- 2

1, EAAT2), glutamate/aspartate transporter (GLAST, EAAT1) and glutamate transporter 3

(EAAT3). GLT-1 is a major glutamate transporter that regulates the uptake of the majority 4 of glutamate (Tanaka et al., 1997, Danbolt, 2001). It has been demonstrated that chronic 5 exposure to morphine can lead to reduction of GLT-1 mRNA expression in the NAc, 6 striatum, thalamus, and hippocampus (HIP) (Ozawa et al., 2001). Relapse to heroin was 7 shown to be associated with increase in the extracellular glutamate concentration in the 8

NAc (Shen et al., 2014). Thus, restoring the glutamate uptake may have beneficial 9 therapeutic effect in attenuating opioid relapse. In regard to GLAST, this protein transports 10 both glutamate and aspartate, and expressed mostly in the cerebellum and spinal cord 11

(Storck et al., 1992). Several studies have suggested that the loss of morphine analgesic 12 effect after repeated exposure to morphine might be due to reduction in GLAST expression 13 as well as glutamate uptake in the spinal cord (Mao et al., 2002, Tai et al., 2006). The 14

EAAT3 is a neuronal glutamate transporter, expressed mainly in the HIP, basal ganglia and 15 cerebellum (Maragakis and Rothstein, 2001). It has been shown that chronic exposure to 16 morphine could downregulate the expression of EAAT3 in the HIP neuronal culture (Guo 17 et al., 2015). In addition, recent report suggested that EAAT3 is important in morphine- 18 induced conditioned place preference (CPP), but not in reinstatement (Wan et al., 2017). 19

Furthermore, cystine/glutamate transporter (xCT) is another transporter that regulates 20 extracellular glutamate through the exchange of cystine with glutamate (Bannai et al., 21

1989, Bassi et al., 2001, Danbolt, 2001). Although there is less known about the role of 22 xCT in opioid relapse, one study has demonstrated that restoring xCT function with N- 23

113 acetylcysteine can attenuate heroin reinstatement in animals (Zhou and Kalivas, 2008). N- 1 acetylcysteine is known to improve the function of xCT, which might attenuate heroin 2 relapse by increasing the glutamatergic tone on the pre-synaptic metabotropic glutamate 3 receptor (mGluR2/3) (Moran et al., 2005). Thus, xCT might be a target candidate for the 4 treatment of opioids dependence. Therefore, in this study, we investigated the effect of 5

HYD reinstatement on the glial glutamate transporters such as GLT-1, xCT and GLAST. 6

In this study, we investigated the effects of HYD in alcohol-preferring (P) rats using the 7

CPP paradigm. We used P rats due to the fact that they have higher density of mu opioid 8 receptors than non-preferring (NP) rats (McBride et al., 1998). Similarly, others have 9 found that alcohol-preferring Alko Alcohol (AA) rats express higher amount of opioid 10 peptides and receptors as compared to alcohol-avoiding Alko Non-Alcohol (ANA) rats in 11 several brain regions, including the NAc and ventral tegmental area (VTA) (de Waele et 12 al., 1995, Marinelli et al., 2000). Also, AA rats have shown to be more susceptible to drug- 13 induced behavioral sensitization in response to morphine than ANA rats (Honkanen et al., 14

1999). Therefore, high ethanol drinking rats may have a potential benefit over low ethanol 15 drinking rats in testing opioids dependence and relapse. In this study, we tested a lower 16 dose of HYD (5 mg/kg, i.p.) in P rats as sensitive bred to induce the conditioning and the 17 reinstatement effects of HYD. 18

The β-lactam compounds, including ceftriaxone (CEF), have shown to attenuate drug- 19 seeking in several drugs of abuse including methamphetamine (Abulseoud et al., 2012), 20 cocaine (Knackstedt et al., 2010, Hammad et al., 2017), nicotine (Alajaji et al., 2013) and 21 morphine (Fan et al., 2012). Moreover, in our laboratory, we have shown that CEF can 22

114 reduce chronic alcohol drinking via upregulating the expression of GLT-1 and xCT in P 1 rats (Alhaddad et al., 2014, Rao and Sari, 2014). Therefore, we hypothesized here that the 2 administration of CEF during the extinction phase would attenuate HYD reinstatement by 3 modulating these transporters. Thus, several important brain rewards regions involved in 4 the glutamatergic transmission were investigated such as the dorsomedial prefrontal cortex 5

(dmPFC), NAc, HIP and amygdala (AMY). 6

3.1. Materials and methods 7

3.1.1. Drugs 8

HYD (+)- bitartrate salt was purchased from Sigma-Aldrich (St. Louis, MO). CEF (Sandoz 9

Inc., Princeton, NJ) was purchased from the pharmacy at the University of Toledo Medical 10

Center. Saline (SAL) solution (0.9% NaCl) was used as vehicle to dissolve both drugs. 11

3.1.2. Animals and drug dosing 12

Male P rats were used to investigate the effects of HYD reinstatement on glutamate 13 transporters: GLT-1, xCT and GLAST using the CPP paradigm. P rats were obtained from 14

Indiana University, School of Medicine, Indianapolis, IN. Rats were housed in single 15 plastic cages with free access to food and water. Housing room temperature was 16 maintained at 21°C and humidity at 50%. Rats were on a 12:12-hour light-dark cycle 17 throughout the whole study. All the proposed experiments were approved by the 18

Institutional Animal Care and Use Committee (IACUC) at The University of Toledo and 19 adhered to the guidelines of the Institutional Animal Care and Use Committee of the 20

National Institutes of Health and the Guide for the Care and Use of Laboratory Animals. 21

115

The HYD dose was selected based on previous study investigated HYD’s reward using 1

CPP in rats (Nazarian et al., 2011) and the similarity between HYD and morphine 2 rewarding effects (Tenayuca and Nazarian, 2012). The use of CEF was based on previous 3 studies demonstrated that this drug attenuated morphine tolerance after its prolonged use 4

(Rawls et al., 2010). The dosing schedule of CEF was chosen based on recent study from 5 our laboratory, which found that β-lactam compounds were able to attenuate cocaine 6 reinstatement when given every other day during the extinction phase in P rats (Hammad 7 et al., 2017). 8

3.1.3. Apparatus 9

The apparatus consists of two chambers (40 cm x 40 cm x 40 cm) separated by smaller 10 middle chamber (30 cm x 40 cm x 40 cm). The first chamber was distinguished with black 11 and white horizontal stripes and textured floor. The second chamber was distinguished 12 with black and white vertical stripes and smooth floor. The middle chamber was neutral. 13

Time spent in either of the chambers was calculated by an observer who was blind to the 14 experimental designs. 15

3.1.4. Experimental procedure 16

The CPP paradigm in this study was performed as described in a recent study from our 17 laboratory (Hammad et al., 2017). For acclimating purposes, animals were handled three 18 days prior to starting the experiments. Rats were divided into three groups: 1) SAL-SAL 19 group; 2) HYD-SAL group; and 3) HYD-CEF group in unbiased manner as shown in 20

(Table 3-1). The CPP was conducted in four phases: habituation, conditioning, extinction 21

116 and reinstatement, as shown in (Figure 3-1). In the habituation phase, animals were 1 allowed to explore the apparatus freely for 20 minutes a day, for three days to minimize 2 stress and initial bias. On Day 4, animals were tested for initial preference (pre- 3 conditioning test); animals were placed in the middle chamber with doors locked for three 4 minutes. Then, both doors were opened for the animals to explore the apparatus for 20 5 minutes. The initial preference was calculated based on the time spent in the first and 6 second chamber. Animals that showed more than 67% preference to one of the chambers 7 were excluded from the study (Fujio et al., 2005). In the conditioning phase, SAL (i.p) 8 was administered in the SAL-SAL group and animals were placed in the assigned chamber 9 alternatively for 30 minutes for a total of four sessions. Animals in the HYD-SAL group 10 were given either HYD (5 mg/kg) or SAL (i.p.), alternatively, in the assigned chamber for 11

30 minutes for a total of four sessions. On Day 13, animals were tested for preference 12

(post-conditioning test). In the extinction phase, SAL (i.p) was administered to the SAL- 13

SAL group in the assigned chamber for 30 minutes for a total of four sessions. In the HYD- 14

SAL group, animals were given SAL alternatively in the assigned chamber for 30 minutes 15 for a total of four sessions. However, in the HYD-CEF group, animals were given CEF 16

(200 mg/kg) or SAL (i.p.), alternatively, in the assigned chamber for 30 minutes for a total 17 of four sessions. On Day 22, animals were tested for preference (extinction test). A 25% 18 reduction in time spent in the HYD-paired chamber was set as a criteria for extinction, and 19 any animal that did not meet that criteria was excluded, as was performed in previous 20 studies from ours and others (Abulseoud et al., 2012, Hammad et al., 2017). In the 21 reinstatement phase, on Day 23, animals were challenged with one single dose of SAL 22

(i.p.) in the SAL-SAL group and HYD (5 mg/kg, i.p.) in both groups (HYD-SAL and 23

117

HYD-CEF) and placed in the assigned chamber for 30 minutes. Then, on Day 24, animals 1 from all groups were given one dose of SAL (i.p.) and placed in the assigned chamber for 2

30 min. On Day 25, animals were tested for preference (reinstatement test) and euthanized 3 on the same day. 4

Table 3-1 Animal groups and treatment during the conditioning, extinction and 5 reinstatement phases. 6

CPP phase SAL-SAL group HYD-SAL group HYD-CEF group

Conditioning SAL HYD/SAL HYD/SAL

Extinction SAL SAL CEF/SAL

Reinstatement SAL HYD/SAL HYD/SAL

Figure 3-1 Timeline of the experimental procedure during the conditioning, extinction and 12 reinstatement phases. 13

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3.1.5. Brain tissue extraction 1

Animals were euthanized, and the brain samples were dissected after the reinstatement test 2 on Day 25. The NAc (core and shell), dmPFC (cingulate cortex and prelimbic cortex), HIP 3

(cornu ammonis, CA, subfield: CA1, CA2 and CA3) and AMY (central amygdala, 4 basomedial amygdala and basolateral amygdala) were extracted using a cryostat machine 5

(Leica CM1950). All brain regions were selected using the Brain Rat Atlas (Paxinos and 6

Watson, 2007). All the samples were stored at -80°C for subsequent immunoblotting. 7

3.1.6. Immunoblots procedure 8

Samples were lysed using lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 9

0.5% NP-40, 1% Triton, 0.1% SDS) with phosphatase and protease inhibitors. Glutamate 10 transporters expression in this study represents the whole cell lysate of both membrane- 11 bound and cytoplasmic protein fractions. Several studies have tested the effect of β-lactam 12 compounds, using the whole cell lysate, on glutamate transporters in several drugs of abuse 13 from ours and others (Abulseoud et al., 2014, Alasmari et al., 2016, Althobaiti et al., 2016, 14

Kim et al., 2016, Alshehri et al., 2017, Hakami and Sari, 2017, Hammad et al., 2017). 15

Protein quantification was performed to measure the amount of protein in the lysed 16 samples. Samples were loaded onto 10-15% Tris-glycine gel to separate the proteins via 17 electrophoresis. Then, proteins were transferred to PVDF membranes. The membranes 18 were then blocked with 3% fat-free milk in 10% Tris-buffered with Tween 20 (TBST) for 19

30-60 minutes. Membranes were incubated overnight at 4°C with the primary antibodies: 20 anti Guinea pig GLT-1 (Millipore Sigma; 1:5000 dilution), anti-Rabbit xCT (Abcam; 21

1:1000 dilution), anti-Rabbit GLAST (Abcam; 1:5000 dilution) and anti-mouse β-tubulin 22

119

(Covance;1:5000 dilution) as a loading control. Membranes were blocked with 3% fat-free 1 milk in TBST for 30 minutes and incubated with the secondary antibodies for 90 minutes. 2

Then, membranes were incubated with a Chemiluminescent kit (SuperSignal West Pico) 3 and developed using X-Ray film processor (Konica SRX101A – Tabletop). Immunoblots 4 were quantified using MCID Digital Imaging Software. The data were presented as a 5 percentage of the ratio of the targeted proteins (GLT-1, xCT or GLAST)/ β-tubulin. The 6 control group was reported as 100% to measure the changes in the expression of GLT-1, 7 xCT and GLAST after HYD and CEF treatment as performed in previous studies from ours 8 and others (Li et al., 2003, Wen et al., 2005, Zhang and Tan, 2011, Simões et al., 2012, 9

Alshehri et al., 2017). 10

3.1.7. Statistical analyses 11

Time spent in the conditioning chambers after each phase in CPP were analyzed using two- 12 way repeated measures ANOVA (Time x Chamber). Tukey’s post hoc test was used 13 whenever a significant effect was found. The immunoblot data of GLT-1, xCT and 14

GLAST were analyzed using one-way ANOVA followed by Newman-Keuls multiple 15 comparison tests. All statistical analyses in this study were performed using GraphPad 16

Prism with p < 0.05 as a level of significance. 17

3.2. Results 18

3.2.1. Effect of SAL administration on animal preference using CPP 19

The effect of SAL administered alone was tested in CPP. Animals were habituated for 20 three days to explore the CPP apparatus. Then, the initial preference was measured and 21

120 considered as baseline. Animals were given SAL (i.p) during the conditioning, extinction 1 and reinstatement phases every other day in chamber one and chamber two, alternately. 2

Two-way repeated measures ANOVA showed no significant difference in time spent when 3

SAL (i.p) was administered during conditioning, extinction and reinstatement (Figure 3- 4

2). There were no significant effects of time [F (3, 18) = 1.289, p = 0.3085], chamber effect 5

[F (1, 6) = 1.194, p = 0.3165], nor time x chamber effect [F (3, 18) = 0.01702, p = 0.9969]. 6

Figure 3-2 Effect of SAL (i.p.) administration alone on CPP. No significant difference 13 was found in time spent in preference test after the conditioning, extinction and 14 reinstatement phases. Values are shown as means ± S.E.M (∗p < 0.05) (n = 7). PRE = pre- 15 conditioning, POST = post-conditioning, EXT = extinction and RE = reinstatement. 16

3.2.2. Effect of CEF on HYD-induced reinstatement using CPP 17

Time spent in the HYD-SAL group was analyzed using two-way repeated measures 18

ANOVA (Figure 3A) with main significant effect of time [F (3, 18) = 18.49, p < 0.0001], 19 significant effect of chamber [F (1, 6) = 399.7, p < 0.0001] and significant interaction 20

121 between time x chamber [F (3, 18) = 15.87, p < 0.0001]. Tukey’s post hoc test showed 1 significant increase in time spent in the HYD-paired chamber following conditioning 2 training with HYD (5 mg/kg, i.p.) (p < 0.05, Figure 3-3A). This difference was eliminated 3 in extinction. Time spent in the HYD-paired chamber was increased significantly after 4 reinstating the animals with one priming dose of HYD (5 mg/kg, i.p.) in the reinstatement 5 phase (p < 0.05, Figure 3-3A). Two-way repeated measures ANOVA analysis in the HYD- 6

CEF group (Figure 3-3B) showed a significant main effect of time [F (3, 24) = 13.73, p < 7

0.0001], a significant effect of chamber [F (1, 8) = 20.5, p = 0.0019] and a significant 8 interaction between time x chamber [F (3, 24) = 30.73, p < 0.0001]. Tukey’s post hoc test 9 showed significant increase in time spent in the HYD-paired chamber after conditioning 10 training with HYD (5 mg/kg, i.p.) (p < 0.05, Figure 3-3B). This effect was again eliminated 11 after extinction. However, no significant effect on time spent in the HYD-paired chamber 12 was observed after reinstating the animals with one priming dose of HYD (5 mg/kg, i.p.) 13 in the reinstatement phase (p > 0.05, Figure 3-3B). 14

122

Figure 3-3 Time spent in the conditioning chamber during pre-conditioning, post- 3 conditioning, extinction, and reinstatement tests. Statistical analysis showed an increase 4 in time spent in the HYD-paired chamber following conditioning compared to pre- 5 conditioning in the HYD-SAL (A) and HYD-CEF (B) groups. Time spent in the HYD- 6 paired chamber decreased in extinction in comparison with post-conditioning in the HYD- 7

SAL and HYD-CEF groups. Time spent in the HYD-paired chamber increased in the 8 reinstatement test in the HYD-SAL group, but not in the HYD-CEF group, in comparison 9 to the extinction test. Values are shown as means ± S.E.M. *p < 0.05, **p < 0.01, and 10

****p < 0.0001. (n = 7-9 for each group). PRE = pre-conditioning, POST = post- 11 conditioning, EXT = extinction and RE = reinstatement. 12

3.2.3. Effect of CEF on the expression of GLT-1, xCT and GLAST in the NAc and 13 dmPFC in HYD-induced reinstatement 14

We investigated the effects of CEF on the expression of GLT-1, xCT and GLAST in the 15

NAc and dmPFC in HYD reinstatement in P rats. One-way ANOVA showed no significant 16 main effect on GLT-1 expression among the SAL-SAL, HYD-SAL and HYD-CEF groups 17 in the NAc [F (2, 18) = 1.342, p = 0.2863, Figure 3-4A] or in the dmPFC [F (2, 18) = 18

123

0.7658, p = 0.4795, Figure 3-5A]. However, one-way ANOVA showed a significant main 1 effect on xCT expression in the SAL-SAL, HYD-SAL and HYD-CEF groups in the NAc 2

[F (2, 18) = 5.007, p = 0.0187, Figure 3-4B], but no effect in the dmPFC [F (2, 18) = 1.18, 3 p = 0.3299, Figure 3-5B]. Further analysis with Newman-Keuls multiple comparison tests 4 showed a significant downregulation in xCT expression in the HYD-SAL group compared 5 to the SAL-SAL group in the NAc (p < 0.05, Figure 3-4B). However, statistical analysis 6 showed a significant upregulation in xCT expression in the HYD-CEF group compared to 7 the HYD-SAL group in the NAc (p < 0.05, Figure 3-4B). One way ANOVA showed no 8 significant main effect on GLAST expression among the SAL-SAL, HYD-SAL and HYD- 9

CEF groups in the NAc [F (2, 15) = 0.713, p = 0.5061, Figure 3-4C] and in the dmPFC [F 10

(2, 15) = 0.8653, p = 0.4409, Figure 3-5C]. 11

124

Figure 3-4 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the 3 expression of GLT-1, xCT and GLAST in the NAc. (A) Upper panel: immunoblots 4 representing GLT-1 expression and β-tubulin. Lower panel: statistical analysis showed no 5 significant difference among all groups. (B) Upper panel: immunoblots representing xCT 6 expression and β-tubulin. Lower panel: statistical analysis showed significant 7 downregulation in xCT expression in the HYD-SAL compared to the SAL-SAL group. 8

However, statistical analysis showed upregulation of the HYD-CEF compared to the HYD- 9

SAL. No statistical difference was found between the SAL-SAL and HYD-CEF groups. 10

(C) Upper panel: immunoblots representing GLAST expression and β-tubulin. Lower 11 panel: statistical analysis showed no significant difference among all groups. Values are 12 shown as means ± SEM (∗p < 0.05) (n = 6-7 for each group). 13

125

Figure 3-5 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the 3 expression of GLT-1, xCT and GLAST in the dmPFC. (A) Upper panel: immunoblots 4 representing GLT-1 expression and β-tubulin. Lower panel: statistical analysis showed 5 no significant difference among all groups. (B) Upper panel: immunoblots representing 6 xCT expression and β-tubulin. Lower panel: statistical analysis showed no significant 7 difference between all groups. (C) Upper panel: immunoblots representing GLAST 8 expression and β-tubulin. Lower panel: statistical analysis showed no significant 9 difference among all groups. Values are shown as means ± SEM (∗p < 0.05) (n = 6-7 for 10 each group). 11

3.2.4. Effect of CEF on the expression of GLT-1, xCT and GLAST in the HIP and 12

AMY in HYD-induced reinstatement 13

We investigated the effects of CEF on the expression of GLT-1, xCT and GLAST in the 14

HIP and AMY in HYD reinstatement in P rats. One way ANOVA revealed no significant 15 main effect on GLT-1 expression among the SAL-SAL, HYD-SAL and HYD-CEF groups 16

126 in the HIP [F (2, 18) = 0.6305, p = 0.5437, Figure 3-6A] or the AMY [F (2, 18) = 0.6376, 1 p = 0.5401, Figure 3-7A]. However, one-way ANOVA showed a significant main effect 2 on xCT expression among the SAL-SAL, HYD-SAL and HYD-CEF groups in the HIP [F 3

(2, 18) = 5.837, p = 0.0111, Figure 3-6B], but no effect in the AMY [F (2, 18) = 0.03411, 4 p = 0.9665, Figure 3-7B]. In addition, Newman-Keuls multiple comparison tests revealed 5 a significant downregulation in xCT expression in the HYD-SAL group compared to the 6

SAL-SAL group in the HIP (p < 0.05, Figure 3-6B). However, statistical analysis showed 7 a significant upregulation in xCT expression in the HYD-CEF group compared to the 8

HYD-SAL group in the HIP (p < 0.05, Figure 3-6B). One way ANOVA showed no 9 significant main effect on GLAST expression among the SAL-SAL, HYD-SAL and HYD- 10

CEF groups in the HIP [F (2, 15) = 0.171, p = 0.8445, Figure 3-6C] and the AMY [F (2, 11

18) = 1.261, p = 0.3072, Figure 3-7C]. 12

127

Figure 3-6 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the 4 expression of GLT-1, xCT and GLAST in the HIP. (A) Upper panel: immunoblots 5 representing GLT-1 expression and β-tubulin. Lower panel: statistical analysis showed 6 no significant difference among all groups. (B) Upper panel: immunoblots representing 7 xCT expression and β-tubulin. Lower panel: statistical analysis showed significant 8 downregulation in xCT expression in the HYD-SAL compared to the SAL-SAL group. 9

However, statistical analysis showed upregulation of the HYD-CEF group compared to the 10

HYD-SAL group. No statistical difference was found between the SAL-SAL and HYD- 11

CEF groups. (C) Upper panel: immunoblots representing GLAST expression and β- 12 tubulin. Lower panel: statistical analysis showed no significant difference among all 13 groups. Values are shown as means ± SEM (∗p < 0.05) (n = 6-7 for each group). 14

128

Figure 3-7 Effects of HYD (5 mg/kg, i.p.) reinstatement and CEF (200 mg/kg, i.p.) on the 3 expression of GLT-1, xCT and GLAST in the AMY. (A) Upper panel: immunoblots 4 representing GLT-1 expression and β-tubulin. Lower panel: statistical analysis showed 5 no significant difference among all groups. (B) Upper panel: immunoblots representing 6 xCT expression and β-tubulin. Lower panel: statistical analysis showed no significant 7 difference among all groups. (C) Upper panel: immunoblots representing GLAST 8 expression and β-tubulin. Lower panel: statistical analysis showed no significant 9 difference among all groups. Values are shown as means ± SEM (∗p < 0.05) (n = 6-7 for 10 each group). 11

3.3. Discussion 12

The CPP paradigm has been used to measure opioid rewards, including heroin (Paul et al., 13

2001, Ashby et al., 2003), morphine (Van Der Kooy et al., 1982, Cavun et al., 2005), HYD 14

(Nazarian et al., 2011) and other drugs of abuse. In this study, the CPP paradigm was 15 adopted from previous work in our laboratory on cocaine reinstatement (Hammad et al., 16

129

2017). Several studies have focused on the association between the glutamatergic system 1 and the reinstatement of morphine and heroin (Do Couto et al., 2005, LaLumiere and 2

Kalivas, 2008). However, to the best of our knowledge, the association between HYD 3 reinstatement and the glutamatergic system has not been thoroughly investigated, 4 especially in P rats. In this study, we used HYD (5 mg/kg, i.p.) to produce the preference 5 and reinstatement in P rats. Importantly, we found that HYD reinstatement was associated 6 with downregulation of xCT in the NAc and HIP and these effects were attenuated with 7

CEF treatment. 8

This study investigated the NAc (both core and shell), since this brain region is involved 9 in opioid rewards (Vaccarino et al., 1985), withdrawal (Stinus et al., 1990) and tolerance 10

(Schmidt et al., 2002). It has been found that blocking the mu-opioid receptor in the NAc 11 diminished heroin rewards in rats (Vaccarino et al., 1985). Moreover, high extracellular 12 concentration of glutamate in the NAc core has been linked to heroin-seeking behavior and 13 relapse (LaLumiere and Kalivas, 2008). Also, it has been shown that injecting mGluR2/3 14 agonist (LY379268) into the NAc shell, but not NAc core, attenuated heroin-seeking 15 behavior (Bossert et al., 2006). Both NAc core and shell receive projections from the 16 dmPFC [for review, see (Moorman et al., 2015)]. It is important to note that glutamatergic 17 projections from the PFC to NAc are suggested to be involved in cocaine- and heroin- 18 seeking behavior (McFarland and Kalivas, 2001, LaLumiere and Kalivas, 2008). 19

In this study, we also focused on the dmPFC, which included the cingulate cortex and 20 prelimbic cortex. The activation of the cortex area in drug addiction was shown in 21 neuroimaging studies during intoxication and craving, but not in withdrawal [for review 22

130 see ref. (Goldstein and Volkow, 2002)]. The mPFC appears to be involved in the 1 acquisition of morphine, but not the reinstatement (Hao et al., 2008). It has been suggested 2 that there is a link between the PFC and cue-induced heroin-seeking (Li et al., 2012). The 3 cingulate cortex was reported to be activated following exposure to psychostimulant drugs 4 such as cocaine (Kilts et al., 2004, Marhe et al., 2013). In addition, it was reported that the 5 cingulate cortex neural activity was altered in heroin users using the functional magnetic 6 resonance imaging (Zhang et al., 2015). The inactivation of the prelimbic cortex was 7 shown to block the reinstatement of heroin (LaLumiere and Kalivas, 2008, Rogers et al., 8

2008) and methamphetamine (Hiranita et al., 2006). Moreover, others have shown that 9 activating the prelimbic cortex could facilitate heroin reinstatement (Schmidt et al., 2005). 10

Moreover, the projections from the VTA to the NAc were suggested to be more important 11 in opioid reinstatement as compared to the PFC (Self and Nestler, 1998), which might 12 explain why this latter brain region was not affected in HYD reinstatement. 13

The HIP was investigated which included (CA1, CA2 and CA3). This brain region is 14 involved in learning and memory function (Postle, 2009). Studies have reported that HIP 15 is implicated in the association between the environmental context and unconditioned 16 stimuli (foot-shock) (Kim and Fanselow, 1992, Phillips and LeDoux, 1992). A number of 17 studies have demonstrated that the HIP is crucial for drug-seeking behavior (Vorel et al., 18

2001, Black et al., 2004, Yang et al., 2004). This brain region is believed to be associated 19 with negative contextual experience associated with withdrawal from several drugs of 20 abuse [for review, see ref (Koob and Volkow, 2010). It has also been found that heroin can 21 increase the amount of polysialic acid-neural cell adhesion molecule expression in glial 22 cells, which could explain the damage found in the HIP area in postmortem heroin addicts 23

131

(Weber et al., 2006). On the other hand, we investigated the AMY which included (central 1 amygdala, basomedial amygdala and basolateral amygdala). It has been suggested that the 2

AMY facilitates the drug reward, learning and seeking behaviors in rats (Everitt et al., 3

1999). In addition, lesions in the AMY were shown to prevent cocaine reinstatement 4

(Whitelaw et al., 1996). Also, It has been found that inactivation of AMY using 5 tetrodotoxin, potent neurotoxin, blocked the heroin-seeking behavior in rats (Fuchs and 6

See, 2002). 7

Several studies have shown that regulation of glutamate homeostasis is critical in relapse 8 to many drugs of abuse (Kalivas, 2009, Knackstedt and Kalivas, 2009). Glutamate is 9 transported by GLT-1, which accounted for about 90% of glutamate clearance from the 10 synaptic cleft (Tanaka et al., 1997, Danbolt, 2001). CEF is known to produce its effects 11 through GLT-1 upregulation. However, in this study, we found that CEF treatment was 12 not associated with changes in GLT-1 expression in all tested brain areas. While no change 13 in GLT-1 expression has been shown, there is a great possibility that CEF could improve 14 the function of GLT-1 without changing its expression. Further studies are warranted to 15 determine the activity of GLT-1 after CEF treatment in HYD-seeking behavior, for 16 example, measuring extracellular glutamate concentrations using microdialysis technique. 17

Another possibility is that the expression of GLT-1 might be different between sub-regions, 18 where the effect in one area could be masked by the other. Future studies are still needed 19 to determine sub-region differential effects in GLT-1 expression. Studies have shown that 20 chronic exposure to morphine reduced the expression of GLT-1 mRNA in the NAc, 21 striatum, and thalamus (Ozawa et al., 2001). In addition, most of morphine studies, with 22 regard to the glutamatergic system, were investigated in the cerebellum and spinal cord, 23

132 which are more related to pain management, but not to relapse (Niederberger et al., 2003, 1

Tai et al., 2007, Gunduz et al., 2011). Thus, studies are required to investigate the effect of 2

HYD with different doses and strains as well to examine the relationship between GLT-1 3 expression and other glutamate transporters in HYD exposure and reinstatement. 4

Although no changes were observed in GLT-1 expression with HYD reinstatement, we 5 found that the xCT expression was downregulated in the NAc and HIP. Cellular 6 mechanistic events involve xCT, GLT-1 and other glutamatergic receptors in several brain 7 regions (e.g. NAc and HIP) to modulate HYD-seeking behavior are demonstrated in 8

(Figure 3-8). To the best of our knowledge, little is known about the function of xCT in 9 opioid dependence and relapse. However, it was reported that N-acetylcysteine can restore 10 the function of xCT system and attenuate drug-seeking behavior in animals (Baker et al., 11

2003a). Dysfunction of xCT can occur after cocaine self-administration, which was 12 associated with a reduction in basal glutamate concentration in the NAc (Baker et al., 13

2003b, Madayag et al., 2007). Similarly, repeated morphine exposure was associated with 14 low basal glutamate concentration in the HIP in mice (Guo et al., 2005). It is important to 15 note that restoring the xCT function can attenuate cocaine (Baker et al., 2003b, Madayag 16 et al., 2007) and heroin-seeking behavior (Zhou and Kalivas, 2008). Moreover, it has been 17 shown that repeated administration of morphine can lead to behavioral sensitization in 18 animals. Thus, morphine-sensitized rats have been shown to have high level of 19 extracellular glutamate in the HIP, when they were challenged with morphine after 20 prolonged abstinence (Farahmandfar et al., 2011). Therefore, restoring xCT function was 21 assumed to improve the glutamatergic tone on mGluR2/3 and attenuate the seeking 22 behavior as previously reported in cocaine (Moran et al., 2005). Here, we assumed that 23

133 the downregulation of xCT expression in the NAc and HIP might be associated with 1 reinstatement to HYD in P rats. Therefore, restoring xCT expression in these brain regions 2 with CEF could, in part, attenuate HYD reinstatement (Figure 3-8). 3

Furthermore, our study investigated another glial glutamate transporter called GLAST. 4

GLAST expression was not changed in HYD reinstatement in all tested groups. This is in 5 accordance with a previous study, which demonstrated that GLAST mRNA expression was 6 not altered after morphine administration in the thalamus, hypothalamus, cerebral cortex, 7

HIP, striatum, midbrain, cerebellum, and pons-medulla (Ozawa et al., 2001). Although 8 other studies have shown that morphine administration is associated with reductions in 9

GLAST and EAAT3 expression in the spinal cord (Mao et al., 2002, Tai et al., 2006). In 10 fact, GLAST is highly expressed in the cerebellum and spinal cord and less expressed in 11 other brain regions (Storck et al., 1992). Together, these data suggest that GLAST is less 12 involved in HYD reinstatement. 13

The third-generation cephalosporin antibiotics can cross the blood brain barrier (BBB) 14

(Fekety, 1990). In addition, several studies have shown that CEF can also cross the BBB 15

(Spector, 1986, 1987) through a facilitated transport process (Spector, 1987). However, 16 due to the fact that CEF has poor bioavailability when taken orally, CEF must be given 17 through the parenteral route for maximum effects. CEF is highly bound to plasma proteins 18

(Steele et al., 1983, Spector, 1987), and most of CFE is eliminated in urine and biliary 19 excretion [for review see Ref. (Richards et al., 1984)]. It is known that CEF can increase 20

GLT-1 and xCT expression in the brain; therefore, different studies have investigated 21 different possible mechanisms behind it. For instance, a study found that CEF treatment 22

134 can facilitate the nuclear P65 translocation and activate the nuclear factor-κB (NF-κB) 1 signaling pathway when tested in primary human fetal astrocytes (Lee et al., 2008). 2

Moreover, it was reported that CEF increased the expression of NF-κB and the 3 phosphorylation of Akt in the NAc and PFC in P rats (Rao and Sari, 2014). In addition, 4

CEF was also found to activate the nuclear factor erythroid 2-related factor2 (Nrf2), which 5 was suggested to increase the expression of xCT in the HIP cell line (Lewerenz et al., 6

2009). 7

In this study, we used CEF (200 mg/kg, i.p.) to attenuate HYD reinstatement. Indeed, 8 studies have found that CEF can attenuate reinstatement of morphine (Fan et al., 2012), 9 heroin (Shen et al., 2014), methamphetamine (Abulseoud et al., 2012), cocaine (Knackstedt 10 et al., 2010) and nicotine (Alajaji et al., 2013). In addition, several studies from our lab 11 revealed that CEF (200 mg/kg, i.p.) attenuated chronic alcohol drinking in P rats (Sari et 12 al., 2011, Alhaddad et al., 2014, Rao and Sari, 2014, Das et al., 2015). Lower doses of 13

CEF were not considered in this study due to the fact that it was administered every other 14 day during the extinction phase. We have tested CEF at lower dose (50 mg/kg, i.p.) to 15 attenuate cue-induced reinstatement to cocaine-seeking behavior (Sari et al., 2009). 16

However, CEF at high dose (200 mg/kg, i.p.) attenuated cue-induced reinstatement to 17 cocaine-seeking behavior and this effect was associated with upregulation of GLT-1 18 expression in the NAc and PFC. In fact, CEF at lower dose (50 mg/kg, i.p.) did not 19 upregulate the expression of GLT-1, which suggests the non-attenuating effect of this dose 20 in cocaine-seeking behavior. Furthermore, it has been suggested that CEF (200 mg/kg, 21 i.p.) is equivalent to 13 g/day in clinical setting, where the normal dose of CEF is around 22

2 g/day (Rasmussen et al., 2011). Also, it has been proposed that this dose could produce 23

135

CNS concentration equivalent to the concentration needed to modulate GLT-1 expression 1

(3.5 uM) in vitro (Rothstein et al., 2005). In general, high doses of CEF may cause 2 unspecific adverse effects when used for a long period of time. However, several studies 3 have used CEF to attenuate the reinstatement of other drugs of abuse for more than five 4 consecutive days without reporting serious adverse effects (Knackstedt et al., 2010, 5

Abulseoud et al., 2012, Fan et al., 2012, Alajaji et al., 2013, Shen et al., 2014). 6

In conclusion, this study showed for the first time that using HYD (5 mg/kg, i.p.) can 7 produce conditioning and reinstatement effects in P rats using the CPP paradigm. Also, 8

HYD reinstatement was associated with reduction in xCT expression in the NAc and HIP, 9 but not in the dmPFC and AMY. Expression of GLT-1 and GLAST were not affected in 10 the HYD reinstatement. CEF (200 mg/kg, i.p.) prevented HYD reinstatement in P rats, in 11 part, through modulating xCT expression in the NAc and HIP. Together, these data 12 demonstrate that xCT has a vital role in HYD reinstatement in P rats. 13

136

Figure 3-8 Proposed mechanistic events associated with changes in the xCT expression in 3 the NAc and HIP for the attenuation of HYD-seeking behavior with CEF treatment. (1) 4

HYD reinstatement was associated with reduction in the xCT expression in the NAc and 5

HIP. This could be due to the effect of repeated exposure to HYD during the conditioning 6 phase and HYD priming during the reinstatement phase. (2) The reduction in xCT 7 expression may decrease the glutamatergic tone on mGluR2/3 and loss of the inhibitory 8 mechanism on glutamate release. (3) High level of extracellular glutamate facilitates HYD 9 reinstatement. (4) CEF treatment during the extinction phase increased xCT expression in 10 the NAc and HIP, which could restore the glutamatergic tone on mGluR2/3. Restoring the 11 inhibitory mechanism on glutamate release through mGluR2/3 could prevent high levels 12 of extracellular glutamate and attenuate HYD reinstatement. 13

137

Disclosure Statements 1

The authors declare no conflict of interest. 2

Acknowledgments 3

This work was supported in part by Award Number R01AA019458 (Y.S.) from the 4

National Institutes on Alcohol Abuse and Alcoholism and also by start-up funds from The 5

University of Toledo. F.S.A. was supported by a scholarship from Umm Al-Qura 6

University, College of Pharmacy & Pharmaceutical Sciences, Makkah, Saudi Arabia. The 7 authors would like to thank Mrs. Charisse Montgomery for editing this paper. 8

138

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152

Chapter 4 4

5 Ceftriaxone Attenuates Alcohol Drinking Behavior and Hydrocodone Reinstatement in 6 Alcohol-Preferring P Rats: A Role of Astroglial Glutamate Transporters 7 8 9 10 Fahad S. Alshehri1, Alqassem Y. Hakami1, Yusuf S. Althobaiti 1 , Youssef Sari 1* 11 12 13 14 15 1Department of Pharmacology & Experimental Therapeutics, College of Pharmacy and 16 Pharmaceutical Sciences, University of Toledo, Health Science Campus, 3000 Arlington 17 Avenue, Toledo, OH 43614, USA 18 19 20

* Corresponding author: 21 Dr. Youssef Sari 22 University of Toledo, College of Pharmacy & Pharmaceutical Sciences 23 Department of Pharmacology & Experimental Therapeutics 24 Health Science Campus, 3000 Arlington Avenue 25 Toledo, OH 43614, USA 26 E-mail: [email protected] 27 Tel: 419-383-1507 28 29

153

Abstract 1

Simultaneous abuse of ethanol and opioids is a common practice among drug users. 2

Several studies have demonstrated that dependence and relapse to several drugs of abuse, 3 including ethanol and opioids, involves the glutamatergic system. Chronic ethanol 4 consumption and chronic opioid increase extracellular glutamate concentrations in several 5 brain regions. High levels of extracellular glutamate are associated with reductions in the 6 expression of glutamate transporter 1 (GLT-1). Glutamate homeostasis is regulated by 7 several glutamate transporters, including GLT-1, the cystine-glutamate transporter (xCT), 8 and the glutamate-aspartate transporter (GLAST). Changes in the expression of these 9 transporters can lead to dysregulation of glutamate clearance and homeostasis. In this 10 study, we investigated the effects of ceftriaxone (CEF) on hydrocodone (HYD) 11 reinstatement and ethanol intake in alcohol-preferring (P) rats using the conditioned place 12 preference (CPP). The CPP procedure was performed with four phases: a habituation 13 phase for three days, a conditioning phase with four HYD pairings (5 mg/kg, i.p.), an 14 extinction phase with four treatments with CEF (200 mg/kg, i.p.), and a reinstatement phase 15 with one priming session of HYD. Animals had free access to ethanol (15% and 30%) for 16 five weeks prior to the CPP paradigm, which continued through the end of the study. One 17 priming dose of HYD (5 mg/kg, i.p.) produced a significant increase in time spent in HYD- 18 paired chamber in the reinstatement test. CEF attenuated HYD-reinstatement and reduced 19 ethanol drinking. Western blot analysis showed a reduction in GLT-1 and xCT expression 20 in nucleus accumbens (NAc), dorsomedial prefrontal cortex (dmPFC) and hippocampus 21

(HIP) in rats simultaneously exposed to ethanol and HYD. CEF treatment attenuated these 22 effects. No changes were observed in the expression of GLAST. These data show that 23

154

CEF attenuated HYD-reinstatement and ethanol drinking, in part, through upregulation of 1

GLT-1 and xCT expression in central brain reward regions. 2

Introduction 3

Alcoholism and opioid addiction are a significant issue worldwide. According to the 4

National Institute on Alcohol Abuse and Alcoholism, 86.4% of people older than 18 had 5 been drunk at a certain point during their lifetime in 2015 (Abuse, 2015). Equally 6 important, the abuse of opioids has climbed sharply in recent years (Becker et al., 2008, 7

Tetrault et al., 2008). Hydrocodone (HYD) is a semisynthetic opioid related to codeine, 8 which is prescribed for mild to moderate pain (Center, 2008). HYD is one of the most- 9 prescribed opioids in recent years (Manchikanti et al., 2005). Furthermore, the 10 concomitant use of ethanol and opioids is common in drug users (Ottomanelli, 1999, 11

Gossop et al., 2000). Adolescents frequently combine non-medical prescription of opioids 12 with other drugs of abuse, including ethanol (McCabe et al., 2012). It has been reported 13 that 18.5% of opioid-related emergency visits and 22.1 % of drug-related deaths are related 14 to concurrent ethanol use (Jones et al., 2014). Opioids and ethanol have overlapping 15 mechanisms of action; ethanol produces its rewarding and reinforcing effects, in part, via 16 endogenous opioid release (Herz, 1997, Oswald and Wand, 2004). 17

Several studies have linked glutamate homeostasis to dependence and relapse for several 18 abused drugs, including ethanol and opioids (Fujio et al., 2005, LaLumiere and Kalivas, 19

2008, Qrunfleh et al., 2013, Hakami et al., 2017). Chronic ethanol drinking increases 20 glutamate concentrations in the nucleus accumbens (NAc) (Das et al., 2015). Also, chronic 21 ethanol exposure has been reported to reduce glutamate clearance by decreasing the 22

155 expression of glutamate transporter 1 (GLT-1) and the cystine-glutamate transporter (xCT) 1

(Goodwani et al., 2015, Hakami et al., 2016). It is important to note that GLT-1 is the 2 transporter that removes the majority of glutamate from the synapse (Danbolt, 2001), while 3 xCT is involved in regulating the extracellular glutamate levels and the biosynthetic 4 process of glutathione production through exchanging extracellular cystine with glutamate 5

(Bassi et al., 2001, Danbolt, 2001). Similarly, administration of opioids, such as morphine, 6 has been proposed to reduce the clearance of glutamate (Mao et al., 2002). In fact, 7 morphine-sensitized rats had high extracellular level of glutamate when challenged with 8 morphine after abstinence (Farahmandfar et al., 2011). Moreover, it has been found that 9 heroin-seeking behavior was associated with high levels of glutamate in the NAc core in 10 rats (LaLumiere and Kalivas, 2008). In addition, it has been shown that glutamate is 11 essential for ethanol seeking behavior (Qrunfleh et al., 2013). Together, glutamate could 12 be a common target for both HYD reinstatement and chronic ethanol drinking. 13

Several studies have shown that ceftriaxone (CEF), a β-lactam antibiotic known to 14 upregulate GLT-1 and xCT, is able to restore glutamate uptake and reduce ethanol drinking 15

(Alhaddad et al., 2014a, Das et al., 2015, Goodwani et al., 2015, Hakami et al., 2016). CEF 16 has been shown to attenuate drug-seeking behavior for several drugs of abuse, such as 17 nicotine (Alajaji et al., 2013), cocaine (Sari et al., 2009, Knackstedt et al., 2010, 18

Sondheimer and Knackstedt, 2011), and methamphetamine (Abulseoud et al., 2012), 19 through modulating the expression of astroglial glutamate transporters. In fact, we reported 20 recently that CEF was able to attenuate HYD reinstatement through modulating the xCT 21 expression in the NAc and hippocampus (HIP) (Alshehri et al., 2018). However, less is 22 known about opioid effects in alcohol exposed animals, as well as astroglial glutamate 23

156 transporters. Therefore, we investigated the effects of HYD exposure during the 1 conditioning phase of conditioned place preference (CPP) aquisition on voluntarily ethanol 2 drinking in alcohol-preferring (P) rats, as well as the effect of chronic ethanol drinking on 3

HYD-induced preference and reinstatement. In addition, we examined the effects of CEF 4 on ethanol drinking and HYD reinstatement when given during the extinction phase of 5

CPP. The expression of GLT-1, xCT and glutamate-aspartate transporter (GLAST) were 6 also investigated after the reinstatement test in different brain reward regions, including the 7

NAc, dorsomedial prefrontal cortex (dmPFC), HIP, and amygdala (AMY). 8

4.1. Materials and methods 9

4.1.1. Drugs 10

HYD (+)- bitartrate salt was obtained from Sigma-Aldrich (St. Louis, MO). CEF (Sandoz 11

Inc., Princeton, NJ) was purchased from the pharmacy at the University of Toledo Medical 12

Center (UTMC). Saline (SAL) (0.9% NaCl) was used as a vehicle for HYD and CEF. 13

Ethanol (190 proof, Decon Labs) was diluted in water and used for animal drinking. 14

4.1.2. Animals and drug dosing 15

Male P rats were obtained from Indiana University, School of Medicine (Indianapolis, IN, 16

USA). P rats have been shown to meet the criteria for an animal model of alcoholism 17 which includes: self-administration, relevant blood alcohol concentrations (BAC), positive 18 pharmacologic effects, metabolic tolerance, withdrawal, and relapse upon abstinence (for 19 review, see Bell et al., 2006). Ethanol and water intake, as well body weight, were 20 measured three times a week and considered as baseline. Rats continued to have free access 21

157 to ethanol (15% and 30%, concurrently) throughout the study. Rats were kept in plastic 1 cages individually in 12:12-hour dark-light cycle; the temperature was kept at 21°C and 2 the humidity at 50%. Rats had free access to water, food and ethanol throughout the whole 3 study. The experiments were conducted with the approval of the Institutional Animal Care 4 and Use Committee (IACUC) at The University of Toledo, conforming to all National 5

Institutes of Health guidelines and the Guide for the Care and Use of Laboratory Animals 6

(Institute of Laboratory Animal Resources, Commission of Life sciences, 1996). HYD (5 7 mg/kg, i.p.) dosing and administration schedules were selected based on a recent study, 8 which showed that HYD can lead to preference in animals using the CPP procedure 9

(Tenayuca and Nazarian, 2012, Alshehri et al., 2018). CEF (200 mg/kg, i.p.) dosing was 10 selected based on several studies which showed that CEF (200 mg/kg, i.p.) can attenuate 11 drug seeking for several drugs of abuse, including morphine (Fan et al., 2012), heroin (Shen 12 et al., 2014) and HYD (Alshehri et al., 2018). CEF administration during the extinction 13 phase was based on the previous studies from our laboratory, which used β-lactam 14 compounds to attenuate HYD and cocaine reinstatement in P rats (Hammad et al., 2017, 15

Alshehri et al., 2018). 16

4.1.3. CPP Apparatus 17

The CPP apparatus consists of three chambers: two main chambers (40 cm x 40 cm x 40 18 cm) separated by smaller chamber (30 cm x 40 cm x 40 cm). The main chambers feature 19 different visual and tactile effects with white and black (vertical or horizontal) stripes and 20

(smooth or textured) floors. The middle chamber had different visual or tactile cues from 21 both of the other chambers. 22

158

4.1.4. Animal groups and experimental procedure 1

CPP was performed based on recent studies from our laboratory that demonstrated 2 attenuation of reinstatement of HYD (Alshehri et al., 2018) and cocaine (Hammad et al., 3

2017) by exposure to β-lactam compounds in P rats. All animals were habituated for three 4 days prior to the apparatus. Animals were divided into four groups as shown in Table 4-1: 5

Group 1 (control: Water (Wt)-SAL-SAL) had free access to water, and then SAL (i.p.) 6 injections during the conditioning, extinction and reinstatement phases. Group 2 (Ethanol 7

(E)-SAL-SAL) received SAL (i.p.) injections during the conditioning, extinction and 8 reinstatement phases. Group 3 (E-HYD-SAL) received HYD (5 mg/kg, i.p.) injections and 9

SAL (i.p.) alternatively every other day for a total of 8 sessions during the conditioning. 10

SAL injections were received every day during the extinction for a total of 8 sessions, and 11 one priming dose of HYD (5 mg/kg, i.p.) was given during the reinstatement phase. Group 12

4 (E-HYD-CEF) received HYD (5 mg/kg, i.p.) injections and SAL (i.p.) every other day 13 for a total of 8 sessions during the conditioning, CEF (200 mg/kg, i.p) and SAL (i.p.) 14 injections every other day for a total of 8 sessions during the extinction phase, and one 15 priming dose of HYD (5 mg/kg, i.p.) during the reinstatement phase. Groups 2, 3 and 4 16 had free access to ethanol (15% and 30% v/v) for five weeks prior to starting the CPP 17 procedure and throughout the study. 18

159

Table 4-1 Classification of groups based on treatments during conditioning, extinction and 1 reinstatement. All groups* were exposed to continuous free access to ethanol (15% and 2

30%) in home cages. 3

CPP phases Wt-SAL- E-SAL-SAL E-HYD-SAL E-HYD-CEF SAL group group* group* group* Conditioning SAL SAL HYD/SAL HYD/SAL

Extinction SAL SAL SAL CEF/SAL

Reinstatement SAL SAL HYD/SAL HYD/SAL

The CPP procedure was performed as described in previous studies (Hammad et al., 2017, 5

Alshehri et al., 2018) and shown in Figure 4-1. Briefly, the CPP procedure consisted of 6 four phases: a) Habituation, animals had free access to explore the apparatus for 20 min for 7 three days. The pre-conditioning test (Day 4) determined baseline for initial preference. 8

Animals showing a preference greater than 67% were excluded from the study, as in 9 previous studies (Fujio et al., 2005, Hammad et al., 2017, Alshehri et al., 2018). b) 10

Conditioning phase, animals were given eight alternating sessions with HYD or SAL, 30 11 min each. A post-conditioning test (Day 13) was performed and the time spent in each 12 chamber was recorded. c) Extinction phase, animals were given four sessions with either 13

CEF or SAL, 30 min each. An extinction preference test (Day 22) was performed; animals 14 must show a reduction of 25% in time spent compared to post-conditioning test to be 15 considered extinguished, as in previous studies (Abulseoud et al., 2012, Hammad et al., 16

2017, Alshehri et al., 2018). d) Reinstatement phase, animals were given one priming dose 17

160 of HYD (5 mg/kg, i.p.) or SAL (Day 23) according to the group. Then, all groups were 1 given SAL (Day 24). The reinstatement test was performed on Day 25. 2

Figure 4-1 Timeline for the chronic ethanol drinking and exposure to HYD in the CPP 6 procedure. A) Overall schedule for the entire experiment. Animals were exposed to 7 ethanol through the end of the experiment, and CPP was started at Week 6. B) Timeline 8 for habituation, conditioning, extinction, and reinstatement during HYD injections (5 9 mg/kg, i.p.) in the CPP apparatus. 10

4.1.5. Brain tissue extraction 11

Rats were euthanized with carbon dioxide after the reinstatement test on Day 25. The brains 12 were collected and kept at -80°C. Brain regions (The NAc (core and shell), dmPFC 13

(cingulate cortex and prelimbic cortex), HIP (cornu ammonis, CA, subfield: CA1, CA2 14 and CA3) and AMY (central amygdala, basomedial amygdala and basolateral amygdala) 15 were extracted using the cryostat machine and with the guidance of The Rat Brain Atlas 16

(Paxinos and Watson, 2007) and stored at -80°C for later immunoblotting. 17

161

4.1.6. Immunoblots procedure 1

The Western blot procedure was performed as described in previous study (Sari et al., 2

2009). Brain samples were lysed using a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 3 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS) mixed with protease and phosphatase 4 inhibitors. Then, the samples were centrifuged, and the supernatants were collected. 5

Protein quantification was performed to identify the amount of the protein in the 6 supernatant of each sample. Polyacrylamide gels (10% to 15%) were used to separate 7 proteins by electrophoresis. The separated proteins were transferred to PVDF membranes. 8

The membranes were then blocked with Tris-buffered SAL with Tween 20 (10%) (TBST) 9 in 3% fat-free milk and incubated with primary antibodies: anti-Guinea pig GLT-1 10

(Millipore Sigma; 1:5000 dilution), Anti-Rabbit xCT (Abcam; 1:1000 dilution), anti- 11

Rabbit GLAST (Abcam1:1000 dilution), and Mouse anti-β-tubulin (Covance;1:5000 12 dilution) overnight at 4°C. Then, the membranes were washed with TBST, blocked with 13

3% fat-free milk, and incubated with secondary antibodies. The membranes were exposed 14 to chemiluminescence and developed in X-ray film processor (Konica SRX101A – 15

Tabletop). The bands were quantified using (MCID Digital Imaging Software). The 16 immunoblots data were presented as a percentage of the proteins (GLT-1, xCT, or 17

GLAST)/ β-tubulin. The Wt-SAL (control) was considered as 100% to show the percent 18 of change in protein expression of the targeted proteins in Wt-SAL, E-SAL-SAL, E-HYD- 19

SAL, and E-HYD-CEF groups as shown in previous studies (Li et al., 2003, Wen et al., 20

2005, Zhang and Tan, 2011, Simões et al., 2012, Alshehri et al., 2017). 21

162

4.1.7. Statistical analyses 2

Two-way repeated measures analysis of variance (ANOVA) were performed for the 3 ethanol, water drinking, and body weight data (Days x Treatment). Bonferroni’s multiple 4 comparisons means tests were performed when statistical significance was observed in the 5

ANOVA. Two-way repeated measures ANOVA were performed to analyze the time spent 6 in pre-conditioning, post-conditioning, extinction, and reinstatement tests in both HYD 7 paired and SAL paired chambers (Time X Chamber). Tukey’s post hoc tests were 8 performed when significant effects were found in the ANOVA. Immunoblots for (GLT-1, 9 xCT and GLAST) were analyzed using a one-way ANOVA followed by a Newman-Keuls 10 multiple comparison tests when the significant factors were observed. All data were 11 analyzed using GraphPad Prism and p < 0.05 was set as a level of significance. 12

4.2. Results 13

4.2.1. Effect of SAL exposure on CPP 14

The effect of SAL on preference was tested during all CPP phases. Animals were given 15 daily injections of SAL (i.p.) and randomly assigned to either chamber one or chamber 16 two. Two-way repeated measures ANOVA showed no significant Time effect in the Wt- 17

SAL-SAL group [F (1, 6) = 1.273, p = 0.3023], no significant Chamber effect [F (1, 6) = 18

0.4442, p = 0.5299], and no significant Time x Chamber effect [F (1, 6) = 0.005607, p = 19

0.9427] (Figure 4-2 A). Two-way repeated measures ANOVA showed a significant effect 20 of Time in the E-SAL-SAL group [F (1, 7) = 16.51, p = 0.0048]. However, there was no 21

163 significant Chamber effect [F (1, 7) = 0.3646, p = 0.5650] nor a Time x Chamber effect [F 1

(1, 7) = 0.06378, p = 0.8079] (Figure 4-2 B). 2

Figure 4-2 Effect of SAL exposure on CPP; no change in time spent due to SAL 6 administration during the pre-conditioning (PRE) and post-conditioning (POST) tests in 7 the Wt-SAL-SAL group (A) or the E-SAL-SAL group (B). Values are shown as means ± 8

S.E.M (∗ p < 0.05) (n = 7-8). 9

4.2.2. The effect of HYD administration on CPP 10

Preference in the E-HYD-SAL group was investigated during the CPP phases. Two-way 11 repeated measures ANOVA showed significant main effects of Time [F (3, 21) = 7.604, p 12

= 0.0013], Chamber [F (1, 7) = 49.45, p = 0.0002], and Time x Chamber [F (3, 21) = 17.12, 13 p < 0.0001] (Figure 4-3 A). Further analysis with the Tukey’s post hoc tests revealed a 14 significant increase in time spent in the post-conditioning test in the HYD-paired chamber 15 as compared to the pre-conditioning test (p < 0.05). Moreover, there was a significant 16

164 decrease in time spent in the extinction test in the HYD-paired chamber as compared to the 1 post-conditioning test (p < 0.05). A priming dose of HYD (5 mg/kg, i.p.) increased the 2 time spent in the HYD-paired chamber, e.g. reinstatement to HYD. 3

4.2.3. Effect of CEF administration on HYD reinstatement 4

We also investigated the effects of CEF (200 mg/kg, i.p.) administered during the 5 extinction phase on reinstatement of CPP in the E-HYD-CEF group. Two-way repeated 6 measures ANOVA showed significant main effects of Time [F (3, 27) = 11.6, p < 0.0001], 7

Chamber [F (1, 9) = 7.511, p = 0.0228], and Time x Chamber [F (3, 27) = 21.5, p < 0.0001] 8

(Figure 4-3 B). Further analysis with Tukey’s post hoc tests revealed a significant increase 9 in time spent on post-conditioning tests in the HYD-paired chamber compared to the pre- 10 conditioning test (p < 0.05). Furthermore, there was significant reduction in preference in 11 the extinction test in the HYD-paired chamber compared to the post-conditioning test (p < 12

0.05). A priming dose of HYD (5 mg/kg, i.p.) did not induce any change in the time spent 13 in the HYD-paired chamber on the reinstatement test compared to the extinction test (p < 14

0.05). 15

165

Figure 4-3 The time spent in each chamber during pre-conditioning (PRE), post- 3 conditioning (POST), extinction (EXT), and reinstatement (RE) tests. Statistical analysis 4 showed an increase in time spent in the HYD-paired chamber in the E-HYD-SAL group 5

(A) and the E-HYD-CEF group (B) in the post-conditioning test. CEF treatment blocked 6

HYD-induced reinstatement in the E-HYD-CEF group, as compared to the E-HYD-SAL 7 group. Values are shown as means ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, and 8

****p < 0.0001. (n = 8-10). 9

4.2.4. Effect of HYD exposure on ethanol and water drinking during the 10 conditioning phase of CPP 11

The effect of HYD administration on ethanol drinking (g/kg/day) was asssessed during the 12 conditioning phase (Figure 4-4 A). Statistical analysis using two-way repeated measures 13

ANOVA showed significant effects of Days x Treatment [F (8, 176) = 2.437, p = 0.0160] 14 and Treatment [F (1, 22) = 11.04, p = 0.0031]. However, there was no effect of Days [F 15

(8, 176) = 1.206, p = 0.2980]. Bonferroni multiple comparison tests showed a significant 16

166 decrease in ethanol drinking in the E-HYD-SAL group on conditioning day 1 (CON 1) (p 1

= 0.0105), CON 3 (p = 0.0068), CON 5 (p = 0.0228), and CON 7 (p = 0.0252) compared 2 to the E-SAL-SAL group. However, statistical analysis showed no significant difference 3 between the E-HYD-SAL and E-SAL-SAL groups on CON 2 (p = 0.2330), CON 4 (p = 4

0.1227), CON 6 (p = 0.3969), and CON 8 (p = 0.2004). We also tested the effect of HYD 5 on water drinking during the conditioning phase (g/kg/day) (Figure 4-5 A). Statistical 6 analysis using two-way repeated measures ANOVA showed no significant effects of Days 7 x Treatment [F (8, 176) = 1.459, p = 0.1752], or Treatment [F (1, 22) = 0.3626, p = 0.5532], 8 but a significant effect of Days [F (22, 176) = 12.28, p < 0.0001]. Furthermore, we tested 9 the effect of HYD on the total fluid intake (water and ethanol) (g/kg/day) during the 10 conditioning phase (Figure 4-6 A). Statistical analysis using two-way repeated measures 11

ANOVA showed no significant effects of Days x Treatment [F (8, 176) = 1.261, p = 12

0.2669], Treatment [F (1, 22) = 0.0611, p = 0.8071] or Days [F (8, 176) = 0.7207, p = 13

0.6730]. 14

4.2.5. Effect of CEF treatment on ethanol and water drinking during the extinction 15 phase 16

The effect of CEF during the extinction phase on ethanol drinking (g/kg/day) was tested 17

(Figure 4-4B). Statistical analysis using two-way repeated measures ANOVA showed a 18 significant interaction of Days x Treatment [F (16, 184) = 7.759, p < 0.0001], and 19 significant main effects of Days [F (8, 184) = 2.649, p = 0.0090] and Treatment [F (2, 23) 20

= 29.51, p < 0.0001]. Bonferroni's multiple comparison tests showed significant decreases 21 in average ethanol consumption in the HYD-CEF group compared with the E-SAL-SAL 22

167 and E-HYD-SAL groups for extinction sessions 1-8 (EXT 1 - 8) (p < 0.05). We also tested 1 the effect of CEF on water drinking during the extinction phase (g/kg/day) (Figure 4-5 B). 2

Statistical analysis using two-way repeated measures ANOVA showed no significant 3 interaction of Days x Treatment [F (16, 184) = 1.52, p = 0.0964], but a significant effect of 4

Days [F (8, 184) = 3.38, p = 0.0012] and Treatment were shown [F (2, 23) = 7.485, p = 5

0.0031]. Bonferroni's multiple comparison tests found a significant increase in average 6 water drinking in the E-HYD-CEF group compared with the E-SAL-SAL and E-HYD- 7

SAL groups. Water intake was increased in the E-HYD-CEF group, as compared to the E- 8

SAL-SAL group, for EXT 1, 2, 4, 5 and 6 (p < 0.05). Also, water intake was increased in 9 the E-HYD-CEF group as compared to the E-HYD-SAL for EXT 2, 3, 4, 5, 6 and 8 (p < 10

0.05). Furthermore, we tested the effect of CEF on total fluid intake (water and ethanol) 11

(g/kg/day) during the extinction phase (Figure 4-6 B). Statistical analysis using two-way 12 repeated measures ANOVA showed no significant interaction of Days x Treatment [F (16, 13

184) = 0.7633, p = 0.7253], a significant effect of Treatment [F (2, 23) = 0.00606, p = 14

0.9940], but no effect of Days [F (8, 184) = 1.025, p = 0.4191]. 15

168

Figure 4-4 A) Effect of HYD exposure on chronic ethanol drinking during the conditioning 6 phase of CPP. Animals had free access to ethanol during the conditioning phase. HYD (5 7 mg/kg, i.p.) was given on conditioning days CON 1, 3, 5, and 7, and SAL was administered 8 on conditioning days CON 2, 4, 6, and 8. Two-way repeated measures ANOVA, followed 9 by Bonferroni multiple comparison post hoc tests, showed a significant decrease in ethanol 10 consumption in the E-HYD-SAL group on conditioning days CON 1, 3, 5, and 7 compared 11 to the E-SAL-SAL group. Values shown as means ± S.E.M. *p < 0.05 and **p < 0.01 (n 12

= 8-16 for each group). B) Effect of CEF treatment on chronic ethanol drinking during the 13 extinction phase. Animals had free access to ethanol during the extinction phase. CEF 14

(200 mg/kg, i.p.) was administered on extinction days EXT 1, 3, 5, and 7, and SAL was 15 administered on the extinction days EXT 2, 4, 6, and 8. Two-way repeated measures 16

ANOVA followed by Bonferroni's multiple comparison post hoc tests showed significant 17 decreases in ethanol consumption in the E-HYD-CEF group on extinction sessions as 18 compared to the E-SAL-SAL and E-HYD-SAL groups. Values are shown as means ± 19

S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (n = 8-9). 20

169

Figure 4-5 A) Effect of HYD exposure on water drinking during the conditioning phase. 14

Two-way repeated measures ANOVA revealed no significant change in water drinking 15 during the conditioning phase (n = 8-16). B) Effect of CEF treatment on water drinking 16 during the extinction phase. Two-way repeated measures ANOVA followed by Bonferroni 17 multiple comparison post hoc tests showed significant increases in water drinking in the E- 18

HYD-CEF group on extinction sessions as compared to the E-SAL-SAL and E-HYD-SAL 19 groups. Values are shown as means ± S.E.M. (*p < 0.05, and **p < 0.01) (n = 8-9). 20

170

Figure 4-6 A) Effect of HYD exposure on total fluid intake during the conditioning phase. 10

Two-way repeated measures ANOVA revealed no significant change in total fluid intake 11 during the conditioning phase (n = 8-16 for each group). B) Effect of CEF treatment on 12 total fluid intake during the extinction phase. Two-way repeated measures ANOVA 13 showed no significant change in total fluid intake. Values shown as means ± S.E.M. (n = 14

8-9) 15

171

4.2.6. Effect of HYD and CEF exposure on weight during conditioning and 1 extinction 2

The effect of HYD on weight was monitored during the conditioning phase. Statistical 3 analysis using two-way repeated measures ANOVA showed a significant interaction of 4

Days x Treatment [F (8, 176) = 11.89, p < 0.0001] and a significant main effect of Days [F 5

(8, 176) = 110, p < 0.0001], but no significant Treatment effect was found [F (1, 22) = 6

0.0008448, p = 0.9771] (Figure 4-7 A). In addition, the effect of CEF on animal’s weight 7 was observed during the extinction phase. Statistical analysis using two-way repeated 8 measures ANOVA showed a significant interaction of Days x Treatment [F (16, 184) = 9

5.242, p < 0.0001], and a significant main effect of Days [F (8, 184) = 125.3, p < 0.0001], 10 but no Treatment effect was found [F (2, 23) = 0.8181, p = 0.4537] (Figure 4-7 B). 11

172

Figure 4-7 Effect of HYD exposure on body weight during the conditioning phase. Two- 8 way repeated measures ANOVA revealed no significant change in body weight during the 9 conditioning phase (n = 8-16 for each group). B) Effect of CEF treatment on body weight 10 during the extinction phase. Two-way repeated measures ANOVA showed no significant 11 change in body weight. Values shown as means ± S.E.M. (n = 8-9). 12

4.2.7. Effect of ethanol, CEF and HYD reinstatement on the expression of GLT-1, 13 xCT, and GLAST in NAc and dmPFC 14

We investigated the effects of CEF on the expression of GLT-1, xCT, and GLAST in the 15

NAc and dmPFC after HYD reinstatement in P rats. One-way ANOVA showed a 16 significant main effect of treatment on GLT-1 expression in the NAc [F (3, 20) = 6.468, p 17

= 0.0031, Figures 4-8 A, B] and the dmPFC [F (3, 24) = 6.453, p = 0.0023, Figures 4-9 A, 18

173

B]. Further analysis with Newman-Keuls multiple comparison tests revealed a significant 1 downregulation of GLT-1 expression in the E-SAL-SAL and E-HYD-SAL groups 2 compared to the Wt-SAL-SAL group in the NAc (p < 0.05). Also, statistical analysis 3 revealed significant upregulation of GLT-1 expression in the E-HYD-CEF group compared 4 to the E-SAL-SAL and E-HYD-SAL groups in the NAc (p < 0.05). Further analysis with 5

Newman-Keuls multiple comparison tests revealed a significant downregulation in GLT- 6

1 expression in the E-HYD-SAL group compared to the Wt-SAL-SAL and E-SAL-SAL 7 groups in the dmPFC (p < 0.05). Moreover, statistical analysis revealed significant 8 upregulation of GLT-1 expression in the E-HYD-CEF group as compared to the E-HYD- 9

SAL group in the dmPFC (p < 0.05). 10

Additionally, one-way ANOVA showed a significant main effect of treatment on xCT 11 expression in the NAc [F (3, 20) = 8.39, p = 0.0008, Fig. 4-8C, D] and in the dmPFC [F (3, 12

24) = 5.088, p = 0.0072, Fig. 4-9C, D]. Further analysis with Newman-Keuls multiple 13 comparison tests revealed a significant downregulation in xCT expression in the E-SAL- 14

SAL and E-HYD-SAL groups compared to the Wt-SAL-SAL group in the NAc (p < 0.05). 15

Also, statistical analysis revealed significant upregulation in xCT expression in the E- 16

HYD-CEF group compared to the E-SAL-SAL and E-HYD-SAL groups in the NAc (p < 17

0.05). Further analysis with Newman-Keuls multiple comparison tests revealed a 18 significant downregulation in xCT expression in the E-SAL-SAL and E-HYD-SAL groups 19 compared to the Wt-SAL-SAL group in the dmPFC (p < 0.05). One-way ANOVA did not 20 show any significant effects on GLAST expression in the NAc [F (3, 20) = 0.3882, p = 21

0.7628, Fig. 4-8E, F] or dmPFC [F (3, 20) = 0.1144, p = 0.9506, Fig. 4-9E, F]. 22

174

Figure 4-8 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and 7

CEF (200 mg/kg, i.p.) on GLT-1, xCT and GLAST protein expression in the NAc. A, C, 8

E immunoblots for GLT-1, xCT, GLAST, and β-tubulin (loading control). B) Statistical 9 analysis showed significant downregulation in GLT-1 expression in the E-SAL-SAL and 10

E-HYD-SAL groups compared with the Wt-SAL-SAL and E-HYD-CEF groups, and 11 significant upregulation in GLT-1 expression in the E-HYD-CEF group as compared with 12 the E-SAL-SAL-SAL and E-HYD-SAL groups. No significant effects differences in GLT- 13

1 expression the Wt-SAL-SAL and E-HYD-CEF groups were found. D) Statistical 14 analysis showed significant downregulation in xCT expression in the E-SAL-SAL and E- 15

HYD-SAL groups compared with the Wt-SAL-SAL and E-HYD-CEF groups, and a 16 significant upregulation in xCT expression in the E-HYD-CEF group compared with the 17

E-SAL-SAL and E-HYD-SAL groups. No significant effects on xCT expression in the 18

Wt-SAL-SAL and E-HYD-CEF groups were found. F) Statistical analysis showed no 19 significant change in GLAST expression between groups. Values are shown as 20 means ± SEM (∗p < 0.05, **p < 0.01) (n = 6). 21

175

Figure 4-9 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and 8

CEF (200 mg/kg, i.p.) on GLT-1, xCT, and GLAST expression in dmPFC. A, C, E show 9 immunoblots for GLT-1, xCT, GLAST, and β-tubulin (loading control). B) Statistical 10 analysis showed significant downregulation in GLT-1 expression in the E-HYD-SAL 11 group compared to the Wt-SAL-SAL group. Significant upregulation of GLT-1 expression 12 in the E-HYD-CEF group compared to the E-HYD-SAL group. No significant difference 13 was found between the Wt-SAL-SAL group and the E-SAL-SAL group and E-HYD-CEF 14 group. D) Statistical analysis showed significant downregulation in xCT expression in the 15

E-SAL-SAL and E-HYD-SAL groups compared to the Wt-SAL-SAL group. F) Statistical 16 analysis showed no significant changes in GLAST expression. Values are shown as means 17

± SEM (n = 6-7). 18

176

4.2.8. Effect of ethanol, CEF and HYD-induced reinstatement on the expression of 1

GLT-1, xCT, and GLAST in HIP and AMY 2

We investigated the effects of CEF and HYD reinstatement on the expression of GLT-1, 3 xCT, and GLAST in the HIP and AMY in P rats. One-way ANOVA showed a significant 4 main effect of treatment on GLT-1 expression in HIP [F (3, 20) = 5.411, p = 0.0069, Figure 5

4-10 A, B], but not in AMY [F (3, 24) = 1.524, p = 0.2337, Figure 4-11 A, B]. Further 6 analysis with Newman-Keuls multiple comparison tests revealed a significant 7 downregulation in GLT-1 expression in the E-SAL-SAL and E-HYD-SAL groups 8 compared to the Wt-SAL-SAL group in HIP (p < 0.05). Also, statistical analysis revealed 9 a significant upregulation in GLT-1 expression in the E-SAL-SAL and E-HYD-SAL 10 groups compared to the E-HYD-CEF group in HIP (p < 0.05). Additionally, one-way 11

ANOVA showed a significant main effect of treatment on xCT expression in HIP [F (3, 12

24) = 7.394, p = 0.0011, Figure 4-10 C, D], but not in AMY [F (3, 24) = 1.149, p = 0.3497, 13

Figure 4-11 C, D]. Further analysis using Newman-Keuls multiple comparison tests 14 revealed a significant downregulation in xCT expression in the E-HYD-SAL group, 15 compared to the Wt-SAL-SAL group in the HIP (p < 0.05). Statistical analysis revealed 16 significant upregulation in xCT expression in the E-HYD-CEF group, compared to the E- 17

SAL-SAL and E-HYD-SAL groups in the HIP (p < 0.05). One-way ANOVA did not show 18 any significant main effect of treatment on GLAST expression in HIP [F (3, 20) = 0.5581, 19 p = 0.6487, Figure 4-10 E, F] or AMY [F (3, 24) = 0.966, p = 0.4249, Figure 4-11 E, F]. 20

177

Figure 4-10 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and 8

CEF (200mg/kg) on GLT-1, xCT, and GLAST expression in HIP. A, C, E show 9 immunoblots for GLT-1, xCT, GLAST, and β-tubulin. B) Statistical analysis showed 10 significant downregulation in GLT-1 expression in the E-SAL-SAL and E-HYD-SAL 11 groups compared to the Wt-SAL-SAL group. Significant upregulation in GLT-1 12 expression was observed in the E-HYD-CEF group compared to the E-SAL-SAL and E- 13

HYD-SAL groups. D) Statistical analysis showed significant downregulation in xCT 14 expression in the E-HYD-SAL group compared to the Wt-SAL-SAL group. Significant 15 upregulation in xCT expression was observed in the E-HYD-CEF group as compared to 16 the E-SAL-SAL and E-HYD-SAL groups. F) Statistical analysis showed no significant 17 change in GLAST expression between groups. Values are shown as means ± SEM 18

(∗p < 0.05, **p < 0.01) (n = 6-7). 19

178

Figure 4-11 Effects of HYD (5 mg/kg, i.p.) reinstatement, chronic ethanol drinking, and 10

CEF (200 mg/kg, i.p.) on GLT-1, xCT, and GLAST expression in AMY. A, C, E show 11 immunoblots for GLT-1, xCT, GLAST, and β-tubulin (loading control). B) Statistical 12 analysis showed no significant change in GLT-1 expression between groups. D) Statistical 13 analysis showed no significant change in xCT expression between groups. F) Statistical 14 analysis showed no significant change in GLAST expression between groups. Values are 15 shown as means ± SEM (n = 7). 16

4.3. Discussion 17

Glutamate homeostasis has been reported to play an essential role in drug dependence and 18 relapse (Kalivas, 2009b). Studies demonstrated that chronic ethanol exposure is associated 19

179 with an increase in extracellular glutamate concentrations in the NAc in P rats (Das et al., 1

2015) and downregulation of astroglial glutamate transporters (GLT-1 and xCT) in P rats 2

(Alhaddad et al., 2014b, Aal‐Aaboda et al., 2015, Goodwani et al., 2015, Hakami et al., 3

2016). Several studies have shown that heroin and morphine-seeking is associated with 4 high levels of glutamate (LaLumiere and Kalivas, 2008, Farahmandfar et al., 2011). 5

Recently, we found that HYD reinstatement in P rats is associated with a reduction in xCT 6 expression in the NAc and HIP (Alshehri et al., 2018). However, less is known about 7 exposure to opioids in alcohol-consuming animals as well as the role of astroglial glutamate 8 transporters in these circumstances. Therefore, in this study, we tested the effects of co- 9 administering HYD (5 mg/kg, i.p.) and showed that the exposure to this drug can lead to 10 reduction in ethanol consumption. Also, a priming dose of HYD (5 mg/kg, i.p.) produced 11 reinstatement; however, CEF treatment during extinction attenuated these effects. In 12 addition, reinstatement was associated with reduction in GLT-1 and xCT expression in 13 different brain regions. Importantly, CEF (200 mg/kg, i.p.) attenuated both ethanol 14 drinking and HYD reinstatement while restoring the expression of GLT-1 and xCT. 15

Glutamate homeostasis affects drug-seeking and relapse behaviors (Kalivas, 2009a). The 16

NAc is one of the most critical areas mediating opioid reward (Vaccarino et al., 1985). 17

Several studies have reported that NAc glutamatergic transmission is essential in the 18 reinstatement of heroin (LaLumiere and Kalivas, 2008) and cocaine (LaCrosse et al., 2016) 19 seeking behavior. Studies have found that the PFC is involved in drug-seeking, binge-like 20 drug-seeking and drug withdrawal behaviors (Goldstein and Volkow, 2002). In addition, 21 it has been suggested that glutamate release from PFC projections to the NAc is vital in 22 drug-seeking behavior (Childress et al., 1999, Goldstein and Volkow, 2002, Capriles et al., 23

180

2003, McFarland et al., 2003). HIP is another vital area where glutamatergic transmission 1 has a role in addictive behavior. In fact, glutamatergic transmission in HIP has been linked 2 to learning and memory function (Postle, 2009). A high level of glutamate has been 3 reported in morphine sensitized animals in HIP (Xu et al., 2003). The AMY is linked to a 4 variety of opioid-mediated effects (Slotnick, 1973, Helmstetter, 1992, Fendt and Fanselow, 5

1999). 6

The effect of vehicle (SAL) administration was tested in animals during the CPP phases. 7

We found that SAL did not change the time spent in any chamber in the post-conditioning 8 test. Therefore, this shows that the vehicle did not have any confounding influence on 9 behavior during the CPP procedure (Bardo and Bevins, 2000). HYD was given during the 10 conditioning phase at a dose of 5 mg/kg (i.p.) every other day four times. During the post- 11 conditioning test, animals showed a significant increase in the time spent in the HYD- 12 paired chamber. This could give a demonstration that HYD can produce preference in 13 alcohol consuming animals when tested in CPP. It has been found that consuming ethanol 14 can result in adaptive changes in the opioid system due to the downregulation of the μ- 15 opioid receptor (Hoffman et al., 1982). Also, chronic ethanol consumption can increase 16

HYD metabolism by increasing the CYP450 enzymes (Das and Vasudevan, 2007, Jana 17 and Paliwal, 2007, Tompkins and Wallace, 2007), which could decrease HYD levels and 18 reinforcing effects. Therefore, the fact that establishing a preference for HYD using CPP 19 in alcohol-consuming animals is important. 20

During the extinction phase, animals were given either CEF/SAL or SAL/SAL every other 21 day for eight sessions. CEF was administered during the extinction phase to modulate 22

181 glutamate transporters, which are proposed to attenuate affect drug reinstatement. It is 1 important to note that CEF (200 mg/kg, i.p.) blocks reinstatement for many drugs of abuse 2 such as nicotine (Alajaji et al., 2013), cocaine (Sari et al., 2009, Knackstedt et al., 2010, 3

Sondheimer and Knackstedt, 2011), and methamphetamine (Abulseoud et al., 2012), in 4 part, through modulating astroglial glutamate transporters (GLT-1 and xCT). Recently, we 5 have shown that CEF attenuates HYD-induced reinstatement and modulates xCT 6 expression in the NAc and HIP in P rats (Alshehri et al., 2018). During the reinstatement 7 phase, a priming dose of HYD (5 mg/kg, i.p.) was given to both the E-HYD-SAL and E- 8

HYD-CEF groups. HYD produced reinstatement in the E-HYD-SAL group. Therefore, 9

HYD reinstatement in alcohol-preferring animals could be challenging due to ethanol 10 additive effects. CEF was able to block HYD reinstatement even in the presence of chronic 11 ethanol drinking. Together, these data show that CEF can attenuate HYD reinstatement in 12 alcoholic P rats. 13

Evidence has shown a link between a risky pattern of ethanol drinking and opioid use (Egli 14 et al., 2012, Larance et al., 2016). Therefore, in this study, P rats were exposed to ethanol 15

(15% and 30%) for five weeks, with average drinking of 6-7 g/kg/day before the CPP 16 procedure that continued until the end of the study. Several studies from our laboratory 17 have shown that five weeks of voluntarily ethanol drinking can produce ethanol addiction 18 and reinstatement after abstinence in P rats (Alhaddad et al., 2014a, Rao and Sari, 2014, 19

Aal‐Aaboda et al., 2015, Das et al., 2015, Alasmari et al., 2016, Hakami et al., 2016, 20

Hakami et al., 2017). HYD was given during the conditioning phase of CPP at a dose of 21

5mg/kg (i.p.) every other day with SAL for a total of eight sessions. Ethanol drinking was 22 reduced on conditioning days 1, 3, 5, and 7. On the other hand, SAL administration did 23

182 not affect the ethanol drinking on conditioning days 2, 4, 6, and 8. This is the first study 1 to test the effects of HYD on voluntarily ethanol drinking in P rats. It is important to note 2 that morphine has been shown to decrease voluntarily ethanol drinking in open access 3 conditions (Sinclair et al., 1973, Sinclair, 1974, Ho et al., 1976). However, other studies 4 have found that morphine use in limited access paradigms, can lead to increases in ethanol 5 drinking (Reid and Hunter, 1984, Hubbell et al., 1987, Linseman and Harding, 1990). In 6 terms of the relationship between the opioid dose and its effect on ethanol drinking, rats 7 that receive low doses of morphine (1- 2.5 mg/kg) have been shown to consume more 8 ethanol (Reid and Hunter, 1984, Hubbell et al., 1987, Wild et al., 1988) than those that 9 receive high doses of morphine (10 mg/kg) (Volpicelli et al., 1991). In our study, the 10 reduction of ethanol consumption was not associated with an increase in water drinking 11 when HYD was administered. Also, it has been reported that opioids (methadone and 12 buprenorphine) can reduce ethanol BAC levels in non-opioid users (Clark et al., 2006). 13

Therefore, in general, the effects of opioid agonists on ethanol drinking could be different 14 based on the doses and frequency of exposure to opioid and history of drug exposure, as 15 well as the consumption conditions, e.g. open access versus limited access. Of note, total 16 fluid intake did not change after HYD treatment during the conditioning phase of CPP. 17

Moreover, we found that CEF administration during the extinction period attenuated 18 ethanol drinking during extinction. This is in agreement with our previous studies showing 19 that β-lactam compounds, including CEF, reduced ethanol consumption in P rats 20

(Alhaddad et al., 2014a, Rao and Sari, 2014, Das et al., 2015, Goodwani et al., 2015, 21

Alasmari et al., 2016, Hakami et al., 2016, Hakami et al., 2017). Of note, water 22 consumption increased with CEF treatment, which has been suggested as a compensatory 23

183 mechanism to the reduction in ethanol drinking, as seen in previous studies from our 1 laboratory (Alhaddad et al., 2014a, Rao and Sari, 2014). In fact, total fluid consumption 2 during extinction did not change, which confirms that P rat compensate with water to 3 maintain overall fluid intake. Body weight did not change. 4

The effect of chronic ethanol drinking on astroglial glutamate transporters was also 5 examined. We found that GLT-1 was downregulated in NAc and HIP, and xCT was 6 downregulated in the NAc and dmPFC in the E-SAL-SAL group. This is in agreement 7 with several studies from our laboratory, which demonstrated that chronic ethanol 8 consumption in P rats was associated with a downregulation in both GLT-1 and xCT 9

(Goodwani et al., 2015, Hakami et al., 2016). GLAST expression was also investigated; 10 however, no changes were shown. It has been reported that chronic ethanol exposure was 11 associated with an increase in extracellular glutamate concentrations due to a deficit in 12 glutamate clearance (Das et al., 2015). Also, the effect of HYD-induced reinstatement and 13 chronic ethanol drinking on astroglial glutamate transporters were also investigated. We 14 found here that GLT-1 expression was also reduced in the NAc, dmPFC, and HIP in E- 15

HYD-SAL group. We have reported recently that HYD reinstatement was associated with 16 a downregulation of xCT expression in P rats (Alshehri et al., 2018). Therefore, the 17 reduction of GLT-1 expression in this study might be due to the additive effect of chronic 18 exposure to ethanol and HYD treatment. It has been shown that GLT-1 expression was 19 reduced in the NAc core and shell after cocaine reinstatement in P rats (Hammad et al., 20

2017). Similarly, we believe here that reinstatement to HYD developed, at least in part, 21 due to the deficits in GLT-1 in the NAc, dmPFC, and HIP. 22

184 xCT was also downregulated in the NAc, dmPFC, and HIP in the E-HYD-SAL group. The 1 combination of HYD and ethanol might further dysregulate the glutamatergic 2 neurotransmission. It has been suggested that the reduction in xCT function could facilitate 3 drug seeking and relapse-like behaviors (Baker et al., 2003). Additionally, several studies 4 suggested that restoring the function of xCT could attenuate the reinstatement of heroin- 5 and cocaine- (Baker et al., 2003, Kau et al., 2008, Zhou and Kalivas, 2008) seeking 6 behavior. The AMY was also investigated; however, no changes in the expression of either 7

GLT-1 or xCT were found in this brain region. Also, the GLAST expression was 8 investigated. However, no changes were observed in any of the tested brain regions. This 9 is in agreement with our recent report showing no effects of HYD reinstatement on GLAST 10 expression (Alshehri et al., 2018). Several studies from our laboratory have reported no 11 changes in GLAST expression with chronic or repeated exposure to different drugs of 12 abuse including ethanol (Alasmari et al., 2016, Hakami et al., 2016), cocaine (Hammad et 13 al., 2017), or methamphetamine (Althobaiti et al., 2016, Alshehri et al., 2017). Thus, this 14 suggests that GLAST expression is not involved in HYD reinstatement or chronic ethanol 15 drinking. 16

β-lactam compounds may have neuroprotective effects through upregulating GLT-1 17

(Rothstein et al., 2005). Studies from our laboratory have shown that β-lactam compounds 18 can reduce ethanol drinking in P rats (Qrunfleh et al., 2013, Alhaddad et al., 2014a, Rao 19 and Sari, 2014, Das et al., 2015, Goodwani et al., 2015, Alasmari et al., 2016, Hakami et 20 al., 2016, Hakami et al., 2017, Hakami and Sari, 2017). This reduction has been suggested 21 to be due to the ability of these compounds to modulate astroglial glutamate transporters. 22

CEF is known to upregulate GLT-1 and xCT; upregulation of these transporters has been 23

185 associated with a reduction in ethanol intake in P rats (Alhaddad et al., 2014a, Hakami et 1 al., 2016, Hakami et al., 2017). Several studies have shown that administering β-lactams 2 compounds can reduce morphine dependence, tolerance, hyperalgesia, and allodynia 3 associated with the chronic exposure of morphine (Yan et al., 2009, Rawls et al., 2010, 4

Chen et al., 2012). Moreover, CEF can attenuate hyperthermia associated with morphine 5

(Rawls et al., 2007). In fact, we have found recently that CEF can attenuate HYD-induced 6 reinstatement in P rats (Alshehri et al., 2018). Several studies have shown that CEF does 7 not produce preference or aversion using CPP (Abulseoud et al., 2012, Alajaji et al., 2013). 8

Importantly, in this study, we found that CEF administration restored the expression of 9

GLT-1 in NAc, dmPFC and HIP; and xCT in NAc and HIP. The ability of CEF to 10 upregulate the expression of GLT-1 and xCT could be one of the factors critical for 11 attenuating HYD reinstatement. It has been reported that β-lactam drugs can block the 12 reinstatement induced by different drugs of abuse (Sari et al., 2009, Knackstedt et al., 2010, 13

Sondheimer and Knackstedt, 2011, Abulseoud et al., 2012, Alajaji et al., 2013, Hammad 14 et al., 2017). Therefore, using β-lactam compounds such as CEF could have potential 15 therapeutic benefits by restoring glutamate homeostasis that might attenuate relapse-like 16 behavior in comorbid drug addiction, such as for opioids and alcohol. 17

In summary, this study brings new insight about the role of astroglial glutamate transporters 18 in HYD reinstatement in animals exposed to chronic ethanol drinking. It is important to 19 consider that many addicts abuse both opioids and alcohol. Thus, finding therapeutic 20 targets to attenuate responses to both drugs of abuse is highly challenging. Here, we show 21 for the first time the ability of CEF to attenuate reinstatement induced by HYD, as well as 22 ethanol drinking. Attenuating the reinstatement of HYD-seeking behavior and ethanol 23

186 drinking via CEF treatment could be mediated by restoring GLT-1 and xCT expression in 1 the NAc, dmPFC and HIP. These data demonstrated the potential therapeutic effects of 2

CEF in attenuating HYD reinstatement and alcohol drinking that may be relevant to 3 treatment of polysubstance abusers. 4

Disclosure Statements 5

The authors declare no conflict of interest. 6

Acknowledgments 7

This work was supported in part by Award Number R01AA019458 (Y.S.) from the 8

National Institutes on Alcohol Abuse and Alcoholism and also by start-up funds from the 9

University of Toledo. F.S.A was supported by a scholarship from Umm Al-Qura 10

University, College of Pharmacy and Pharmaceutical Sciences, Makkah, Saudi Arabia. A. 11

Y. H. was supported by a scholarship from King Saud bin Abdulaziz University for Health 12

Sciences College of Medicine, Jeddah, Saudi Arabia. 13

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Chapter 5 4

Summary 6

In this project, we investigated the effects of HYD on the astroglial glutamate transporters 7

(GLT-1, xCT and GLAST) in three different studies. First, we investigated the effect of 8 repeated exposure of HYD using primary astrocyte cell cultures. We found that HYD 9 treatment for five days led to a reduction in GLT-1 and xCT expression. Second, we 10 investigated the effects of CEF on HYD-induced reinstatement using CPP in P rats. We 11 found that HYD induced reinstatement, was associated with reduction in xCT expression 12 in the NAc and HIP, but no effect was seen in the dmPFC and AMY. CEF treatment during 13 the extinction phase blocked HYD-induced reinstatement and restored xCT expression. 14

Finally, we investigated the effect of chronic ethanol drinking and HYD-induced 15 reinstatement in P rats. HYD-induced reinstatement and chronic ethanol drinking were 16 associated with reduction in GLT-1 and xCT expression in the NAc, dmPFC and HIP, but 17 no effect was seen in the AMY. CEF treatment during the extinction phase reduced ethanol 18 drinking and blocked HYD-induced reinstatement. Also, CEF treatment restored GLT-1 19 and xCT expression in the NAc and HIP. 20

199

5.1. Experimental designs 1

In this project, we used three different experimental designs. In first study, we used the 2 primary astrocytes to examine the HYD effects on the expression of GLT-1, xCT and 3

GLAST. In the second study, we investigated the effects of HYD reinstatement using the 4

CPP paradigm. In third study, we examined the effect of chronic ethanol drinking and 5

HYD reinstatement in P rats. Summary of experimental study 1, 2 and 3 are shown below 6 in figure 5-1 A, B and C respectively. 7

200

1 A 2

B 3

C 5

Figure 5-1 Summary of all experimental designs for the studies used in this project. A) 8

HYD was tested in primary astrocytes cell culture in different concentrations and time 9 points. B) Experimental design for testing HYD reinstatement using the CPP paradigm. 10

C) Experimental design for chronic ethanol drinking and HYD reinstatement in P rats. 11

Upper panel overall schedule for the entire experiment. The animals were exposed to 12 ethanol through the end of the experiment, and CPP experiment was started in Week 6. 13

Lower panel CPP paradigm for testing HYD reinstatement in P rats exposed to chronic 14 ethanol drinking. 15

201

5.2. Outcomes 1

In this project, we found that treating primary astrocytes with HYD for five days was 2 associated with downregulation of GLT-1 and xCT expression, but no effect was observed 3 on the GLAST expression. In addition, we found that HYD reinstatement was associated 4 with reduction in xCT expression in NAc and HIP. CEF treatment attenuated the 5 reinstatement effect of HYD and restored xCT expression in the NAc and HIP. Finally, 6

HYD and ethanol led to downregulation of both GLT-1 and xCT. However, CEF 7 attenuated the HYD-reinstatement and reduced ethanol drinking. CEF treatment restored 8 the GLT-1 and xCT expression in the NAc, dmPFC and HIP. Summaries of the 9 mechanisms that are thought to underlie these project outcomes are shown in figures 5-2, 10

5-3, and 5-4. 11

A B 13

Figure 5-2 Schematic representation showing the effects of repeated exposure to HYD on 18 primary astrocyte cultures. A) Primary astrocytes prior to HYD exposure. B) Primary 19 astrocytes after repeated HYD exposure. HYD repeated exposure for five days led to 20 reduction in GLT-1 and xCT expression, which can lead to reductions in glutamate uptake. 21

202

Figure 5-3 Proposed mechanistic events associated with changes in the xCT expression in 3 the NAc and HIP underlying the attenuation of HYD-seeking behavior after CEF treatment. 4

(1) HYD reinstatement was associated with a reduction in xCT expression in the NAc and 5

HIP. This could be due to the effect of repeated exposure to HYD during the conditioning 6 phase or HYD priming during the reinstatement phase. (2) The reduction in xCT 7 expression may lead to decreases in the glutamatergic tone on mGluR2/3 and reductions 8 of this inhibitory mechanism on glutamate release. (3) High levels of extracellular 9 glutamate facilitate HYD reinstatement. (4) CEF treatment during the extinction phase 10 increased xCT expression in the NAc and HIP, which could restore the glutamatergic tone 11 on mGluR2/3. Restoring this inhibitory mechanism on glutamate release through 12 mGluR2/3 may attenuate high levels of extracellular glutamate as well as attenuate HYD 13 reinstatement. 14

203

Figure 5-4 Proposed mechanistic events associated with changes in the xCT and GLT-1 3 expression in the NAc, dmPFC and HIP underlying the attenuation of HYD-seeking 4 behavior and ethanol drinking by CEF treatment. (1 and 2) HYD reinstatement and ethanol 5 drinking were associated with reductions in GLT-1 and xCT expression in the NAc, 6 dmPFC and HIP. This could be due to the effect of repeated exposure to HYD during the 7 conditioning phase or HYD priming during the reinstatement phase, as well as chronic 8 ethanol drinking. (3) The reduction in xCT expression may decrease glutamatergic tone 9 on mGluR2/3 and reduce this inhibitory mechanism regulating glutamate release. Also, 10 the reduction of GLT-1 expression may reduce glutamate uptake. (4) High levels of 11 extracellular glutamate facilitate HYD reinstatement. (5 and 6) CEF treatment during the 12 extinction phase increased both GLT-1 and xCT expression in the NAc, dmPFC and HIP, 13 which may restore glutamatergic tone on mGluR2/3 and increase the uptake of glutamate, 14 which may attenuate HYD reinstatement and ethanol drinking. 15

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