Glutamate Transporter 1 and Cystine-Glutamate Antiporter As Potential Targets

A Thesis

Entitled

Glutamate Transporter 1 and Cystine-Glutamate Antiporter as Potential Targets

for Attenuating Alcohol Consumption in Male P Rats

Munaf Aal-Aaboda

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

Master of Science Degree in Pharmaceutical Sciences

______Dr. Youssef Sari, Committee Chair

______Dr. Ezdihar Hassoun, Committee Member

______Dr. Surya Nauli, Committee Member

______Dr. Zahoor Shah, Committee Member

______Dr. Patricia Komuniecki, Dean College of Graduate Studies

The University of Toledo. August-2014

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

Glutamate Transporter 1 and Cystine-Glutamate Antiporter as Potential Targets for Attenuating Alcohol Consumption in Male P Rats

Munaf Aal-Aaboda

Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the Master of Science Degree in Pharmaceutical Sciences

The University of Toledo, OH. August-2014

Alcohol abuse is associated with dysfunction of glutamatergic system along with other neurotransmitter systems in mammalian central nervous system. Studies have shown that both behavioral effects of acutely administered alcohol and neuroadaptation associated with chronic ethanol intake are mediated by glutamatergic neurotransmission in key regions of brain reward circuitry. Extracellular glutamate level has been reported to be elevated following alcohol consumption. The role of several glutamate transporters in restoring glutamate homeostasis has been well established. Among these, glutamate transporter 1 (GLT1) and cystine-glutamate antiporter (xCT) are key players in regulating extracellular glutamate levels. Previous studies from our lab have reported that ceftriaxone, a β-lactam antibiotic known to upregulate GLT1, attenuated ethanol consumption in alcohol preferring (P) rats in chronic ethanol-drinking paradigm. This effect was associated in part with GLT1 upregulation in central brain reward regions. In the present study, we investigated the short and long lasting effects of MS-153, GLT1 activator, on ethanol intake and glutamate transporters’ expression in male P rats. We further examined the effects of MS-153 and Augmentin combination on ethanol

iii consumption, body weight and water intake in P rats. P rats were exposed for five weeks to continuous free-choice ethanol drinking paradigm. On the first day of week 6, P rats were injected intraperitoneally with MS-153 (50 mg/kg), or a combination of MS-153 (50 mg/kg) and Augmentin (100 mg/kg), or vehicle for five consecutive days. Interestingly, we found a significant reduction in ethanol intake in P rats treated with MS-153 starting

24 hours after the first injection, which lasted up to ten days after the last injection as compared to vehicle-treated animals. This long lasting effect on ethanol consumption was associated with significant upregulation of GLT1 levels in the nucleus accumbens (NAc) but not in the prefrontal cortex (PFC). Additionally, one day after the last MS-153 injection, xCT levels were significantly upregulated in amygdala and hippocampus.

Furthermore, treatment with combination of MS-153 and augmentin resulted in reduction of ethanol consumption as compared to vehicle-treated rats. Our study demonstrates that modulating GLT1 and xCT expression may be a promising therapeutic target for treating alcohol dependence.

Acknowledgments

First of all, I would like to express my sincere gratitude to my advisor, Dr.

Youssef Sari, who was extremely helpful and offered invaluable guidance throughout this project. Without his immense knowledge and patience, this thesis would not have been possible. I would also like to thank my committee members Dr. Ezdihar Hassoun, Dr.

Surya Nauli and Dr. Zahoor Shah. I thank Dr. Ezdihar Hassoun for her timely advice and encouragement. I would also like to take this opportunity to thank Dr. Surya Nauli and his lab members for their patience and assistance.

I also express my greatest appreciation to Dr. Shantanu Rao, Sujan Chandra Das and Sunil Goodwani for their endless support in completion of this project.

I would also like to acknowledge the financial support of Higher Committee for

Education Development in Iraq (HCED). I also thank the faculty members at the

Department of Pharmacology and the University of Toledo-College of Pharmacy and

Pharmaceutical Sciences for their support and assistance during my Master’s degree.

Last, but by no means the least, I am inordinately thankful to my friends and family for their constant support and understanding.

Contents

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

Acknowledgments ……………………………….………………………….…..……..... v

Contents ………………………………………………………………………………… vi

List of Figures.….……………………….………………….……………………………. x

List of Abbreviations.…….……….………..……………….…...……….…………….. xii

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

1.1 Overview…………………………………………………………………………….1

1.2 Glutamate…………………………………………………………………………....2

1.2.1 Glutamate Transporters…………………………………………………….….4

1.2.1.1 Vesicular Glutamate Transporters………...………………………………...4

1.2.1.2 Excitatory Amino Acid Transporters…………………………….………….4

1.3 Reward Circuitry Involved in Drugs Addiction ……………………………….…....5

1.3.1 Nucleus Accumbens (NAc)…………………...………………………….…....6

1.3.2 Prefrontal Cortex (PFC)………………………………………….………...….7

1.3.3 Amygdala (AMG)……………………………………………………………..7

1.3.4 Hippocampus (Hipp)……………………………………………………….....8

1.4 Glutamate Transporters and Alcoholism……………………………………………9

1.4.1 GLT1 and Alcoholism……………………………………………………...…9

1.4.2 Cystine-glutamate Antiporter and Alcoholism……………………….……...10

1.5 Glutamate Receptors and Alcoholism……………………………….…….………12

1.5.1 Ionotropic Glutamate Receptors………………………………………...…...12

1.5.1.1 NMDA Receptors and Alcoholism………………………………...………12

1.5.1.2 Non-NMDA Receptors and Alcoholism………………………………...…13

1.5.2 Metabotropic Receptors and Alcoholism…………………………...………..14

1.5.2.1 Group I Metabotropic receptors…………………………………...……….14

1.5.2.2 Group II Metabotropic receptors……………………………………..……16

1.5.2.3 Group III Metabotropic receptors……………………………………...…..16

1.6 Other Neurotransmitter Systems and alcoholism……………………………..…...17

1.7 Aims and Objectives…………………………………………………………….....19

2. Materials and Methods ……………………………………………………………...20

2.1. MS-153 .………………………………………………………………………..…20

2.2. Augmentin………………………………………………………..………..……...21

2.3.Animals………………………………………………………………………….....21

2.4.Behavioral Drinking Paradigm………………………………………………….....22

2.4.1. Effect of MS-153 on chronic ethanol drinking paradigm…………….....23

2.4.2. Effect of MS-153 on xCT and pAkt levels in Amygdala and

Hippocampus……………………………………………..…………….…24

2.4.3. Effect of Combination of MS-153 and Augmentin on Chronic Ethanol

Drinking Paradigm……………………………………………………….24

2.5.Brain Tissue Harvesting……………………………………………………….….25

2.6. Protein Tissue Extraction Protocol…………………………………………….…25

2.6.1. Tissue lysate ………………………………………………………....…..25

vii

2.7.Protein Quantification Assay……………………………………………..…...….26

2.8.Western Blot Procedures………………………………………………………...... 27

2.8.1. Gels preparation ………………………………………………...... …27

2.8.2. Samples Preparation……………………………………………….…27

2.8.3. Running and Transfer of Proteins…………………………………....28

2.8.4. Blocking………………………………………………………..….…28

2.8.5. Incubations with Primary Antibodies……………………………..…29

2.8.6. Incubations with Secondary Antibodies…………………………..…29

2.8.7. Developing Membranes…………………………………………...…30

2.9. Statistical Analyses……………………………………………………………...30

2.9.1. Behavioral Data of MS-153 study……………………………….…..30

2.9.2. Behavioral Data of Augmentin and MS-153 combination study…....30

2.9.3. Western Blot Data……………………………………………………31

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

3.1. Effect of MS-153 on Chronic Ethanol Drinking Paradigm………………...….....32

3.1.1. Effect of MS-153 Treatment on Ethanol Consumption in Male P

Rats………………………………………………..……………....…32

3.1.2. Effect of MS-153 Treatment on Water Intake in Male P

Rats……………………………………………….……………….....34

3.1.3. Effect of MS-153 Treatment on Body Weight in Male P Rats .….….35

3.1.4. Long Lasting Effect of MS-153 on GLT1 Expression in NAc……...37

viii

3.1.5. Long Lasting Effect of MS-153 on GLT1 Expression in PFC………38

3.2. Effect of MS-153 on xCT and pAkt Levels in Amygdala and Hippocampus...... 40

3.2.1. Effect of MS-153 on xCT Expression in Amygdala..……………..…40

3.2.2. Effect of MS-153 on pAkt Expression in Amygdala ………..……....41

3.2.3. Effect of MS-153 on xCT Expression in Hippocampus……. ………43

3.2.4. Effect of MS-153 on pAkt Expression in Hippocampus…..………..44

3.3.Effect of Combination of MS-153 and Augmentin on Chronic Ethanol

Drinking Paradigm……………………………………………………………….46

3.3.1. Effect of Combination of MS-153 and Augmentin on Ethanol

Consumption in Male P rats……………………...…………………..46

3.3.2. Effect of Combination of MS-153 and Augmentin on Water Intake

of Male P Rats…………….………………………………………….48

3.3.3. Effect of Combination of MS-153 and Augmentin on Male P rats’

Body Weight………..………………………………………………..50

4. Discussion…………………………………………………………………………...52

4.1.Effect of MS-153 on Chronic Ethanol Drinking Paradigm…………………....54

4.2.Effect of MS-153 on xCT and pAkt levels in Amygdala and Hippocampus.....57

4.3.Effect of Combination of MS-153 and Augmentin on Chronic Ethanol

Drinking Paradigm……………………………………………………………..61

References ……………………………………………………………………………….63

List of Figures

1-1 Neurocircuitry involved in drugs addiction including alcohol……..……….….9

2-1 Chemical Structure of MS-153 ………...………………………………….…..20

2-2 Chemical structure of amoxicillin and clavulanic acid……………….………..21

3-1 Effects of MS-153 treatment on average daily ethanol intake (g/kg/day)

in male P rats exposed to five weeks of continuous free choice of

ethanol and water……………………………………………………………..33

3-2 Effects of MS-153 treatment on average daily water intake (ml/kg/day) in

male P rats exposed to five weeks of continuous free choice of ethanol

and water …………………………………………………………………...35

3-3 Effects of MS-153 treatment on body weight (grams) of male P rats exposed

to five weeks of continuous free choice access to ethanol and

water………………………………………………………………………..36

3-4 Ten-days post treatment effect of MS-153 on GLT1 expression in NAc……..38

3-5 Ten-days post treatment effect of MS-153 on GLT1 expression in PFC……..39

3-6 Effect of MS-153 on xCT expression in amygdala…………………...... 41

3-7 Effect of MS-153 on pAkt expression in amygdala……………………….…...42

3-8 Effect of MS-153 on xCT expression in Hippocampus……………..………...44

3-9 Effect of MS-153 on pAkt expression in hippocampus………………………..45

3-10 Effects of combination of MS-153 and augmentin treatment on average daily

ethanol intake (g/kg/day) in male P Rats…………………………………..47

3-11 Effects of combination of MS-153 and augmentin treatment on

average daily water intake (ml/kg/day) in male P rats……………………49

3-12 Effects of combination of MS-153 and augmentin treatment on

Body Weight of male P Rats……………………………………………….51

List of Abbreviations

ANOVA ……………………...Analysis of Variance AMG………………………….Amygdala AMPA………………………..α-Amino-3-Hydroxy-5-Hethyl-4-Isoxazole-Propionic acid

CNS…………………………..Central Nervous System

EAATs………………………..Excitatory Amino Acids Transporters

GLM………………………….General Linear Model GLT1…………………………Glutamate Transporter 1 GLAST……………………… L-glutamate/L-aspartate transporter

Hipp…………………………..Hippocampus iGluRs…………………………Ionotropic Glutamate Receptors

KA……………………………. Kainate mGluRs………………………..Metabotropic Glutamate Receptors

NMDA…………………………N-methyl-D-Aspartate NAc……………………………Nucleus Accumbens pAKT ………………………….Phosphorylated-Akt PFC ……………………………Prefrontal Cortex

xii

VTA …………………………..Ventral Tegmental Area VGLUTs………………………Vesicular Glutamate Transporters xCT…………………………....Cystine-Glutamate Antiporter

xiii

Chapter 1

Introduction

1.1 Overview

Drug addiction can be defined as a chronically relapsing disorder identified by compulsive seeking for the drug despite negative consequences associated with taking the drug. Drugs of abuse can be divided into different classes, inclusive of narcotics like opiates, depressants like ethanol, cannabinoids like marijuana, stimulants like amphetamine and nicotine, and hallucinogens like ecstasy (Feltenstein and See, 2008).

One of the major problems with drugs of abuse is dependence, which can be defined as consuming a substance initially for its pleasurable or rewarding (positive reinforcement) effect and then dependence will develop as a result of this repeated drive for reward

(Wise, 1980). Dependence can be characterized by seven criteria: first criterion is tolerance, which is defined as the urge to consume higher dose of the substance to get the desired rewarding effect of the substance. The second criterion is withdrawal, which can be defined as defective cognitive and physiological symptoms that happened after the reduction in consuming the substance (negative reinforcement) (Feltenstein and See,

2008, Quintero, 2013). The third criterion is the compulsive use of the substance. The

1 fourth criterion is manifested as the failure of the subject to stop using the substance. The fifth criterion is defined as spending longer time to obtain and consume the substance or alleviating its effects. Furthermore, the subject becomes careless about socioeconomic activities (criterion 6). The last criterion is defines as the subject will keep using the substance in spite of its negative consequences (Quintero, 2013). Out of six or seven persons who had tried alcohol once in their life, at least one of them will develop dependence to alcohol (Wagner and Anthony, 2002). The world Health Organization reported in 2004 that alcohol abuse is responsible for about 5% of the global disease burden and result in 2.5 million death each year (Janeczek and Lewohl, 2013). It has been hypothesized that all drugs of abuse, including alcohol, are able to activate the mesocorticolimbic dopaminergic (DA) system (Wise, 1980, 2002, Feltenstein and See,

2008). However, compelling evidences suggest that changes in glutamatergic transmission within different brain reward regions are important in alcohol dependence, tolerance, and withdrawal (Sari, 2013). Additionally, glutamatergic neurotransmission are involved in mediating the brain adaptive changes that occur following ethanol exposure in animal models and in human (Dodd et al., 2000). Accordingly, I will focus in this chapter on the role of glutamatergic system in alcohol dependence.

1.2 Glutamate

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system(CNS) and is involved in most aspects of brain physiological functions, including learning, memory, and cognition (Headley and Grillner, 1990). By

2 acting on its receptors located on the target cells’ surfaces, glutamate exerts its excitatory effect, which is crucial for normal physiology and pathology of the brain (Fonnum,

1984). Glutamate induces its excitatory effects by acting on two main families of receptors; (1) ionotropic (iGluRs) receptors which are ligand-gated ion channels, including N-methyl-D-aspartate(NMDA) receptors, 2-carboxy-3-carboxymethyl-4- isopropenylpyrrolidine or kainate (KA) receptors, and α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA) receptors and this group of receptors mediate the fast transmission; and (2) metabotropic (mGluRs) receptors or G-protein coupled receptors, which mediate the slow transmission of glutamate (Chandrasekar, 2013, Zorumski et al.,

2014). Glutamate is synthesized within the cytoplasm and then stored in synaptic vesicles by the vesicular glutamate transporters (VGLUTs 1-3) (Shigeri et al., 2004) (see

Section1.2.1.1). Glutamatergic neurotransmission is responsible for about 70% of synaptic transmission within the CNS (Gass and Olive, 2008). However, the key factor that determines the extent of glutamate receptors stimulation is its extracellular concentration. Extracellular glutamate concentration should be maintained at low level because excess of glutamate can cause oxidative stress leading to neuronal death

(Headley and Grillner, 1990, Danbolt, 2001). There are no enzymes able to significantly metabolize extracellular glutamate as yet; therefore, the only major way to remove glutamate from extracellular fluid is cellular uptake process (Danbolt, 2001). This uptake process is executed by a family of sodium-dependent glutamate transporters (Excitatory

Amino Acid Transporters, EAATs 1-5), which are located on the neurons and astrocytes

(see Section1.2.1.2) (Shigeri et al., 2004). After the release of glutamate, then it will be taken up by pre- or post-synaptic neurons or astrocytes surrounding the synaptic cleft.

Glutamate is then converted to glutamine, non-toxic form, by glutamine synthetase in astrocytes and then released to be taken up by the neurons where is recycled into glutamate by glutaminase, and this process called glutamine-glutamate cycle (Danbolt,

2001). It is important to know that glutamate can also be released to the extracellular space by the astrocytes’ cystine-glutamate antiporter (xCT) (Kalivas, 2009).

1.2.1 Glutamate Transporters

1.2.1.1 Vesicular Glutamate Transporters

VGLUT1, VGLUT2 and VGLUT3 are the three identified subtypes of VGLUTs

(Bellocchio et al., 2000, Fremeau et al., 2001). It has been found that VGLUTs 1 and 2 are localized mostly in glutamatergic neurons, while VGLUT3 are located within glutamatergic and non-glutamatergic neurons (Gras et al., 2002, Takamori et al., 2002).

1.2.1.2 Excitatory Amino Acid Transporters

This family of sodium-dependent glutamate transporters includes five different transporters: EAAT1 (its rodent homologue is L-glutamate/L-aspartate transporter,

GLAST), EAAT2 (its rodent homologue is glutamate transporter 1, GLT-1), EAAT3 (its rodent homologue is Excitatory Amino Acid Carrier, EAAC1), EAAT4 and EAAT5

(identified in both human and rodents). GLAST is expressed in astrocytes throughout the

CNS and at high levels in the cerebellum (Beart and O'Shea, 2007). GLT1 is almost

4 exclusively expressed by the astrocytes and it is the most abundant glutamate transporter in the CNS (Kanai and Hediger, 2004, Beart and O'Shea, 2007). EAAT3 and EAAT4 are localized mainly in the neurons with EAAT3 distributed throughout the CNS, while

EAAT4 is more specific to cerebellar Purkinje cells (Danbolt, 2001, Beart and O'Shea,

2007). However, EAAT5 is localized in rod photoreceptor and bipolar cells of the retina

(Pow and Barnett, 2000). Increased extracellular glutamate levels in different brain reward regions have been directly linked to the development of drugs dependence, including alcohol. Accordingly, with their ability to regulate extracellular glutamate levels, glutamate transporters and particularly GLT1 have been and continue to be a promising target for treating drug abuse problems (Rao and Sari, 2012).

1.3 Reward Circuitry Involved in Drugs Addiction

All drugs of abuse have the ability to enhance dopaminergic activity, although they interact at different levels, within the mesocorticolimbic system. This circuitry includes both the mesolimbic and mesocortical pathways. The mesolimbic pathway includes dopaminergic projections from ventral tegmental area (VTA) to the ventral pallidum (VP), amygdala (AMG), hippocampus (Hipp) and the nucleus accumbens

(NAc). Alternatively, the mesocortical pathway consists of VTA dopaminergic projections into cortical structures, including the prefrontal cortex (PFC), the anterior cingulate and the orbitofrontal cortex. Although these two pathways act in a parallel manner, studies have shown that they have different roles in drug addiction. Importantly, the primary reinforcing effects associated with drugs of abuse have been attributed to the

NAc and VP, while the conditioned learning, which is necessary for addiction, has been linked to the AMG and Hipp. Accordingly, drug craving and compulsive drug seeking are controlled by the mesocortical pathway, while the mesolimbic pathway has been linked to the acute reinforcing effects and relapse to drugs of abuse (Feltenstein and See,

2008). Glutamate has a pivotal role in drug addiction by modulating the activity of DA neurons both directly and indirectly. Studies have shown that both cell bodies of DA neurons within VTA and their synaptic terminals within the NAc receive glutamatergic input from the PFC, AMG and Hipp (Kelley et al., 1982, Christie et al., 1987,

Groenewegen et al., 1987, Gorelova and Yang, 1997).

1.3.1 Nucleus Accumbens (NAc)

It is widely accepted that the NAc plays a central role in reward. The NAc is localized within the ventral striatum and it consists of two main parts, including the core and shell (Quintero, 2013, Gipson et al., 2014) . Glutamatergic neurotransmission within the NAc is directly linked to drug seeking behavior. Alternatively, both the behavioral effects of acutely administered alcohol and the neuroadaptations associated with chronic ethanol intake have been linked to glutamate excitatory neurotransmission (Eckardt et al.,

1998, Krystal et al., 2003, Sari et al., 2011b). More recently, it has become evident that changes in glutamate transmission within the NAc are responsible for the switch from intermittent use of drug to dependence (Gipson et al., 2014). It is important to mention that glutamatergic input into NAc from other brain regions has a major role in regulating addictive behavior (Gass and Olive, 2008). Importantly, it is well documented that NAc

6 receives glutamatergic afferents from PFC, the basolateral amygdala (BLA), and the ventral hippocampus (vHipp) (Gipson et al., 2014).

1.3.2 Prefrontal Cortex (PFC)

PFC is responsible for a wide range of functions like regulating goal-directed behaviors, executing behavioral responses, emotion and cognition (Goldstein and

Volkow, 2011, Gipson et al., 2014). Studies have shown that drugs addiction associated with dysfunction of PFC and then losing the control over drug taking. Additionally, neuroimaging studies have shown that various PFC regions, namely the orbitofrontal cortex, anterior cingulate cortex and dorsolateral prefrontal cortex, are involved in drugs addiction (Goldstein and Volkow, 2011). Activating the glutamatergic projections from the PFC to NAc, during cocaine or heroin reinstatement, has been shown to result in elevated extracellular levels of glutamate and this increase in glutamate can be prevented by inhibiting these projections (McFarland et al., 2003, LaLumiere and Kalivas, 2008). It is of interest to know that AMG and Hipp send glutamatergic fibers into the PFC (Gass and Olive, 2008).

1.3.3 Amygdala (AMG)

AMG has been extensively examined for its role in addiction (Di Ciano and

Everitt, 2004), anxiety (projections from basolateral AMG into the Hipp also involved in regulating anxiety), memory (particularly aversive learning), and emotional behavior

(Lalumiere, 2014). Recently, it has been found that glutamatergic input from PFC into

NAc releases less glutamate as compared to projections from basolateral AMG to NAc.

Also, it has been shown in the same study that activating glutamatergic projections from basolateral AMG into the NAc core and shell was sufficient and necessary to induce a motivated behavioral response (Stuber et al., 2011, Britt et al., 2012). Both sensitization and drug seeking behaviors require glutamate release into the NAc and the source of this glutamate is from the AMG and PFC (Kalivas et al., 2009).

1.3.4 Hippocampus (Hipp)

More attention has been paid recently to the Hipp because it has an important role in reward learning and drug-context memory (Fuchs et al., 2005, Adcock et al., 2006,

Hernandez-Rabaza et al., 2008, Delgado and Dickerson, 2012). Studies have shown that

Hipp sends glutamatergic fibers into the NAc and that activating or inhibiting these projections induces addiction like behavior or inhibits drug –induced locomotion, respectively (Gipson et al., 2014). Furthermore, there are glutamatergic connections between Hipp, PFC and AMG (Russo and Nestler, 2013).

In summary, the NAc serves as a gateway to integrate and process the information from other brain reward regions before sending them to the motor system as a specific behavioral response (figure 1.1).

Figure 1.1 Neurocircuitry involved in drugs addiction, including alcohol

1.4 Glutamate transporters and Alcoholism

1.4.1 GLT1 and Alcoholism

Studies have shown that GLT1 downregulation resulted in reduced glutamate uptake and elevated extracellular glutamate levels in different brain regions in certain diseases such as ischemia and Alzheimer (Li et al., 1997, Martin et al., 1997).

Alternatively, upregulating GLT1 has been shown to result in enhanced glutamate uptake and consequently reduced extracellular levels of glutamate (Castaldo et al., 2007).

Reduced expression of GLT1 have been found following nicotine self-administration

(Knackstedt et al., 2009), cocaine self-administration (Knackstedt et al., 2010) and in

9 alcoholic persons (Kryger and Wilce, 2010). Additionally, recent studies from our lab showed downregulation of GLT1 in rats’ brain following chronic exposure to ethanol

(Sari and Sreemantula, 2012). However, ceftriaxone, an FDA-approved antibiotic known to upregulate GLT1 (Rothstein et al., 2005), treatment was found to be associated with reduction in cocaine reinstatement (Sari et al., 2009, Knackstedt et al., 2010), and ethanol consumption in male P rats exposed to five weeks continuous free-choice ethanol drinking paradigm (Sari et al., 2011b). Similarly, studies from our lab also found reduction in ethanol consumption after treating P rats with the GLT1 upregulator GPI-

1046 (Sari and Sreemantula, 2012).

1.4.2 Cystine-glutamate Antiporter and Alcoholism

xCT is a cystine-glutamate antiporter belongs to the family of heteromeric amino acid transporters (HATs) (Lo et al., 2008). Functionally, xCT is expressed as a heterodimer consisting of a heavy chain called 4F2hc linked to a light chain (catalytic subunit) via a disulphide bond (Lo et al., 2008, Bridges et al., 2012b). The transport activity of cystine-glutamate antiporter is attributed to its catalytic subunit, while the heavy chain serves in the trafficking of the light chain (Bridges et al., 2012b). xCT was first described in human fibroblasts as a sodium-independent transporter for cystine and glutamate (Bannai and Kitamura, 1980). Previous studies have shown that xCT is localized also on astrocytes within the CNS (Cho and Bannai, 1990, Gochenauer and

Robinson, 2001). Studies have shown that xCT acts in an electroneutral, chloride- dependent manner and it has a role as an antiporter mediating the exchange of

10 intracellular glutamate with extracellular cystine in 1:1 stoichiometry (McBean, 2002,

Bridges et al., 2012b). xCT is responsible for transporting cystine into astrocytes and this cystine is reduced intracellularly into cysteine which is then released into the extracellular space as either glutathione (GSH) or cysteine itself. Cysteine, by being a source for neuronal synthesis of GSH because mature neurons have a little or no xCT activity, and

GSH are both important for protection against oxidative damage within the CNS (Bridges et al., 2012b).

xCT has been extensively studied in animal models, as it is considered as a major source of extrasynaptic, non-vesicular release of glutamate. The released glutamate through xCT activates group II metabotropic glutamate receptors (mGluR2/3) resulting in a decrease of synaptic glutamate release (Moussawi and Kalivas, 2010). Studies have shown that xCT is downregulated in central brain reward regions following nicotine self- administration (Knackstedt et al., 2009) and cocaine self-administration (Knackstedt et al., 2010). Additionally, recent study from our lab showed that xCT is downregulated in different reward regions following continuous five weeks exposure to ethanol but not after relapse-like ethanol drinking paradigm (Alhaddad et al., 2014).

1.5 Glutamate Receptors and Alcoholism

1.5.1 Ionotropic Glutamate Receptors

1.5.1.1 NMDA Receptors and Alcoholism

NMDA receptors are heterodimeric complexes consisting of NR1 subunit linked to at least one NR2 (A, B, C, D) and may be NR3 (A, B) subunits (Kaniakova et al.,

2012). It has been thought for a long time that ethanol mediates its action specifically through potentiating GABAergic neurotransmission, while recent studies showed that ethanol also acts by inhibiting NMDA receptors after acute and chronic exposure

(Moykkynen and Korpi, 2012). Studies have shown that NMDA receptors containing

NR2B are more sensitive to the inhibitory effect of ethanol as compared to NR2C or NR3 containing receptors (Gass and Olive, 2008). Ethanol inhibits NMDA receptors in various brain reward regions, including NAc (Nie et al., 1994), cortex (Randoll et al., 1996),

AMG (Roberto et al., 2004) and Hipp (Simson et al., 1993). NMDA receptors antagonists augment the effect of ethanol and produce ethanol-like stimulus effects in humans and experimental animals (Sanger, 1993, Schechter et al., 1993, Kotlinska and Liljequist,

1997, Krystal et al., 1998, Shen and Phillips, 1998). Chronic ethanol exposure associated with adaptive upregulation of NR1, NR2A and NR2B subunits of NMDA receptors in

Hipp and cerebral cortex (Trevisan et al., 1994, Hu et al., 1996, Devaud and Morrow,

1999, Henniger et al., 2003, Nelson et al., 2005, Sircar and Sircar, 2006). Chronic ethanol exposure also associated with increased NMDA receptors functionality, i.e. conductance.

Acute withdrawal from ethanol has been reported to result in seizure-like effect which can be prevented by NMDA receptor antagonists (Gass and Olive, 2008).

1.5.1.2 Non-NMDA Receptors and Alcoholism

Non-NMDA receptors include AMPA and KA receptors. AMPA receptors are tetrameric receptors with four different subunits (GluR1-4, recently termed as GluA1-4)

(Moykkynen and Korpi, 2012). AMPA receptors localized post-synaptically and widely distributed throughout the brain (Ozawa et al., 1998). AMPA receptors are also inhibited by ethanol but not as sensitive as NMDA receptors (Gass and Olive, 2008). Ethanol- mediated inhibition of AMPA receptors has been found in rat cortical neurons (Wirkner et al., 2000), and Hipp (Martin et al., 1995). Chronic exposure to ethanol reported to be associated with upregulation of several subunits of AMPA receptors, including GluR1 subunit within the VTA (Ortiz et al., 1995), and GluR2-3 subunits in the Hipp and cortical neurons (Bruckner et al., 1997, Chandler et al., 1999).

KA receptors are tetrameric receptors with five different subunits (GluR5, GluR6,

GluR7, KA1, and KA2, recently named as GluK1-5). GluR5-GluR7 represented the low- affinity, while the KA1 and KA2 represented the high-affinity binding sites for KA receptors (Ozawa et al., 1998, Moykkynen and Korpi, 2012). Studies have shown that physiological function of KA receptors in CA3 area of Hipp is the regulation of neurotransmitter release (autoreceptors located pre-synaptically) and synaptic plasticity

(Moykkynen and Korpi, 2012). Recent studies showed that KA receptors are dual function receptors and they can work as ligand-gated or metabotropic receptors and they are located both pre- and post-synaptically (Lerma and Marques, 2013). KA receptors are also inhibited by ethanol, though less sensitive compared to NMDA receptors, in AMG,

Hipp and NAc (Nie et al., 1994, Weiner et al., 1999, Carta et al., 2003, Lack et al., 2008).

The inhibitory effect of ethanol on KA receptors in AMG was linked to the short term anxiolytic effect of ethanol (Lack et al., 2008). Both non-NMDA receptors have been reported to be involved in the rewarding actions of ethanol within the AMG (Zhu et al.,

2007).

1.5.2 Metabotropic Receptors and Alcoholism

Eight metabotropic receptors have been identified and subdivided based on their amino acids homology into three major groups: Group I includes mGlu1 and mGlu5 receptors, group II includes mGlu2 and mGlu3 receptors, and group III includes mGlu4, mGlu6, mGlu7, and mGlu8 receptors. The metabotropic receptors are involved in regulating neuronal excitability, synaptic plasticity and presynaptic regulation of glutamate release (Ozawa et al., 1998).

1.5.2.1 Group I Metabotropic receptors

This group includes mGluR1 and mGluR5, which are located pre and post- synaptically (mostly post-synaptically). Group I metabotropic receptors are located on both neurons and astrocytes. mGlu1 receptors are ubiquitously distributed with different expression levels throughout different brain regions, including Hipp (except CA1 part),

AMG, NAc, cerebral cortex and striatum (Olive, 2009). Besheer et al have used mGluR1 antagonist in P rats and found that antagonizing mGluR1 was associated with reduction in ethanol self-administration and reinforcing effect of ethanol but there was also

14 significant impairment in motor activity and sucrose self-administration and they suggested that mGluR1 do not pay an important role in regulating ethanol self- administration (Besheer et al., 2008). Another mGluR1 antagonist, CPCCOEt, reported to reduce the glutamate and dopamine release induced by acute exposure to ethanol as well as ethanol self-administration (Lominac et al., 2006). Alternatively, another study using

CPCCOEt did not show the same effect (Hodge et al., 2006).

mGlu5 receptors are highly expressed in forebrain and limbic areas, including the

NAc, dorsal striatum, Hipp, cerebral cortex, and AMG (Olive, 2009). Mice lacking mGluR5 consumed less amount of ethanol and showed reduced ethanol preference and were more sensitive to ethanol (Bird et al., 2008). Antagonizing mGluR5 by MPEP has been found to reduce ethanol consumption, reinforcement and relapse to drinking in different animal models (Backstrom et al., 2004, McMillen et al., 2005, Olive et al.,

2005, Hodge et al., 2006, Lominac et al., 2006). Ethanol-stimulated releases of glutamate and dopamine in the NAc and the conditioned place preference induced by ethanol have been reduced after using MPEP (Lominac et al., 2006). Ferraro et al have found that allosteric modulation of mGluR5 by GET73 was associated with reduction in ethanol consumption and an anxiolytic effect (Ferraro et al., 2013). All the previously mentioned evidences indicate the importance of mGluR5 in alcohol abuse.

1.5.2.2 Group II Metabotropic receptors

This group includes mGluR2 and mGluR3, which are localized pre- (act as inhibitory autoreceptors) and post-synaptically. Studies have found that this group of metabotropic receptors distributed with different levels within the cerebral cortex, NAc,

AMG, and Hipp (Olive, 2009). Zhao et al have found that activating group II metabotropic receptors within the AMG and Hipp resulted in attenuating ethanol seeking behavior induced by stress and drug cues (Zhao et al., 2006). The mGlu2/3 receptor agonist (LY379268) was shown to be effective in reducing ethanol self-administration and reinstatement but that was associated with reducing locomotor activity as well

(Backstrom and Hyytia, 2005). However, it was found in preclinical studies that with chronic use, tolerance will develop to the effect of LY379268 on locomotor activity but not to its effect on mGlu2/3 receptors (Imre, 2007). This suggests that activating mGluR2/3 receptors seems to be promising for treating alcohol dependence.

1.5.2.3 Group III Metabotropic receptors

This group includes pre-synaptically located mGluR4, mGluR6, mGluR7 and mGluR8 (Kenny and Markou, 2004). Knockout of mGluR4 in mice was linked to the loss of ethanol stimulatory effect on motor activity without affecting ethanol consumption or preference, as compared to the wild type mice, which led to the suggestion that mGluR4 has no effect on ethanol consumption (Blednov et al., 2004). mGluR6 does not seem to play a role in alcohol addiction because it is localized only in retina (Nakajima et al.,

1993). Bahi et al have found that knockdown of mGluR7 within the NAc of rats was associated with increased ethanol consumption and preference (Bahi, 2013).

Alternatively, activating mGluR7 was linked to reduction in ethanol intake without affecting fluid intake or locomotor activity; and it was suggested that targeting mGluR7 is promising as a target for treating alcohol abuse (Bahi et al., 2012). Furthermore,

Backstrom et al have found that activating mGluR8 decreased ethanol consumption at doses that were associated with reduction in locomotor activity (Backstrom and Hyytia,

2005).

1.6 Other Neurotransmitter Systems and alcoholism

Rewarding effects of ethanol has been linked to other neurotransmitters systems, including GABAergic system, opioid system, and serotonergic system along with dopaminergic and glutamatergic systems (Koob et al., 1998).

GABA (γ-aminobutyric acid) is the main inhibitory transmitter within the CNS and functions as a regulator of neuronal excitability. There are two classes of GABA receptors, including GABAA (ligand-gated ion channels) and GABAB (G-protein coupled) receptors. Activity of GABAA receptors increases following acute exposure to alcohol while chronic alcohol abuse has opposite effects (Mhatre et al., 1993). Studies have shown that ethanol increases GABAergic activity either pre-synaptically by increasing

GABA release or post-synaptically by enhancing GABAA receptors activity. Blocking

GABAA receptors within the NAc and AMG has been shown to attenuate ethanol consumption (Gilpin and Koob, 2008).

Endogenous opioids have been linked to the rewarding effects of ethanol. Three classes of endogenous opioids have been identified, including endorphins, enkephalins and dynorphins and exert their effects by interacting with three types of opioid receptors termed µ, κ and δ (Gilpin and Koob, 2008). Studies have shown that alcohol rewarding effects mediated in part by releasing endogenous opioids within the brain (Ulm et al.,

1995). On the other hand, the opioid receptor antagonist, naltrexone, works by preventing the binding of endorphins to its receptors, and it is effective in attenuating heavy alcohol drinking in human [for review see ref. (Gilpin and Koob, 2008)].

It is widely accepted that serotonergic system is involved in alcoholism. Serotonin depletion has been linked to impulsivity and drinking behavior of alcohol in animals and human [for review see ref. (Gilpin and Koob, 2008)]. Inhibition of serotonin reuptake has been shown to be effective in attenuating the voluntary consumption of alcohol in animals (Johnson, 2008). Selective serotonin reuptake inhibitors are effective in attenuating alcohol consumption, depression and anxiety after alcohol withdrawal in some alcoholic people [for review see ref. (Sari et al., 2011a)].

1.7 Aims and Objectives

MS-153, a novel neuroprotective compound, has been shown to enhance glutamate uptake (Shimada et al., 1999). Recent studies from our lab demonstrated the effectiveness of treating P rats, after exposing them to five weeks free-choice ethanol drinking paradigm, with ceftriaxone in attenuating ethanol consumption. The reduction in ethanol intake was found to be associated with upregulation of GLT1 levels in central brain reward regions including NAc and PFC (Sari et al., 2011b). In this study, we investigated the effect of MS-153 treatment on ethanol consumption, body weight and water intake. We then examined the long lasting effect (ten days after the last dose) of

MS-153 on GLT1 levels (using western blot analysis) in NAc and PFC. Additionally, we studied the effect of MS-153 (24 hours after the last dose) on xCT and phosphorylated

AKT (pAkt) levels in AMG and Hipp. Furthermore, we investigated the behavioral effects after treatment with a combination of MS-153 and augmentin (β-lactam antibiotic) on ethanol consumption, body weight and water intake in P rats.

Chapter 2

Materials and Methods

2.1 MS-153

(R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) (fig. 2.1) has been synthesized in Dr. James Leighton’s lab, Columbia University, Department of Chemistry,

New York, NY. The compound was dissolved in vehicle (1%DMSO in phosphate buffer saline (PBS)) and then injected intraperitoneally (i.p.) to the animals according to their body weight at a dose of 50 mg/kilogram.

Figure 2.1 Chemical structure of MS-153

2.2 Augmentin

Augmentin (Fig. 2.2) is a fixed dose combination of amoxicillin and clavulanic acid. Augmentin was purchased from (GSK, France) and reconstituted with 0.9% saline and then i.p. injected into the rats at a dose of 100 mg/kg.

Figure 2.2 Chemical structure of amoxicillin and clavulanic acid

2.3 Animals

Male Alcohol-preferring (P) rats were used in this study to test the effect of MS-

153 on ethanol drinking. P rats were received from Indiana University School of

Medicine (Indianapolis, USA) at the age of 21-30 Days. We used this strain of rats because it is an established model for alcoholism (Sari et al., 2006). In the Department of

Laboratory Animal Resources (The University of Toledo, Health Science Campus), rats were individually housed at age of 90 days in bedded plastic tubs. All animals had ad lib

21 access to food and water. P rats were accustomed to a temperature of 250C, 50% humidity, and a 12-hour light-dark cycle. All of these experimental and animal housing procedures were ratified by the Institutional Animal Care and Use committee of The

University of Toledo in consonance with the guidelines of the Institutional Animal Care and Use Committee of the National Institutes of Health and the Guide for the Care and

Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on

Life Sciences, 1996). The Institution is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALACI).

2.4 Behavioral Drinking Paradigm

Male P rats were divided into three experimental groups at the age of 90 days.

The ethanol vehicle (ethanol control) group and the ethanol treatment groups were exposed to uninterrupted free choice access to food, water and two bottles with different concentrations of ethanol (15% and 30% v/v). In addition, ethanol-naïve control (water control) group had access to food and water only. Experimental procedures started on all animals at the same time and for five weeks prior to the start of treatment. Ethanol has been prepared by diluting 190-proof ethyl alcohol (95%) with distilled water to make two concentrations (15% and 30%). 190-Proof ethanol was purchased from PHARMCO-

AAPER (Shelbyville, KY). During the last two weeks, body weight, ethanol intake and water intake were measured three times per week. Both ethanol and water intake were measured by subtracting the bottle weight from its initial weight containing ethanol or water, respectively. Both ethanol and water had been changed three times per week.

Using densitometry formula, ethanol consumption and water intake measurements were converted into either grams of ethanol or milliliter of water consumed per kilogram of animal body weight per day. The average measurements of ethanol consumption, water intake and body weight during the last two weeks were used as a baseline. Animals drank less than 4 g of ethanol/kg/day were excluded as previously performed (Sari et al.,

2011b). During week 6, ethanol-naïve vehicle and ethanol vehicle groups were i.p. injected with the vehicle. However, ethanol treatment groups received intraperitoneal

(i.p) injections of the specified treatment as explained later. All animals were i.p. injected for five consecutive days around 11:00 A.M. Ethanol intake, water intake and body weight were also measured every day during treatment and post treatment days.

2.4.1 Effect of MS-153 on Chronic Ethanol Drinking Paradigm

To examine the long lasting effect (ten-days after the last dose) of MS-153 on ethanol drinking, we have divided P rats into three groups: (1) Ethanol-naïve vehicle group (naïve) had free access to food and water only and received i.p. injections of vehicle solution (1% DMSO in PBS) (n=5); (2) Ethanol vehicle group (vehicle) received i.p. injections of the same vehicle solution (n=9); and (3) Ethanol MS-153 group received i.p. injections of MS-153 (50 mg/kg body weight) (n=8). Similarly, all animals were i.p. injected daily for five consecutive days on the first day of week six and then euthanized and decapitated ten days after the last i.p. injection.

2.4.2 Effect of MS-153 on xCT and pAKt Levels in Amygdala and

Hippocampus

To test the effect (one-day post treatment) of MS-153 on ethanol consumption, P rats were divided into three experimental groups: (1) Ethanol-naïve vehicle group (naïve), which is used as a water control group, had free access to food and water only and received i.p. injections of vehicle solution (1% DMSO in PBS) (n=7); (2) Ethanol vehicle group (vehicle), which is considered as ethanol control group, received i.p. injections of the same vehicle solution (n=7); and (3) Ethanol MS-153 group received i.p. injections of

MS-153 (50 mg/kg body weight) (n=5). All animals were injected daily on the first day of the sixth week with either vehicle solution or MS-153, as described above, for five consecutive days. All animals were euthanized with CO2 and then decapitated 24 hours after the last injection.

2.4.3 Effect of Combination of MS-153 and Augmentin on Chronic

Ethanol Drinking Paradigm

We have tested the effects of MS-153 alone, augmentin alone and in combination on ethanol consumption. Four experimental groups have been tested as follows: (1)

Ethanol vehicle group (vehicle) received i.p. injections of saline and 1% DMSO in PBS

(n=5); (2) Ethanol augmentin group received i.p. injections of augmentin alone (100 mg/kg body weight) (n=5); (3) Ethanol MS-153 group received i.p. injections of MS-153

(50 mg/kg) (n=5); (4) Ethanol Augmentin+MS-153 group received i.p. injections of

24 augmentin (100 mg/kg) and MS-153 (50 mg/kg) daily during treatment days (n=5).

Animals treated every day for five consecutive days and then euthanized and decapitated

24 hours after the last i.p. injections.

2.5 Brain Tissue Harvesting

After animals were decapitated using guillotine, brains were immediately kept on dry ice and then stored at -700C. Brain regions were then stereotaxically microdissected, in accordance with Paxinos and Watson (2007) atlas for the rat brain, in the cryostat machine, which was maintained at -200C to keep the brain tissues frozen. Extracted brain regions (AMG, Hipp, NAc and PFC) were then kept at –700 C for western blot analysis to examine protein expression levels.

2.6 Protein Tissue Extraction Protocol

2.6.1 Tissue lysate

The brain regions samples were homogenized using regular filtered lysis buffer

(2.5mL 1M Tris HCL, 2.5mL 3 M NaCl, 0.1mL 0.5M EDTA, 2.5mL 10% NP-40, 5mL

10% Triton, 0.5mL 10% SDS (sodium dodecyl sulfate), 5 mL of protease inhibitor solution, and 31.9 mL Millipore water) with 0.5 ml of phosphatase inhibitor. We further added 180-250 μL of lysis buffer to each sample in eppendorf tubes (1.5 ml size) and the tissue samples were then homogenized using pestles. The tissue samples were then kept on ice for half an hour with vortexing each 10 minutes. The samples were then

25 centrifuged (Centrifuge 5415R, Eppendorf Inc.) for 15 minutes at 13,200 RPM and 4ºC, and the supernatant was collected and instantly stored at -70°C for western blot .

2.7 Protein Quantification Assay

The Lowry protein quantification assay has been performed in 96 well plates on one aliquot of each sample to determine the amount of protein present in the samples.

Four wells had been used for each sample. Bovine serum albumin (BSA) (New England

Bio labs) was used for the standard curve and regression line equation by plotting the optical density against the diluted concentrations of BSA (1.48mg/ml). The proteins samples were assayed by adding 1μL of each sample into 4 μL of lysis buffer (four wells for each sample). Reagents were purchased from BioRad Laboratories (Reagents A, B, and S). A mixture of 3 ml of reagent A and 60 μL of reagent S was prepared and 25μL of this mixture was added to all of the BSA’s and the samples’ wells. Furthermore, a 200μL of reagent B was added to each well and the 96-well plate was maintained in a dark place for 15 minutes at room temperature. Multiskan FC spectrophotometer (Thermo

Scientific) was used to measure the absorbance at a wavelength of 750 nm. For all samples, the average of the blank optical density values was subtracted from the average of the samples’ optical density values. Finally, the samples’ optical density and the standard curve’s line equation were used to determine the protein concentration for each sample in order to use equal amounts of protein from all samples for western blot.

2.8 Western Blot Procedures

2.8.1 Gels preparation

Two different gels had been used for western blot. The first one is 10-20% Tris- glycine gel (Invitrogen). The other one was prepared as 10 % Tris-glycin gel. After assembling the minigel apparatus (BioRad), 10 % resolving or separating gel was prepared by mixing the following reagents in specified ratios according to the number of gels to be prepared: Deionized water, 30 % Acrylamide/Bis solution (BioRad), 1.5 M

Tris Buffer pH 8.8, 10 % SDS, 10% Ammonium Persulfate (APS, Fisher Scientific), and

TEMED (N,N,N’,N’-tetramethylethylene-diamine, BioRad). The resulting mixture was immediately transferred into the minigel apparatus until it solidified. After about 30 minutes, the 6 % stacking gel was prepared by mixing deionized water, 30%

Acrylamide/Bis solution, 0.5 M Tris Buffer pH 6.8, 10 % SDS, 10% APS, and TEMED and then immediately transferred into the minigel apparatus over the separating gel. After about an hour, the gels were used for western blot.

2.8.2 Samples Preparation

Equal amounts of protein from each sample (according to their protein quantification values) were diluted with lysis buffer. The diluted samples were then mixed with 5 μL of 5X Laemmli dye (1M Tris HCL, 100 % Glycerol, SDS, b- mercaptoethanol, bromophenol blue) and vortexed thoroughly. All tubes containing the final samples mixtures were heated at 98°C for 5 minutes in a digital dry bath (Labnet

International Inc.). Finally, the heated samples mixtures were centrifuged at 13,200 RPM and 4°C for 3 minutes.

2.8.3 Running and Transfer of Proteins

After preparing the samples, 20 μL of each sample mixture was loaded into a well of the gels. The gels were first placed in the running apparatus (XCell SureLock™ Mini-

Cell, Invitrogen) and immersed with 1X Laemmli buffer (prepared by diluting 10X

Laemmli buffer). The 10X Laemmli buffer consists of 30.2 Tris Base (SIGMA), 144 g

Glycine (SIGMA), 10 g SDS, and distilled water up to 1 L. The proteins were then separated by electrophoresis (100 volts for 90 minutes or 200 volts for 1 hour). After running is done, the protein-containing gels were detached from the gels’ cassettes and transferred onto an Immuno-Blot® PVDF membrane (Polyvinylidene fluoride, BioRad) or an immobilon-P membrane (Millipore, Fisher Scientific, Inc.). The transfer of proteins has been done in a transfer apparatus (Idea Scientific Company, MN) after filling it with the transfer buffer (28.8 g Glycine and 5.9 g Tris Base dissolved in 3.2 L distilled water, after that 800 mL methanol was added) for 3 hours at 24 volts. After the transfer is complete, the membranes were placed in square petri dishes and washed twice (one minute each) with deionized water.

2.8.4 Blocking

The membranes were then blocked in blocking buffer at room temperature for

0.5-1 hour depending on the antibody to be used later. The blocking buffer was prepared

28 by dissolving the desired amount of Non-Fat dry milk (Labscientific) in TBST (50 mM

Tris HCL, 150 mM NaCL (pH adjusted to 7.4), 0.1 % Tween20).

2.8.5 Incubations with Primary Antibodies

The membranes were further incubated with one of the following antibodies:

Rabbit anti-xCT antibody (Novus; 1:1000 dilution), guinea pig anti-GLT1 (Millipore;

1:5000 dilution), rabbit anti-pAKT (1:5000; Cell Signaling Technology), or mouse anti-

Akt (1:5000; Cell Signaling Technology). Similarly, mouse anti β-tubulin was also used to detect β-tubulin (as a loading control). The membranes were then incubated overnight on the shaker at 40C.

2.8.6 Incubations with Secondary Antibodies

On the following day, the blocking buffer containing the primary antibodies was removed and the membranes were washed with TBST 5 times (5 minutes each). The membranes were then blocked for 30 minutes before adding either Horseradish peroxidase (HRP) labeled anti-guinea pig (Jackson ImmunoResearch Laboratories, Inc.) or anti-rabbit (thermo scientific) secondary antibodies at 1:5000 dilutions. Regarding β- tubulin and total Akt, HRP labeled anti-mouse was used (1:5000; Cell signaling technology). Incubation with secondary antibodies was done for 90 minutes at room temperature. After that, the membranes were washed 5 times with TBST (5 minutes each) before developing.

2.8.7 Developing Membranes

After washing the membrane, chemiluminescent substrate has been used to detect the proteins (SuperSignal® West Pico). Kodak BioMax MR Films (Thermo Fisher

Scientific) have been used to capture the signals from HRP. SRX-101A machine was used to develop the films. B-tubulin has been used as a loading control. Subsequently, the bands on the films were digitized and analyzed using the MCID system. Finally, the data were reported as GLT1/β-tubulin, xCT/β-tubulin and pAkt/Total Akt ratios.

2.9 Statistical Analyses

2.9.1 Behavioral Data of MS-153 study

General Linear Model (GLM) repeated measures analyses were used to analyze the behavioral data related to ethanol consumption, water intake and body weight followed by independent t-test (SPSS) to determine the daily effect of treatment. All statistical results were based on p<0.05 level of significance.

2.9.2 Behavioral Data of Augmentin and MS-153 combination study

Two-way repeated measures ANOVA followed by Bonferroni’s multiple comparisons test as a post-hoc test to observe the daily effects of each treatment on ethanol drinking, water intake, and body weight and also to compare different treatments.

All statistical results were based on p<0.05 level of significance.

2.9.3 Western Blot Data

One-way ANOVA was used to analyze western blot data (GLT1/ β-tubulin, pAkt/Total Akt and xCT/ β-tubulin ratio) followed by Newman-Keuls multiple comparisons post-hoc test for comparing the protein expression levels of ethanol-naïve, ethanol vehicle and ethanol MS-153 treated groups. All statistical data were based on p<0.05 level of significance.

Chapter 3

Results

3.1 Effect of MS-153 on Chronic Ethanol Drinking Paradigm

3.1.1 Effect of MS-153 Treatment on Ethanol Consumption in Male P

Rats

We examined the effect of MS-153 (50 mg/kg daily for five consecutive days) on ethanol consumption over a period of 14 days starting 24 hours after the first injection with either vehicle solution (i.p.) or MS-153 (50 mg/kg, i.p.). The average daily ethanol consumption (g/kg/day) was measured daily starting 1 day after the first injection up to

10 days after the last injection. The average daily ethanol intake of P rats during the treatment and post treatment periods was compared to the baseline ethanol intake. The baseline ethanol consumption values were considered as the average ethanol consumed over the period of two weeks preceding the treatment.

General Linear Model (GLM) repeated measures of ethanol consumption data revealed a significant main effect of day [F (1,14)=19.159, p<0.0001) and significant Day

32 x Treatment interaction effect [F(1,14)=3.03, p<0.0001]. Regarding the daily effect of

MS-153 treatment, independent t-test revealed a statistically significant reduction, as early as 24 hours after the first injection, in ethanol consumption of MS-153-treated animals compared to ethanol vehicle-treated animals (p<0.05) (Fig. 3-1).

Vehicle

MS-153 50 mg/kg

6 * ** ** * ** ** * **

4 * (g/kg/day) ** ** ** **

2 ** Average Ethanol Intake Ethanol Average

DAY1 DAY2 DAY3 DAY4 DAY5 DAY6 DAY7 DAY8 DAY9 DAY10 DAY11 DAY12 DAY13 DAY14 Baseline Treatment period Post treatment period

Figure 3-1 Effects of MS-153 treatment on average daily ethanol intake (g/kg/day) in male P rats exposed to five weeks of continuous free choice of ethanol and water. Statistical analyses demonstrated a significant difference between MS-153 treated group (n=8) and ethanol vehicle group (n=9). Additionally, independent t-test revealed a significant decrease in ethanol intake with MS-153 (50 mg/kg, i.p.) treated group from Day 1 (24 hrs after the first i.p. injection) through Day 14 (10 days after the last injection) as compared to ethanol vehicle group. Data are shown as mean ± SEM. (* p<0.05; ** p<0.01)

3.1.2 Effect of MS-153 Treatment on Water Intake in Male P Rats

We also monitored the effect of MS-153 treatment (50 mg/kg daily for five consecutive days) on daily water intake during treatment and post treatment periods (for

14 days starting 24 hours after the first injection). The average water intake of male P rats was measured daily starting 24 hours after the first injection with either MS-153 (50 mg/kg/day for five consecutive days) or vehicle solution (regarding ethanol vehicle animals) and then compared to the baseline value. The baseline values were measured as the average daily water intake during the two weeks period preceding the treatment.

Statistical analysis using GLM repeated measures of water intake data revealed a significant main effect of day [F(1,14)=4.052, p<0.0001) and significant Day x Treatment interaction effect [F(1,14)=2.225, p<0.01]. Furthermore, independent t-test showed that water intake of MS-153 treated animals was significantly higher than ethanol vehicle treated animals starting on Day 3 through day 8 (p<0.05) (Fig. 3-2).

Vehicle MS-153 50 mg/kg 50

** * 40 * ** * * 30

(ml/kg/day) Average WaterAverage Intake 10

DAY1 DAY2 DAY3 DAY4 DAY5 DAY6 DAY7 DAY8 DAY9 DAY10 DAY11 DAY12 DAY13 DAY14 Baseline Treatment period Post treatment period Figure 3-2 Effects of MS-153 treatment on average daily water intake (ml/kg/day) in male P rats exposed to five weeks of continuous free choice of ethanol and water Statistical analysis revealed a significant difference between treatment and control animals. Further, Independent sample t-test showed a significantly increased water consumption in MS-153 treated P rats (50mg/k, i.p.; n=8) as compared to ethanol vehicle treated P rats (n=9), starting from day 3 through day 8. Data are presented as mean ± SEM. (* p<0.05; ** p<0.01).

3.1.3 Effect of MS-153 Treatment on Body Weight in Male P Rats

Figure 3-3 represents the effect of MS-153 treatment on P rats’ body weight during the treatment and post treatment periods. We monitored the body weight of P rats, after treating them with either MS-153 (50 mg/kg, i.p.) or vehicle (i.p.) for five days.

Body weight measurements during the treatment and post treatment days were compared to the baseline body weight values. Baseline values were calculated as the average body weight measurements during the last two weeks before starting the treatment.

GLM repeated measures of body weight data revealed a significant main effect of day [F(1,14)=5.968, p<0.0001] and a significant Day x Treatment interaction effect

[F(1,14)=20.172, p<0.0001]. However, Independent t-test did not show any significant difference in body weight between the MS-153-treated animals and ethanol vehicle animals through the entire period of the study (p>0.05).

Vehicle

MS-153 50 mg/kg 550 500 450 400 350 300

250 (grams) 200 150 Average Body Weight Body Average 100 50 0

DAY1 DAY2 DAY3 DAY4 DAY5 DAY6 DAY7 DAY8 DAY9 DAY10 DAY11 DAY12 DAY13 DAY14 Baseline Treatment period Post treatment period

Figure 3-3 Effects of MS-153 treatment on body weight (grams) of male P rats exposed to five weeks of continuous free choice access to ethanol and water. Statistical analysis of animals’ body weight data revealed no significant difference in body weight between the ethanol MS-153 treated group (50 mg/kg; n=8) and the ethanol vehicle group (n=9) during the entire period of the study (treatment and post treatment periods). Data are shown as mean ± SEM.

3.1.4 Long Lasting Effect of MS-153 on GLT1 Expression in NAc

The post treatment effect of MS-153 (10 days after the last i.p. injection) on

GLT1 levels in the NAc was examined using western blot. Statistical analysis of western blot data related to GLT1, using one-way ANOVA, revealed a significant difference amongst MS-153 treated, ethanol vehicle and ethanol-naïve groups [F(2,12)=6.920, p=0.010]. Furthermore, a Newman-Keuls , multiple comparisons, post-hoc test showed significant upregulation of GLT1 levels following MS-153 treatment as compared to ethanol vehicle animals (p<0.05). Alternatively, statistical analysis also demonstrated a significant downregulation of GLT1 level in ethanol vehicle group as compared to ethanol-naïve vehicle group (p<0.05). Statistical analysis revealed no significant difference between ethanol-naïve and MS-153-treated groups (Fig. 3-4).

Figure 3-4 Ten-days post treatment effect of MS-153 on GLT1 expression in NAc. Upper panel: Representative western blots of β-tubulin, used as a loading control, and GLT1 in NAc. Lower panel: Quantitative analysis of Western blot data revealed a significant upregulation of GLT1 levels on day 14 (10 days after the last injection) in MS-153 treated animals (50 mg/kg; n=5) as compared to ethanol vehicle animals (n=5). Additionally, statistical analysis exhibited significant decrease of GLT1 levels in ethanol vehicle group compared to ethanol-naïve group (n=5). Data are shown as mean ± SEM. (* p<0.05; ** p<0.01).

3.1.5 Long Lasting Effect of MS-153 on GLT1 Expression in PFC

We further explored the effect of MS-153 on GLT1 levels in PFC on day 14 (10 days after the last injection) (Fig. 3-5). One-way ANOVA analysis of immunoblots followed by Newman-Keuls post-hoc test demonstrated no significant difference amongst

38 the MS-153 treated, ethanol vehicle and ethanol-naïve groups [F(2,12)=0.2935, p=0.7508].

Figure 3-5 Ten-days post treatment effect of MS-153 on GLT1 expression in PFC. Upper panel: Representative western blots of β-tubulin, used as a loading control, and GLT1 in PFC. Lower panel: Quantitative analysis of Western blot data revealed no significant changes on GLT1 levels on day 14 (10 days after the last injection) in MS-153 treated animals (50 mg/kg; n=5) as compared to ethanol vehicle animals (n=5). Furthermore, no significant difference on GLT1 levels was found between ethanol vehicle group and ethanol-naïve group (n=5). Data are expressed as mean ± SEM.

3.2 Effect of MS-153 on xCT and pAKT levels in Amygdala and Hippocampus

3.2.1 Effect of MS-153 on xCT Expression in Amygdala

Western blot was performed to examine the effect (24 hours after the last dose) of

MS-153 (50 mg/kg, i.p. daily for five days) on xCT expression in AMG as compared to vehicle treated group. One-way ANOVA analysis of western blot data demonstrated a significant difference between the ethanol-naïve, ethanol vehicle and ethanol MS-153 treated groups [F (2,12) =9.698, p=0.0031). Additionally, Newman-Keuls multiple- comparisons post-hoc test demonstrated a significant upregulation of xCT expression in

MS-153 treated group as compared to ethanol vehicle-treated group (p<0.01).

Furthermore, Newman-Keuls post-hoc test revealed a significant downregulation of xCT levels in ethanol vehicle animals as compared to ethanol-naïve vehicle animals (p<0.05).

However, statistical analysis did not show any significant difference in xCT expression between ethanol-naïve vehicle and MS-153-treated groups (Fig. 3-6).

Figure 3-6 Effect of MS-153 on xCT expression in amygdala. Upper panel: Representative immunoblots of xCT and β-tubulin, a loading control, in AMG. Lower panel: Quantitative analysis of the immunoblots demonstrated significant upregulation of xCT in MS-153 treated group (50 mg/kg, i.p.; n=5) as compared to ethanol vehicle group (n=5). Alternatively, statistical analysis revealed a significant downregulation of xCT in ethanol vehicle group as compared to ethanol-naïve vehicle group. Data are shown as mean ± SEM. (*p<0.05; **p<0.01).

3.2.2 Effect of MS-153 on pAkt Expression in Amygdala

Recent study from our lab (unpublished findings) showed that GLT1 is significantly upregulated in AMG following treatment with MS-153 (50 mg/kg, i.p. for five consecutive days). Since evidences have shown that Akt signaling pathway is involved in regulating GLT1 expression (Li et al., 2006). We explored the effect (24

41 hours after the last dose) of MS-153 treatment on Akt expression in AMG. Surprisingly,

One-way ANOVA analysis of western blot data did not demonstrate any significant difference between the ethanol-naïve, ethanol vehicle and ethanol MS-153 treated groups

[F(2,12)=1.088, p=0.368) (Fig. 3-7).

Figure 3-7 Effect of MS-153 on pAkt expression in amygdala. Upper panel: Representative immunoblots for pAkt, total Akt and β-tubulin, a loading control, in AMG. Lower panel: Quantitative analysis of the immunoblots did not show any significant difference among the ethanol-naïve (n=5), ethanol vehicle (n=5) and ethanol MS-153 (50mg/kg, i.p.; n=5) treated groups. Data are shown as mean ± SEM.

3.2.3 Effect of MS-153 on xCT Expression in Hippocampus

We further explored the effect of MS-153, one day after the last dose, on xCT level in Hipp using western blot (Fig. 3-8). Statistical analysis of western blot data using one-way ANOVA demonstrated a significant difference amongst the ethanol-naïve, ethanol vehicle and ethanol MS-153 treated groups [F (2,12)=7.167, p=0.009). The

Newman-Keuls , multiple-comparisons, post-hoc test revealed a significant upregulation of xCT expression in MS-153 treated group as compared to ethanol-vehicle treated group

(p<0.01). Additionally, xCT was significantly downregulated in the ethanol vehicle group as compared to ethanol-naïve vehicle group (p<0.05). Statistical analysis did not show significant difference in xCT expression between ethanol-naïve vehicle and MS-153- treated groups.

Figure 3-8 Effect of MS-153 on xCT expression in Hippocampus. Upper panel: Representative immunoblots of xCT and β-tubulin, a loading control, in Hipp. Lower panel: Quantitative analysis of the immunoblots demonstrated significant upregulation of xCT in MS-153 treated group (50 mg/kg, i.p.; n=5) as compared to ethanol vehicle group (n=5). Alternatively, statistical analysis showed a significant downregulation of xCT in ethanol vehicle group as compared to ethanol-naïve vehicle group. Data are shown as mean ± SEM. (*p<0.05; **p<0.01).

3.2.4 Effect of MS-153 on pAkt Expression in Hippocampus

Furthermore, we explored the effect of MS-153 (one day after the last i.p. injection) on pAkt levels in Hipp. Surprisingly, statistical analysis using one-way

ANOVA did not show any significant difference between the ethanol-naïve, ethanol vehicle and ethanol MS-153 treated groups [F(2,12)=3.864, p=0.0506]. (Fig. 3-9)

Figure 3-9 Effect of MS-153 on pAkt expression in hippocampus. Upper panel: Western blots for pAkt, total Akt and β-tubulin, a loading control, in Hipp. Lower panel: Quantitative analysis of the immunoblots did not show any significant difference amongst the ethanol-naïve (n=5), ethanol vehicle (n=5) and ethanol MS- 153(50 mg/kg, i.p.; n=5) treated groups. Data are shown as mean ± SEM.

3.3 Effect of Combination of MS-153 and Augmentin on

Chronic Ethanol Drinking Paradigm

3.3.1 Effect of Combination of MS-153 and Augmentin on Ethanol

Consumption in Male P rats

P rats were given uninterrupted access to 15% and 30% ethanol solutions, food and water for five weeks prior to starting the treatment. The average daily ethanol consumption was measured three times per week during the last two weeks before starting the treatment and considered as a baseline value. On the first day of week six, we treated the animals with augmentin alone (100 mg/g, i.p.; n=5), MS-153 alone (50 mg/kg, i.p.; n=5), or a combination of MS-153 (50 mg/kg, i.p.) and augmentin (100 mg/kg, i.p.)

(n=5). Alternatively, ethanol control animals were injected at the same time with both vehicle solution and saline (i.p.) (n=5).

Statistical analysis using two-way repeated measures ANOVA revealed a significant main effect of day [F (5,80)=45.39, p<0.0001] and a significant effect of treatment [F(3,16)=32.69, p<0.0001] on ethanol intake. Furthermore, Bonferroni’s post- hoc test revealed a significant reduction in ethanol consumption of drug treated animals as compared to ethanol vehicle treated animals starting 24 hours after the start of the treatments through the end of the study (p<0.001). Surprisingly, Bonferroni’s post-hoc test demonstrated no significant differences among ethanol augmentin, ethanol MS-153, and the ethanol Augmentin+MS-153 treated group (Fig. 3-10).

Vehicle Augmentin 8 MS-153 Augmentin+MS-153

4 # # # # (g/kg/day) # # # # # # # 2

# # # # Average Ethanol Intake Ethanol Average

Day1 Day2 Day3 Day4 Day5 Baseline Treatment period

Figure 3-10 Effects of combination of MS-153 and augmentin treatment on average daily ethanol intake (g/kg/day) in male P rats. Two-way ANOVA demonstrated a significant difference in ethanol consumption between drugs treated groups and ethanol vehicle group. Additionally, statistical analysis revealed a significant decrease in ethanol intake with MS-153 (50 mg/kg, i.p.), augmentin (100mg/kg, i.p.), and augmentin-MS-153 combination (augmentin 100mg/kg i.p, and MS-153 50 mg/kg i.p.) treated groups from Day 1 (24 hrs after the first i.p. injection) through the end of the study as compared to ethanol vehicle group. There were no significant differences between all treatment groups. Data are shown as mean ± SEM. (# p<0.001). [Vehicle group, n=5; Augmentin group, n=5; MS-153 group, n=5; Augmentin+MS-153 group, n=5]

3.3.2 Effect of Combination of MS-153 and Augmentin on Water Intake of Male P rats

Two-way repeated measures ANOVA revealed a significant main effect of day

[F(5,80)=9.927, p<0.0001], and a significant effect of treatment [F(3,16)=4.308, p=0.0208]. Regarding the daily effect of each treatment on water intake, Bonferroni’s multiple-comparisons post-hoc test revealed a significant increase (as compared to ethanol vehicle group) in water consumption on days 2 and 4 for augmentin treated rats, on days 4-5 for MS-153 treated rats, and on days 3-4 for augmentin+MS-153 group

(p<0.05) (Fig. 3-11). Additionally, Bonferroni’s post-hoc test revealed no significant differences among ethanol augmentin, ethanol MS-153, and ethanol augmentin+MS-153 treated groups throughout the study.

Vehicle Augmentin MS-153 Augmentin+MS-153

70 # ** 60 ** ** * 50 * 40

(ml/kg/day) 30

20 Average WaterAverage Intake 10

Day1 Day2 Day3 Day4 Day5 Baseline Treatment period

Figure 3-11 Effects of combination of MS-153 and augmentin treatment on average daily water intake (ml/kg/day) in male P rats. Statistical analysis demonstrated a significant difference in daily water intake between drugs treated groups and ethanol vehicle group. Additionally, statistical analysis revealed a significant increase in water intake after treatment with MS-153 (50 mg/kg, i.p.) on days 4-5, augmentin (100mg/kg, i.p.) on days 2 and 4, and augmentin-MS-153 combination (augmentin 100mg/kg i.p, and MS-153 50 mg/kg i.p.) on days 3-4, as compared to ethanol vehicle group. There were no significant differences between all treatment groups. Data are shown as mean ± SEM. (* p<0.05; ** p<0.01, # p<0.001). [Vehicle group, n=5; Augmentin group, n=5; MS-153 group, n=5; Augmentin+MS-153 group, n=5]

3.3.3 Effect of Combination of MS-153 and Augmentin on Male P rats’

Body Weight

Two-way repeated measures ANOVA revealed a significant main effect of day on body weight [F(5,80)=24.78, p<0.0001]. However, two-way ANOVA did not show a significant effect of treatment on body weight [F (3,16)=0.7231, p=0.5528]. Additionally,

Bonferroni’s multiple-comparisons post-hoc test revealed no significant effect on body weight among all treated groups as compared to ethanol vehicle group (Fig. 3-12).

Furthermore, Bonferroni’s post-hoc test revealed no significant differences among ethanol augmentin, ethanol MS-153, and ethanol augmentin+MS-153 treated groups.

Vehicle Augmentin MS-153 Augmentin+MS-153 600

500

400

300 (grams) 200

100 Average Body Weight Body Average

Day1 Day2 Day3 Day4 Day5 Baseline Treatment period

Figure 3-12 Effects of combination of MS-153 and augmentin treatment on Body Weight of male P rats. Two-way ANOVA revealed no significant difference in body weight between drugs treated groups and ethanol vehicle group. Additionally, statistical analysis did not show any significant difference in body weight between MS-153 (50 mg/kg, i.p.), augmentin (100mg/kg, i.p.), and augmentin-MS-153 combination (augmentin 100mg/kg i.p, and MS-153 50 mg/kg i.p.) treated groups. Body weight data are shown as mean ± SEM. [Vehicle group, n=5; Augmentin group, n=5; MS-153 group, n=5; Augmentin+MS-153 group, n=5]

Chapter 4

Discussion

Studies have found that MS-153 treatment associated with significant reduction in extracellular glutamate concentration and the size of cerebral infarct in rats (Umemura et al., 1996). Alternatively, studies have shown that MS-153 enhanced glutamate uptake through GLT1 in COS-7 cells (Shimada et al., 1999). Interestingly, MS-153 was effective in inhibiting the development of tolerance and physical dependence to morphine

(Nakagawa et al., 2001). Additionally, the conditioned rewarding effects of morphine, methamphetamine and cocaine were also inhibited with MS-153 treatment at a dose of 10 mgin mice models (Nakagawa et al., 2005). Studies also reported that MS-153 was effective in blocking behavioral sensitization to phencyclidine through attenuating glutamatergic transmission at a dose of 10 mg/kg (Abekawa et al., 2002).

Regarding the use of augmentin in our study, augmentin is a mixture of amoxicillin and clavulanic acid. Both amoxicillin and clavulanic acid have β-lactam structures. Interestingly, β-lactam antibiotics have been reported to increase GLT1 expression and amoxicillin was one of the drugs tested in the previous study and proved to be effective in upregulating GLT1 (Rothstein et al., 2005). Additionally, a recent unpublished study from our lab found that augmentin was effective in reducing ethanol

52 consumption in P rats and this reduction in alcohol consumption was associated with increasing GLT1 expression in various brain reward regions. Thus, we tested both MS-

153 and augmentin in this study.

We have used P rats because evidences showed that alcohol preferring P rats meet all the criteria for an animal model of alcoholism (Lester and Freed, 1973, Bell et al.,

2006a). In summary, P rats were shown to voluntarily self-administer more than 5 grams of ethanol per kilogram of body weight per day following chronic free choice exposure to alcohol (Li et al., 1987, McBride et al., 2013), and consumption of this amount of ethanol results in blood-alcohol concentration (BAC) ranging from 50 to 200 mg% (Rodd-

Henricks et al., 2001, Bell et al., 2006b). In addition, P rats exert effort to get access to ethanol for its reinforcing and post-ingestive effects and not only for its taste (Bell et al.,

2006a). Also, chronic ethanol consumption in P rats has been shown to result in metabolic tolerance (Lumeng and Li, 1986) and functional tolerance (Gatto et al., 1987,

Stewart et al., 1991). Furthermore, following a chronic ethanol intake by P rats, it was found that P rats develop physical signs of withdrawal after terminating ethanol access

(Kampov-Polevoy et al., 2000).

4.1 Effect of MS-153 on chronic ethanol drinking paradigm

In this study, we found that MS-153 administration associated with significant attenuation in daily ethanol consumption starting 24 hours after the first dose of MS-153 as compared to vehicle treated animals, and this effect lasts 10 days after the last i.p. injection. Additionally, we found that, along with this long lasting reduction in ethanol intake, GLT1 expression was significantly upregulated in the NAc but not in the PFC.

Water intake by the treated group was significantly higher as compared to the control animals starting from day 3 till day 8. This could be explained as a behavioral compensatory mechanism due to reduction in ethanol intake. The effect of MS-153 treatment on water consumption is in agreement with previous studies from our lab (Sari et al., 2011b, Sari and Sreemantula, 2012, Rao and Sari, 2014). Alternatively, statistical analyses did not show any significant effect on body weight of the treated animals during the study.

Elevated glutamate transmission has been linked to drug addiction (Sari, 2013).

Importantly, extracellular glutamate levels have been reported to increase following exposure to ethanol. Melendez et al. have reported an elevation of the extracellular glutamate concentration in NAc of male Sprague Dawley rats that were exposed to ethanol (Melendez et al., 2005). Similarly, studies have shown elevated extracellular glutamate levels in cortical slices of P rats (McBride et al., 1986). Extracellular glutamate levels are regulated by several glutamate transporters and more importantly by GLT1

(Danbolt, 2001, Sari, 2013). Downregulation of GLT1 level in NAc has been reported following five weeks of ethanol exposure (Sari et al., 2011b, Sari and Sreemantula, 2012,

Sari et al., 2013), nicotine self-administration (Knackstedt et al., 2009) and cocaine self- administration (Knackstedt et al., 2010). Accordingly, in this study, we found significant downregulation of GLT1 level in NAc. However, we did not find any downregulation in

PFC, which is also in agreement with previous studies in our lab (Sari and Sreemantula,

2012, Sari et al., 2013).

Studies have shown that both behavioral effects of acutely administered alcohol and the neuroadaptations associated with chronic ethanol intake are mediated by glutamate excitatory neurotransmission in several brain reward regions (Eckardt et al.,

1998, Krystal et al., 2003, Sari et al., 2011b, Bahi et al., 2012). Glutamatergic transmission has been linked to the development of addiction to cocaine (Sari et al., 2009,

Moussawi et al., 2011), and heroin (Bossert et al., 2012). It has been suggested that glutamate neurotransmission in the NAc mediates drug seeking behavior, and the changes in glutamate transmission of this brain region are thought to mediate the switch from intermittent use of drug to dependence (Kalivas and Volkow, 2011). Additionally, NAc receives glutamatergic afferents from PFC (Papp et al., 2012, Stefanik et al., 2013).

Furthermore, activation of these projections has been implicated in goal-directed behaviors and in executing an adaptive behavioral response (Gipson et al., 2014).

Evidences have shown that upregulating GLT1 in NAc is an effective approach for preventing cocaine relapse (Sari et al., 2009, Knackstedt et al., 2010). Previous studies from our lab have shown that Neuroimmunophilin GPI-1046 (Sari and

Sreemantula, 2012) and ceftriaxone (Sari et al., 2013) were effective in reducing ethanol consumption, and this was associated with upregulation of GLT1 level in NAc.

Interestingly, we report here that MS-153 is effective in reducing ethanol consumption

55 starting at 24 hours after the first injection through day 14 (ten days after the last injection). This reduction in ethanol intake was found associated in part with upregulation of GLT1 level in NAc but not PFC. However, we did not notice downregulation of GLT1 level in PFC in vehicle-treated animals neither upregulation of this transporter after MS-

153 treatment. The lack of upregulation of GLT1 level in PFC could be explained by the fact that systemically administered compound do not equally distribute throughout the different brain regions (Alavijeh et al., 2005). Another possible explanation is the difference in blood flow, availability of transporter proteins and the presence of metabolizing enzymes (Kornhuber et al., 2006) between different brain regions.

However, to further confirm that, it is warranted to measure MS-153 level in PFC.

One major advantage in using MS-153 is that this compound, unlike other β- lactam antibiotics which are known as GLT1 upregulators, is devoid of antibacterial activity. Thus, the problem of bacterial resistance could be avoided with using this compound. However, it will be interesting to examine the long lasting effects of MS-153 on xCT expression levels in brain reward areas and to test the effect of MS-153 treatment in relapse-like ethanol drinking paradigm.

We conclude here that MS-153 is an effective drug in reducing ethanol consumption (an effect which lasts ten days after the last dose) in male P rats exposed to five weeks of free choice ethanol drinking paradigm. The effect of MS-153 on ethanol consumption was associated, in part, with GLT1 upregulation in NAc. These findings suggest that MS-153 could be a promising drug for treating alcohol dependence, and possibly other drugs of abuse.

4.2 Effect of MS-153 on xCT and pAkt levels in Amygdala and

Hippocampus

Recently in our lab, it was found that treating P rats with MS-153 for five consecutive days resulted in attenuating ethanol drinking without affecting body weight or sucrose intake (appetitive control) and this was associated with upregulating GLT1 levels (one day after the last dose) in AMG and Hipp (unpublished data). In this study, we report that exposing P rats to five weeks of uninterrupted free choice ethanol causes a significant downregulation of xCT in both AMG and Hipp. Alternatively, MS-153 treatment significantly upregulated xCT levels in AMG and Hipp.

It is widely accepted that NAc has a central role in reward learning and addiction

(Koob and Volkow, 2010). Glutamatergic input into NAc from other brain regions has been shown to play a key role in regulating addictive behavior. Importantly, it is well documented that NAc receives glutamatergic afferents from BLA (Stuber et al., 2011,

Papp et al., 2012), and the vHipp (Britt et al., 2012, Papp et al., 2012). Interestingly, each of these glutamatergic projections has a role in addictive behavior. For example, Stuber et al. found that selectively activating glutamatergic projections from BLA to NAc promotes motivated behavioral response (Stuber et al., 2011). Additionally, Vorel et al. have shown that activating glutamatergic projections from Hipp promotes addiction like behavior and relapse to cocaine (Vorel et al., 2001). Importantly, alcohol consumption is associated with alterations in the extracellular glutamate levels in various brain reward regions (reviewed by (Rao and Sari, 2012). For example, it has been found that extracellular glutamate concentration is elevated in the Hipp of male Wistar rats after

57 ethanol withdrawal (Dahchour and De Witte, 1999) and of binge drinking Wistar rats

(Ward et al., 2009, Chefer et al., 2011) and in AMG of male Sprague Dawley rats after chronic ethanol exposure (Roberto et al., 2004).

Disruptions in glutamate homeostasis are not limited to changes in GLT1 levels only but also xCT, where studies have shown that these two transporters play a key role in maintaining glutamate homeostasis (Bridges et al., 2012a). xCT was found to be downregulated in NAc after nicotine self-administration (Knackstedt et al., 2009), and after cocaine self-administration (Knackstedt et al., 2010). Similarly, our lab has recently shown that xCT is downregulated after five weeks of continuous ethanol exposure in both

NAc and PFC in P rats (Alhaddad et al., 2014). Additionally, recent studies from our lab have also shown that ceftriaxone treatment upregulated xCT levels in both NAc and PFC

(Alhaddad et al., 2014) and AMG (Rao and Sari, 2014). Based on these evidences, our central hypothesis in this study was to modulate the elevated extracellular glutamate levels after chronic ethanol exposure using MS-153, which is known to enhance glutamate uptake (Shimada et al., 1999).

Preliminary studies in our lab have shown that MS-153 treatment resulted in significant upregulation of GLT1 level in AMG and Hipp (unpublished data). We therefore tested the effect of MS-153 on pAkt levels, as Akt signaling pathway has been implicated in regulating GLT1 level (Li et al., 2006). Surprisingly, there were no significant changes in pAkt levels in both AMG and Hipp (although there was a trend similar to GLT1 changes in AMG but it failed to be significant), which may indicate the presence of other signaling pathways that regulate GLT1 expression.

In this study, we found significant downregulation of xCT level in AMG and

Hipp of P rats that subjected to five weeks continuous ethanol exposure as compared to ethanol-naïve rats. It is well known that the reduction in xCT levels causes a decrease in extracellular, non-synaptic glutamate levels, and consequently losing the glutamatergic tone on presynaptic mGLU2/3 receptors causing an increase in synaptic glutamate release

(Moran et al., 2005, Javitt et al., 2011). Moran et al. have found that restoring xCT activity by N-acetylcysteine prevented cocaine seeking (Moran et al., 2005).

Alternatively, activating mGLU2/3 receptor was shown to be effective in attenuating cue- induced ethanol seeking (Zhao et al., 2006). Additionally, chronic exposure to ethanol induced an inhibition of mGLU2/3 receptor function (Moussawi and Kalivas, 2010). It is noteworthy that modafinil attenuated cocaine reinstatement by activating mGLU2/3 receptor and it requires xCT for its action (Mahler et al., 2014). In this study, we found that MS-153 treatment for five days caused an upregulation in xCT levels. Restoring xCT level will eventually lead to reduction in extracellular glutamate level and hence reducing ethanol drinking.

Evidences have shown that Hipp is an important region in addiction and drug- context memory (Adcock et al., 2006, Meyers et al., 2006, Shen et al., 2006, Hernandez-

Rabaza et al., 2008), and in relapse (Vorel et al., 2001, Fuchs et al., 2005). Alternatively, evidences have shown that chronic alcohol exposure associated with impairment of hippocampal neurogenesis (Herrera et al., 2003, He et al., 2005), and this reduction in neurogenesis could be prevented by antioxidant (Herrera et al., 2003). Reduction in hippocampal neurogenesis has been linked to cocaine addiction (Noonan et al., 2010), and it is well known that astrocytic xCT is an important source of glutathione that can

59 protect against oxidative damage and neurodegeneration (Griffith, 1999, Bridges et al.,

2012b, Lewerenz et al., 2012). Additionally, it has been shown that glutamate excitotoxicity involves inhibition of cystine exchange and eventually neuronal cell death via oxidative stress (Murphy et al., 1989). These indirect evidences support the idea that upregulating xCT is important to decrease extracellular glutamate and possibly decrease the neuronal loss associated with chronic ethanol exposure.

Studies have found that AMG is an important region in addiction and drug reinforcement (Baxter and Murray, 2002, Sinclair et al., 2012, Christian et al., 2013).

Alternatively, Roberto et al. have found that chronic ethanol exposure associated with increased presynaptic glutamate release (Roberto et al., 2004) which further supports our finding regarding the downregulation of xCT. Accordingly, upregulating xCT level by

MS-153 might be important for targeting the elevated glutamate level in AMG, and consequently reducing ethanol consumption.

In summary, we report here that chronic exposure to ethanol in male P rats associated with downregulation of xCT levels and the use of MS-153 resulted in upregulation of xCT levels in both amygdala and hippocampus. Further experiments are warranted to verify the effect of MS-153 on xCT, which can be achieved through local perfusion of xCT blocker such as (S)-4-carboxyphenylglycine (4-CPG). However, our findings in this study, along with our unpublished data regarding the effect of MS-153 on

GLT1, indicate that MS-153 could be a promising treatment for alcohol dependence.

4.3 Effect of Combination of MS-153 and Augmentin on

Chronic Ethanol Drinking Paradigm

Our aim in this study was to compare the effects of MS-153 alone, augmentin alone and a combination of both drugs on ethanol consumption in male P rats exposed to five weeks of continuous free choice ethanol drinking paradigm. We report here that all of these drugs, either alone or in combination, resulted in significant reduction in ethanol consumption as compared to ethanol vehicle P rats. Additionally, treated animals consumed significantly higher amount of water as compared to the control group. It is important to note that neither one of these drugs, alone or in combination, cause reduction in body weight of P rats.

The observed increase in water intake by treated animals is consistent with previous studies from our lab and it has been explained as a compensatory mechanism for the reduction in fluid (ethanol) intake (Sari et al., 2011b, Sari and Sreemantula, 2012,

Rao and Sari, 2014).

Previous studies from our lab have shown that treatment of male P rats with either ceftriaxone (Sari et al., 2011b), GPI-1046 (Sari and Sreemantula, 2012), augmentin

(unpublished) or MS-153 (unpublished) was associated with attenuation of ethanol intake. Additionally, studies from our lab also have shown that this reduction in ethanol intake was associated with upregulation of GLT1 level in several regions of the reward circuitry (Sari et al., 2011b, Sari and Sreemantula, 2012, Rao and Sari, 2014).

According to our previous findings about the effects of augmentin and MS-153 on ethanol consumption in P rats, we hypothesized that the combination of MS-153 and augmentin would be more effective than either drug given alone. Surprisingly, we found that the combination of MS-153 and augmentin has a comparable effect, on ethanol consumption, to either compound given alone. Additionally, Bonferroni’s multiple- comparisons post-hoc test revealed significant reduction in ethanol intake (compared to vehicle treated group) among all treated groups, but it did not show any significant difference among the treated P rats during the entire study. This may indicate that these drugs act at the same site of action and as a result the observed effect on ethanol drinking in augmentin+MS-153 group may be due to either MS-153 or augmentin, since the augmentin+MS-153 group starts consuming higher amount of alcohol (although not a significant difference) than other treated groups. However, further studies are warranted to determine the lack of any additive effect with the combination of MS-153 and augmentin. Also, pharmacological study is warranted to explore the difference in proteins expressions in brain reward regions to evidently determine whether the combination of the two drugs has a comparable or better effect relative to either drug given alone.

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