AAV MEDIATED UPREGULATION OF GLT-1 DOES NOT ATTENUATE THE REINSTATEMENT OF COCAINE SEEKING

By

CARLY N. LOGAN

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

© 2017 Carly N. Logan

To my mother and father, Lori and Patrick Logan

4

ACKNOWLEDGMENTS

This research was funded by DA 033436 from NIDA awarded to Lori Knackstedt.

I thank Lizhen Wu for her hard work and assistance with each step of this project. I

acknowledge Brooke Jackson, Brianna Parlette, Brianna Yaffe for their skills and

dedication. I thank Dr. Lori Knackstedt for guidance and support through this project, as

well as Dr. Gonzalo Torres and Dr. Marek Schwendt for lending their expertise to my

committee.

5

TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF FIGURES .......................................................................................................... 7

LIST OF ABBREVIATIONS ............................................................................................. 5

ABSTRACT ..................................................................................................................... 9

CHAPTER

1 INTRODUCTION .................................................................................................... 11

Operant Self-administration Extinction-reinstatement Animal Model ...................... 11 Neurobiology Underlying Cue-primed Reinstatement of Cocaine-seeking ............. 12

Glutamate Homeostasis .......................................................................................... 14 Cocaine Disrupts Glutamate Homeostasis ............................................................. 16 Glutamatergic Alterations Associated with Prevention of Reinstatement to Drug-

seeking ................................................................................................................ 17

2 MATERIALS AND METHODS ................................................................................ 19

Subjects .................................................................................................................. 19

Surgical Procedures ............................................................................................... 19

Viral Vectors ........................................................................................................... 20 Cocaine Self-administration Extinction training, and Reinstatement ....................... 20 Histology and Tissue Preparation ........................................................................... 21

Immunohistochemistry ............................................................................................ 21 Western Blotting ..................................................................................................... 22

Statistical Analysis .................................................................................................. 23

3 RESULTS ............................................................................................................... 25

Self-administration and Extinction ........................................................................... 25

Cue Primed Reinstatement ..................................................................................... 26

AAV-GFAP-GLT-1 Increases GLT-1 Protein Expression to Levels of Cocaine Naïve Control ....................................................................................................... 26

AAV-GFAP-GLT-1 Expression does not Alter xCT Protein Amount ....................... 27

4 DISCUSSION ......................................................................................................... 37

LIST OF REFERENCES ............................................................................................... 41

BIOGRAPHICAL SKETCH ............................................................................................ 48

6

LIST OF TABLES

Table page 2-1 Antibody concentrations and catalog numbers .................................................. 24

7

LIST OF FIGURES Figure page 3-1 His-tagged AAV-GFAP-GLT-1, GFAP and overlay ............................................. 28

3-2 Spread of the His-tagged, GFAP and overlay .................................................... 28

3-3 Inactive lever presses during self-administration ................................................ 29

3-4 Active lever presses during self-administration................................................... 29

3-5 Infusions attained during self-administration ...................................................... 30

3-6 Lever presses on the previously active lever during the first twelve days of extinction training ................................................................................................ 30

3-7 Cue primed reinstatement test............................................................................ 31

3-8 Rats analyzed in the western blot data inactive lever presses during self-administration ..................................................................................................... 32

3-9 The rats analyzed in the western data active lever presses during self-administration ..................................................................................................... 33

3-10 Rats analyzed in the western blot data infusions during self-administration ....... 34

3-11 Lever presses on the previously active lever during extinction training for rats that were analyzed for the western blot data. ..................................................... 35

3-12 Western blot data of GLT-1 expression for each virus group and cocaine naïve controls. .................................................................................................... 36

3-13 Western blot data of xCT expression for each virus group ................................. 36

8

LIST OF ABBREVIATIONS

AAV Adeno-associated virus

CNS Central Nervous System

GFAP Glial fibrillary acidic protein

GFP Green fluorescent protein

mGluR2/3 Group II metabotropic glutamate receptors

NAc Nucleus accumbens core

PBS Phosphate buffered Saline

9

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

AAV MEDIATED UPREGULATION OF GLT-1 DOES NOT ATTENUATE THE

REINSTATEMENT OF COCAINE SEEKING By

Carly N. Logan

May 2017

Chair: Lori Knackstedt Major: Psychology

Cocaine addiction is a serious and highly prevalent issue in the United States.

Treatments of cocaine addiction are complicated by high rates of relapse. Animal

models of relapse, such as the operant self-administration extinction-reinstatement

paradigm, are useful tools to study relapse prevention. In this model, the operant

response required for cocaine delivery is established, then extinguished and reinstated

with cues previously paired with drug delivery or a dose of the drug itself. Previous

research has shown several neurobiological changes occur in the nucleus accumbens

with repeated exposure to cocaine such as the downregulation of the cystine-glutamate

exchanger and its catalytic subunit xCT and downregulation of GLT-1 glutamate

transporters. The antibiotic ceftriaxone has been shown to upregulate the cystine-

glutamate exchanger (xCT), and GLT-1 expression after cocaine use, ultimately

preventing reinstatement of drug-seeking behaviors. To determine which

neurobiological consequences of ceftriaxone treatment prevents the reinstatement of

drug-seeking, here we used an adeno-associated virus (AAV) to upregulate GLT-1

transporters alone without altering xCT expression. Rats self-administered cocaine for

two weeks, receiving injections of either AAV-GFAP-GLT-1a or a control AAV (AAV-

10

GFAP-eGFP) in the nucleus accumbens immediately following the last day of self-

administration. The animals then underwent three weeks of extinction training (during

which the virus overexpression occurred) before undergoing a cue primed reinstatement

test. Rats that had received the AAV-GFAP-GLT-1a reinstated cocaine-seeking in a

similar manner as the rats that had received AAV-GFAP-eGFP. These results indicate

that the upregulation of GLT-1 transporters alone is not sufficient to prevent the

reinstatement of cocaine seeking behaviors.

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CHAPTER 1 INTRODUCTION

Cocaine addiction is a compulsive, chronic and uncontrollable disease that

affects approximately 1.5 million Americans each year (National Institute on Drug

Abuse, 2015). Drug addiction is associated with uncontrollable motivation to seek drugs

and a decreased need to seek non-drug rewards (Goldstein & Volkow, 2002).

Treatment and rehabilitation plans for cocaine addiction are complicated by high rates

of relapse even after long periods of abstinence (O'Brien, 2001). Current research is

striving to better understand the neurobiological mechanisms involved in addiction to

produce pharmacological therapies and more effective treatments for addiction and

relapse prevention.

Operant Self-administration Extinction-reinstatement Animal Model

Animal models of substance abuse have been developed to examine the cellular

and molecular processes involved in acquisition, maintenance, and relapse to cocaine-

seeking. The operant self-administration extinction-reinstatement paradigm is

considered a valid animal method for studying the relapse of drug-seeking (Epstein et

al., 2006). This paradigm includes a period of self-administration during which the

animal performs an operant behavior (such as pressing a lever) to receive an infusion of

a drug. The drug infusion is paired with a conditioned stimulus, such as illumination of a

light above the lever, and a tone. An inactive lever is present that results in no drug

infusion or conditioned stimuli presentation when pressed. This inactive lever

demonstrates the animal’s preference for the active lever and serves as a locomotor

control. Once a pre-set criterion of drug intake is met, animals enter a period of

extinction training in the operant chamber during which both levers are extended, but

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presses on neither lever result in programmed consequences. When the drug-seeking

behavior of lever pressing has been extinguished to a criterion (i.e. less than 20 lever

presses on the previously active lever), animals undergo a reinstatement test.

Reinstatement tests consist of reintroduction of conditioned stimuli from self-

administration (light and tone) when the active lever is pressed, or reintroduction of the

unconditioned stimuli (a low dose of the drug). Reinstatement to drug seeking occurs

when the animal presses the previously active lever significantly more during the

reinstatement test than during the previous days of extinction training (Katz & Higgins,

2003; O’Brien & Gardner, 2005).

Neurobiology Underlying Cue-primed Reinstatement of Cocaine-seeking

The neurocircuitry of relapse has been thoroughly studied for decades and is well

understood. Corticostriatal glutamate projections from the prefrontal cortex to the

nucleus accumbens core (NAc) are responsible for reinstatement to drug seeking

(Mcfarland, Davidge, Lapish, & Kalivas, 2004). Interruption of glutamate homeostasis in

the NAc alters communication between the prefrontal cortex and the nucleus

accumbens (Kalivas, 2009). Cue primed reinstatement is blocked by pharmacological

(McLaughlin and See, 2003) and optical (Stefanik, Kupchik & Kalivas, 2016) inactivation

of the dorsal prefrontal cortex. Inactivation of the lateral orbitofrontal cortex blocks cue-

induced reinstatement, whereas inactivation of the medial orbitofrontal cortex had no

effects on cue-primed reinstatement (Fuchs, Evans, Parker, & See, 2004). Cue primed

reinstatement also relies heavily on glutamatergic activation of the basolateral

amygdala, and dopaminergic projections to the NAc (Everitt & Wolf, 2002; Kalivas &

McFarland, 2003). Pharmacological inactivation of the rostral basolateral amygdala

(Kantak et al., 2002), and optical inactivation of the basolateral amygdala (Stefanik et

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al., 2013) blocks cue-primed reinstatement. Inhibiting glutamatergic binding in the

ventral tegmental area, which projects to the nucleus accumbens, basolateral amygdala

and the prefrontal cortex (Kalivas & McFarland, 2003), blocks cue-primed reinstatement

(Mahler et al., 2013). Inactivation of the NAc specifically prevents cue-primed

reinstatement, whereas inactivation of the nucleus accumbens shell did not attenuate

cue-primed reinstatement (McFarland & Kalivas, 2001). The projections from the

prefrontal cortex, basolateral amygdala, and ventral tegmental area to the NAc play vital

roles in cue-primed reinstatement, and thus the NAc is a key area to examine in regards

to addiction and relapse prevention.

Within the NAc, there are several receptors that play a role in addiction and

relapse. Glutamate binding in the NAc is necessary for reinstatement as reinstatement

behaviors were blocked with an intra-accumbens infusion of AMPA/kainate receptor

antagonist CNQX, whereas NMDA antagonists did not attenuate reinstatement to

cocaine-seeking (Backstrom & Hyytia, 2007). There is an increase of AMPA receptors

in the NAc after one month of cocaine withdrawal (Wolf & Tseng, 2012). Administration

of mGluR5 antagonists (Wang, Moussawi, Knackstedt, Shen & Kalivas, 2012) or

infusion of negative allosteric modulators into the nucleus accumbens core attenuate

cue-primed reinstatement (Kumaresan, Yuan, Yee, et al., 2009). Interestingly, infusions

of mGluR5 negative allosteric modulators into the nucleus accumbens core does not

prevent the increase of synaptically released glutamate although it does prevent

reinstatement to drug-seeking (Smith et al., 2017). These various findings suggest

neurotransmitter binding to specific receptors in the NAc is necessary for cue-primed

reinstatement.

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With consideration of certain receptor antagonist preventing reinstatement, it can

be postulated that glutamate plays an important role in reinstatement to drug-seeking.

There are numerous alterations specifically in glutamate regulatory systems in the NAc

following cocaine use. After chronic cocaine use, there is a significant decrease in basal

extracellular accumbens glutamate (Baker et al., 2003) and a decrease of glutamate

uptake (Knackstedt et al., 2010). Glutamate spillover due to increased synaptically

released glutamate and decrease of glutamate uptake, stimulates mGluR5, producing

nitric oxide which contributes to cue-primed reinstatement (Smith et al., 2017). These

findings have narrowed relapse prevention research to glutamatergic alterations in the

nucleus accumbens for possible pharmacological treatments.

Glutamate Homeostasis

Glutamate is the major excitatory neurotransmitter in the central nervous system

(CNS) and plays a vital role in brain functions and diseases. Extracellular levels of

glutamate are regulated by well-developed cellular mechanisms, and provide tone on

transporters and receptors (Reissner & Kalivas, 2010). Basal levels of nonvesicular

glutamate in the nucleus accumbens are regulated by the cystine-glutamate exchanger

(xc-), which exchanges intracellular glutamate for extracellular cystine (Baker, Xi, Shen,

Swanson, & Kalivas, 2002). The cystine-glutamate exchanger exchanges one

extracellular cystine with intracellular glutamate at a 1:1 ratio (McBean et al., 2002). The

cystine-glutamate exchanger is a sodium independent exchanger (Baker et al., 2002)

and is expressed on glial cell membranes (Lehre et al., 1995). The cystine-glutamate

exchange is a heterodimer containing a light chain, xCT, which is unique to this

exchanger, and a heavy chain, 4F2, that is common in many transporters (Sato, Tamba,

Ishii, & Bannai, 1999). The cystine-glutamate exchange is primarily located in glial cells

15

and is a substrate, not energy, dependent system (McBean & Flynn, 2001; Pow, 2001).

Blockade of cystine-glutamate exchange results in a decrease of basal extracellular

glutamate levels in the NAc (Baker et al., 2002).

Extracellular basal glutamate homeostasis provides tone on receptors and

transporters, such as group II metabotropic glutamate receptors (mGluR2/3) (Baker et

al., 2002: McBean & Flynn, 2001). Glutamatergic tone on is regulated by the cystine-

glutamate exchange (Baker et al., 2002), and both extrasynaptic and vesicular

glutamate are regulated by mGluR2/3 in the nucleus accumbens core (Baker et al.,

2002; Conn & Pin, 1997). Levels of synaptically released glutamate are dependent on

glutamatergic tone on mGluR2/3 (Moran, Mcfarland, Melendez, Kalivas, & Seamans,

2005; Cartmel & Schoepp, 2000). Infusion of mGluR2/3 agonists significantly decrease

cystine uptake and decrease extracellular glutamate levels (Baker et al., 2002).

Glutamate is a potent neurotoxin, and prompt removal from the synapse is

essential for avoidance of excitotoxicity (Haugeto et al., 1996). Glutamate transporters

are located on glial cells and neurons throughout the brain (Tzingounis & Wadiche,

2007). Sodium dependent glutamate transport from the extracellular space into glial

cells plays an important role in regulating extracellular glutamate levels (Danbolt, 2001).

Glutamate transporters regulate the activation of nearby metabotropic receptors, control

cross-talk between synapses, and shape the kinetics of excitatory postsynaptic currents

(Rimmele & Rosenberg, 2016). Removal of glutamate from the synapse is achieved by

six glutamate transporters; EAAT1 (rodent GLAST), EAAT2 (rodent GLT-1) are the

most two most abundant transporter types, and EAAT3 (EAAC1), EAAT4, and EAAT5

are less abundant (Huang & Bergles, 2004). Of the glutamate transporters, GLT-1 is

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responsible for removing 90% of synaptically released glutamate from the synaptic cleft

to avoid excitotoxicity from over stimulation (Haugeto et al., 1996). GLT-1 is

predominately located in the membrane of astrocytes with increased amounts of

expression adjacent to synapses, however there is evidence supporting GLT-1

expression in hippocampal and cortical neuronal membranes as well (Chaudhry et al.,

1995; Chen et al., 2004; Danbolt, 2001; Murphy-Royal, Dupuis, Groc, & Oliet, 2017).

There are two variants of GLT-1 in the rat brain, GLT-1a and GLT-1b. GLT-1a is the

more prominent form found throughout the brain (Berger et al., 2005). The two variants

differ in that GLT-1b is a C-terminal splice variant of GLT-1a, and have been observed

coexisting in regions, as well as separately (Reye, Sullivan, Scott &Pow, 2002). GLT-1b

expression is only approximately 6-10% of the expression of GLT-1a, and is not located

in spines or nerve terminals (Furness, Danbolt and Zhou, 2016; Holmseth et al., 2009).

GLT-1b is located more proximal to the soma, whereas GLT-1a is located near the

synapse (Sullivan et al., 2004). Approximately 10% of GLT-1 expression is pres-

synaptic (see Furness, Danbolt and Zhou 2016 for review). Glutamate homeostasis in

the nucleus accumbens depends on these various glutamate systems, and drug use,

abstinence, and reinstatement rely on the disruption of these systems.

Cocaine Disrupts Glutamate Homeostasis

Glutamate regulatory systems within the nucleus accumbens are altered from

chronic cocaine use, including a decrease in basal extracellular glutamate levels (Baker

et al., 2003) . A decrease in basal extracellular nonvesicular glutamate is related to

down regulation of the cystine-glutamate exchange function following cocaine use

(Baker et al., 2003). The cystine-glutamate exchanger exchanges one extracellular

cystine for one intracellular glutamate (McBean et al., 2002). The catalytic subunit of the

17

cystine-glutamate exchanger, xCT, is significantly reduced following cocaine self-

administration (Knackstedt, Melendez, & Kalivas, 2010). There is a downregulation of

GLT-1 transporter proteins and a decrease in sodium dependent glutamate uptake after

cocaine use in the nucleus accumbens core (Knackstedt et al., 2010).

Glutamatergic Alterations Associated with Prevention of Reinstatement to Drug-seeking

Following cocaine use, there is a down-regulation of xCT and GLT-1, and a

decrease in basal extracellular glutamate. The β-lactam antibiotic ceftriaxone was first

identified as having the ability to increase the transcription and expression of GLT-1 in a

mouse model of ALS (Rothstein et al., 2005). Intraperitoneal injections (100-200 mg/kg)

of ceftriaxone for six days following cocaine self-administration increases protein

expression of GLT-1 and xCT in the nucleus accumbens (Knackstedt et al., 2010;

LaCrosse et al., 2016; Lewerenz et al., 2009). Ceftriaxone avoids the increase of

extracellular synaptically released glutamate during cocaine primed reinstatement

(Trantham-Davidson, LaLumiere, Reissner, Kalivas, & Knackstedt, 2012). The

upregulation of xCT, GLT-1 and restored glutamate homeostasis in the NAc prevents

reinstatement to drug seeking with cue (Sari, Smith, Ali, & Rebec, 2009) and drug

primes (Knackstedt et al., 2010). Ceftriaxone treatment attenuated reinstatement to

cocaine-seeking for weeks after administration has ceased (Sondheimer & Knackstedt,

2011).

Systemic injections of N-acetylcysteine, a cysteine pro-drug, delivers large

amounts of cysteine to the brain, and elevates extracellular glutamate by increasing

cystine-glutamate exchange and thereby increasing glutamate export into the

extrasynaptic space (Baker al., 2003). N-acetylcysteine prevents cocaine-primed

18

increases in synaptically released glutamate and prevents reinstatement to cocaine-

seeking (Baker et al., 2003). The prevention of reinstatement to drug-seeking by N-

acetylcysteine treatment is mediated by GLT-1 as N-acetylcysteine does not attenuate

reinstatement to drug-seeking when GLT-1 expression is decreased in the NAc

(Reissner et al., 2014).

While upregulation of both xCT and GLT-1 has been observed to attenuate

reinstatement to cocaine-seeking, the role of GLT-1 transporters alone has not been

investigated. Here, we upregulate GLT-1a expression using an adeno-associated virus

and examine cue-primed of reinstatement of cocaine-seeking. We hypothesized

upregulation of GLT-1a would increase the re-uptake of glutamate, thus preventing the

increase of synaptically released glutamate that has been observed during

reinstatement of cocaine-seeking, and therefore prevent reinstatement.

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CHAPTER 2 MATERIALS AND METHODS

Subjects

Twenty five adult male Sprague Dawley rats (Charles River, 300–350 g; Raleigh,

NC, USA) were housed in a temperature and humidity controlled vivarium. Rats were

maintained on a 12hr-reversed light cycle and all procedures were carried out during the

dark phase of the cycle. Animals were provided 20 grams of standard lab chow daily

and water ad libitum.

Surgical Procedures

Ketamine (87.5 mg/ kg, i.p.) and xylazine (5 mg/kg, i.p.) were administered to

anesthetize the animals prior to surgery. Keterolac was administered following surgery

to provide analgesia. Catheters (SILASTIC silicon tubing, ID 0.51 mm, OD 0.94 mm,

Dow Corning, Midland, MI) were implanted and secured into the jugular vein and

passed subcutaneously through the shoulder blades and exited the back. The catheter

tubing was connected to a cannula (Plastics One, Roanoke, VA, USA) embedded in a

rubber harness (Instech, Plymouth Meeting, PA, USA) worn by the rat for the duration of

the self-administration. Immediately following catheter implantation, guide cannulas

(Plastics One, Roanoke, VA, USA) were implanted directly above the nucleus

accumbens core (AP+1.2 mm, ML +1.6 mm, DV -5.5 mm) and secured using dental

cement and stainless steel skull screws. Cefazolin (1 µl) was administered for three

days after surgery as an antibiotic. Catheters were flushed with heparinized saline (1 µl,

100 U/ml) prior to and following self-administration sessions.

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Viral Vectors

We employed two adeno-associated viruses (AAV) constructed with an adeno

helper plasmid (pF6), and AAV helper encoding serotype 8, and the AAV packaging

vector containing the GFAP promoters to permit the specific targeting of glial cells. The

vectors contained either eGFP or GLT-1a gene sequences. AAV-GFAP-eGFP was

purchased from the UNC Vector Core. The his-tagged AAV-GFAP-GLT-1a plasmid (Li

et al., 2014) and was a kind gift from David Poulson (SUNY Buffalo) and was amplified

and packaged into an AAV-8 by the UNC Viral Vector Core. Either AAV-GFAP-GLT-1a

or AAV-GFAP-eGFP was injected into the nucleus accumbens core using Harvard

Apparatus pump at 0.25 µL/min. Injectors extended 1mm below the cannula into the

NAc.

Cocaine Self-administration Extinction training, and Reinstatement

Rats were allowed five days to recover from surgery before self-administration

began. Animals were trained to self-administer cocaine in standard 2-lever operant

chambers under a FR1 schedule during two hour sessions. When the active lever was

pressed, intravenous infusion cocaine (0.35 mg/infusion) was delivered. Infusions were

paired simultaneously with a 5 second tone (2900 Hz tone) and a light directly above

the lever and were followed by a 20 second time out period, during which time lever

presses were counted, but did not results in delivery of the drug or cues. Lever presses

on the inactive lever were not reinforced but the lever presses were recorded. The

criteria for self-administration was ten or more infusions for twelve days.

Immediately following the conclusion of the 12-day self-administration period,

rats began extinction training, during which time both levers were presented but presses

on neither lever resulted in programmed consequences. Rats received bilateral intra-

21

NAc AAV-GFAP-GLT-1 (n=14) or the control AAV-GFAP-eGFP (n=16) immediately

following the first session of extinction training. Rats in both groups were either

subjected to a reinstatement test (n=18) or were killed for western blotting (n=12).

Following three weeks of extinction training, a 1-hr cue-primed reinstatement test

was conducted. During this test, the cues that were previously associated with cocaine

self-administration were once again associated with presses on the previously active

lever. The amount of lever presses on the previously active lever and inactive lever

during the reinstatement test were compared to the average number of presses during

the final two days of extinction training.

Histology and Tissue Preparation

Animals were deeply anesthetized with pentobarbital (100 mg/kg, i.p.), and were

transcardially perfused with phosphate-buffered saline (PBS) and then 4%

paraformaldehyde (PFA). Brains were extracted and preserved in 4% PFA for 24 hours

following the perfusion. They were then stored in 20% sucrose PBS solution for 48

hours. Brains were frozen and stored at -80°C until they were sliced to 30 microns using

a cryostat. Brains were sliced using a cryostat within 24 hours of beginning

immunohistochemistry on the slices and stored in PBS

Immunohistochemistry

Immunohistochemistry to detect the his-tagged AAV-GFAP-GLT-1 virus was

completed. Brain slices were washed in PBST (PBS and 0.2% Triton) for three washes.

The slices were then blocked in 10% Normal Goat Serum in PBS before being probed

with rabbit anti-his tag antibody overnight at 4° C. The slices were then washed in 3%

Normal Goat Serum (4 x 10min) before they were probed with appropriate AlexaFluor

secondary antibodies. The slices were washed (4 x 10min) in PBS, then washed (3 x

22

10min) in PB (monobasic sodium phosphate and dibasic sodium phosphate in H20)

before being placed on slides and coverslipped using citifluor mounting solutions. See

Table 1 for antibody dilution and product information.

Immunohistochemistry was also used to detect the AAV-GFAP-eGFP virus. Brain

slices were washed three times in PBS containing potassium (KPBS). The slices were

then blocked in a solution of 2% Normal Goat Serum in PBS and subsequently

incubated in rabbit anti-GFP at 4° C overnight. The slices were then washed in KPBS (3

x 10in), before being incubated in goat anti-rabbit secondary anti-body. The slices are

then washed three additional times in KPBS before being transferred to slides and

coverslipped.

Images were acquired on a fluorescent microscope to confirm that viral

overexpression was confined to the NAc. Rats were eliminated from the behavioral

analysis if there was improper spread (e.g. up the cannula track) or improper placement

(not within the nucleus accumbens core).

Western Blotting

A separate group of rats (n=18), underwent surgery, self-administration,

extinction training and AAV infusion as described above but were not tested for

reinstatement. Immediately following the final day of extinction training, brains were

extracted and NAc region was dissected on ice. Cocaine naïve rats were included also.

These rats underwent surgeries, and remained in their home-cages through-out the

experiment until their brains were extracted. Following the brain extraction , the tissue

was homogenized and frozen at minus 80. Western blotting was performed for GLT-1a,

and xCT expression. Proteins were separated in 10% glycine Criterion gel (Bio-rad

Hercules, CA, USA) and then transferred to PVDF membrane (Bio-rad Hercules, CA,

23

USA). The membrane was blocked with 5% milk in TBST (Tris/TrisHCl 25mM, NaCl

0.13M, KCl 0.0027M, 1ml Tween20; 4 x 10min). For GLT-1a protein quantification, the

rabbit anti-GLT-1a antibody diluted in 5% milk in TBST was incubated overnight at 4° C.

The membrane was washed in TBST (4 x 10min) before goat anti-rabbit secondary

antibody was incubated for two hours at room temperature. After being washed in TBST

(4 x 10min), the membrane was exposed to Amersham ECL Prime Western Blotting

Detection Reagent (GE Healthcare Limited, Little Chalfont, United Kingdom) and were

developed using High Performance chemiluminescence film (GE Healthcare Limited,

Little Chalfont, United Kingdom). Digitized images of immunoreactive proteins were then

analyzed using ImageJ software. To quantify xCT proteins, the same procedures were

followed with the exception of the use of rabbit anti-xCT primary antibody for overnight

incubation. See Table 1 for antibody dilutions and product information.

Statistical Analysis

Behavioral data (self-administration, extinction and reinstatement test) was

analyzed using SPSS (IBM, Armonk, NY). An alpha level of p<0.05 was set for all

statistical analyses. Greenhouse-Geisser adjustments were used when sphericity

assumptions were not met. Bonferroni post-hoc tests were used when necessary.

Repeated measure (RM) analyses of variance (ANOVAs) were used to compare the

number of lever presses and infusions during self-administration, lever presses during

extinction and reinstatement between Groups, with Time as the RM. Western blot data

was analyzed using GraphPad Prism (version 5.00, GraphPad Software, La Jolla, CA).

This data was normalized for the density of calnexin immunoreactivity within the same

sample and was analyzed using one-way ANOVA (Fig. 3-11) or independent samples t-

tests (Fig. 3-12).

24

Table 2-1. Antibody concentrations and catalog number

Antibod-y Company Catalog number

Lot number Concentration

Rabbit anti-his tag

Cell Signaling Technology

2365S 3 1:100

Mouse anti-GFAP

UC Davis NIH NeuroMab Facility

75-240 447-2JH-75d 1:250

Alexa Fluor 488 goat anti-mouse

Life Technologies A11073 1637243 1:200-500

Alexa Fluor 594 goat anti-rabbit

Life Technologies A11076 1611307 1:200

Rabbit anti-GFP

Abcam Ab290 GR278073 1:20,000

Rabbit anti-GLT-1a

Paul Rosenburg, Harvard

1:80,000

Goat anti-rabbit

Jackson Immunohistochemistry Research Laboratory Inc.

111-035-003

113534 1:50,000

Rabbit anti-Calnexi

Millipore AB2301 2587261 1:40,000

Rabbit anti-xCT

Novus NB300-

318 I-01 1:50,000

25

CHAPTER 3 RESULTS

Self-administration and Extinction

For rats later tested for reinstatement, repeated measures ANOVA of the inactive

lever presses (Fig. 3-3) during self-administration revealed no significant effect of Group

(F(1.34,16)=1.75, p=.202). There a significant effect of Group x Time for the inactive

lever presses during self-administration (F(1.34,16) =4.116, p=.045). There was no

significant effect of Group (F(1,16)=.916, p=.353). Repeated measures ANOVA

conducted on the active lever presses (Fig.3-4) during self-administration revealed no

significant Group x Time difference for rats later assigned to received GLT-1 AAV or

GFP AAV (F(3.3,16)=1.452, p=.145). There was a significant effect of Time on active

lever presses during self-administration (F(3.5,62.9)=3.42, p=.018). There was no

significant effect of Group (F(1,16)=0.056, p=0.815).

Repeated measures ANOVA conducted on mean infusions (Fig. 3-5) received

during self-administration revealed no significant differences between groups that were

then assigned to receive GLT-1-AAV or GFP-AAV (F(4.6,16)=.935 , p=.458). A

significant effect of Time was detected as there was an increase in infusions received

through the 12 days of self-administration (F(4.6,16) =10.4, p<.000). There was no

significant effect of Group (F(1,16)=1.08, p=.312).

There was no significant effect of Group x Time on the previously active lever

presses during extinction training (Fig. 3-6) (F(1,28)=.537, p=.572). There was a

significant effect of Time on active lever presses during the first twelve days of

extinction training, showing a decrease in lever presses (F(1,28)=19.72, p=.000). There

was not a significant effect of Group x Time on inactive lever presses during the first

26

twelve days of extinction between groups (F(3.57,25)=.489, p=.723). There was an

effect of Time during extinction training for the previously inactive lever that was inactive

during self-administration (F(3.57,25)= 19.72, p=.000). There was no significant effect of

Group (F(1,16)=1.083, p=.323).

Cue Primed Reinstatement

The animals were tested for cue primed reinstatement to drug seeking behaviors

during a one-hour test during which the active lever presses during the reinstatement

test revealed significantly more presses when compared to active lever presses during

extinction for both groups (Fig. 3-7) [GLT-1: (T(1,8)=-3.7, p=.006), GFP: (T(1,8)=-3.84,

p=.005)]. A repeated measure ANOVA revealed no effect of Group x Time

(F(1,16)=28.24, p=.77) indicating reinstatement behaviors for GLT-1-AAV and GFP-

AAV were not significantly different. There was an effect of Time indicating both groups

increased lever presses during the reinstatement test compared to lever presses during

extinction (F(1,16)=28.24, p=.000).

AAV-GFAP-GLT-1 Increases GLT-1 Protein Expression to Levels of Cocaine Naïve Control

Protein expression was measured in a separate group of animals. Repeated

measures ANOVA of lever presses on the inactive lever (Fig. 3-8) during self-

administration for the rats that were later administered GLT-1-AAV or GFP-AAV

revealed no effect of Group x Time (F(3.9,46.9)=0.49, p=0.73) or Time (F(3.9,46.9)=1.7,

p=0.17). There was no significant effect of Group on inactive lever presses during self-

administration (F(1,16)= )There was not a significant effect of Time for active lever

presses (Fig. 3-9) during self-administration (F(11,121)=1.23, p=0.28). There was not a

significant effect of Group x Time on active lever presses during self-administration

27

(F(11,121)=0.6, p=0.83) and no significant Group effect (F(1,16)=3.92, p=0.44). There

was not a significant effect of Group x Time on infusions (Fig. 3-10) received during self-

administration between the rats that were later administered GLT-1-AAV or GFP-AAV

(F(11,121)=1.04, p=0.95). There was a significant effect of Time on infusions received

during self-administration (F(11,121)=4.83, p=0.000) and no significant effect of Group

(F(1,16)=12.17, p=.95). There was a significant effect of Time on presses on the

previously active lever during extinction (Fig 3-11) (F(11,121)=15.85, p=.000) and no

significant effect of Group (F(1,16)=6.95, p=0.595). There was not an effect of Group x

Time between rats that had received GLT-1-AAV and rats that had received GFP-AAV

during extinction (F(11,121)=1.05, p=.0.40).

A one-way ANOVA comparing GLT-1 protein amounts in rats that received GLT-

1AAV, GFP-AAV and cocaine naïve control rats (Fig. 3-12) revealed a significant overall

effect (F(2,15)=4.25, p=.03). Bonferroni post hoc tests revealed a significant difference

between GFP-AAV and Control rats (t(1,11)=2.84, p<0.05), suggesting rats that

received GFP-AAV had significantly less GLT-1 protein than cocaine naïve control rats.

There was no significant difference in GLT-1 expression between GLT-1-AAV and

Control rats (t(1,10)=0.82, p>0.05). This indicates the protein expression in GLT-1-AAV

animals is comparable to that of a cocaine naïve control animal.

AAV-GFAP-GLT-1 Expression does not Alter xCT Protein Amount

An independent samples t-test on rats that received GLT-1-AAV and GFP-AAV

(Fig. 3-13) revealed no differences in xCT protein amounts between groups

(t(1,10)=1.59, p=.144). This indicates that upregulation of GLT-1 does not alter

expression of cystine-glutamate exchange.

28

Figure 3-1. His-tagged AAV-GFAP-GLT-1, GFAP and overlay. A) His-tagged AAV-GFAP-GLT-1 in the nucleus accumbens core. B) GFAP stain of glial cells in the nucleus accumbens. C) A red-green merge of his-tagged virus and GFAP stained glial cells.

Figure 3-2. Spread of the His-tagged, GFAP and overlay. A) Spread of the His-tagged AAV-GFAP-GLT-1 in the nucleus accumbens core below the cannula track. B) GFAP stain of glial cells in the nucleus accumbens core below the cannula. C) A red-green merge of his-tagged virus and GFAP stained glial cells below the cannula track. D) Spread of AAV-GFAP-eGFP below the cannula track, above anterior commissure.

A.C.

29

Figure 3-3. Inactive lever presses during self-administration. Inactive lever presses during self-administration did not differ between rats later administered GLT-1-AAV (n=9) or GFP-AAV (n=9) (F(3.57,25)=.489, p=.723). There was a significant effect of Time on inactive lever presses during self-administration (F(1.34,16) =4.116, p=.045).

Figure 3-4. Active lever presses during self-administration. Active lever presses during self-administration did not significantly differ between rats later administered GLT-1-AAV (n=9) or GFP-AAV (n=9). (F(3.3,16)=1.452, p=0.145). There was a significant effect of Time on active lever presses during self-administration (F(3.5,62.9)=3.42 p=0.018).

30

Figure 3-5. Infusions attained during self-administration. The mean number of infusions attained during self-administration increased over time, and did not differ between rats later administered GLT-1-AAV (n=9) or GFP-AAV (n=9). [Group x Time: F(4.6,16)=.935 , p=0.458, Time: (F (4.6,16) =10.4,p=0.000)]

Figure 3-6. Lever presses on the previously active lever during the first twelve days of extinction training. The mean number of lever presses on the previously active lever during the first twelve days of extinction training did not differ between the virus groups. (F(1,28)=.537, p=0.572). There was a significant effect of Time on presses on the previously active lever (F(1,28)=19.72, p=0.000).

31

Figure 3-7. Cue primed reinstatement test. Rats that were administered GLT-1-AAV (n=9) and GFP-AAV (n=9) showed significantly greater lever presses during cue primed reinstatement test compared to the amount of lever presses during extinction [GLT-1: (T(1,8)=-3.7, p=0.006), GFP: (T(1,8)=-3.84, p=0.005)] A Bonferroni correction was used to account for multiple t-tests and familywise error inflation (therefore α=0.025). There was no significant effect of Group x Time (F(1,16)=28.24, p=0.77), indicating both GLT-1-AAV and GFP-AAV reinstated to lever presses equally. There was an effect of Time indicating both groups increased lever presses during the reinstatement test compared to lever presses during extinction (F(1,16)=28.24, p=0.000).

Extinction

Cue test

32

Figure 3-8. Rats analyzed in the western blot data inactive lever presses during self-administration. Rats analyzed in the western blot data did not show a significant effect of Group x Time on inactive lever presses during self-administration for rats that were later administered GLT-AAV (n=5) or GFP-AAV (n=7) (F(3.9,46.9)=0.49, p=0.73). There was no significant effect of Time on the inactive lever during self-administration (F(3.9,46.9)=1.7, p=0.17).

33

Figure 3-9. The rats analyzed in the western data active lever presses during self-administration. The rats analyzed in the western data did not have a significant effect of Group x Time on active lever presses during self-administration for rats that were later administered GLT-1-AAV (n=5) or GFP-AAV (n=7) (F(11,121)=0.6, p=0.83). No significant effect of Time for active lever presses during self-administration was detected (F(11,121)=1.23, p=0.28).

34

Figure 3-10. Rats analyzed in the western blot data infusions during self-administration. Rats analyzed in the western blot data did not have a significant effect of Group x Time for infusions during self-administration between rats that later were administered GLT-1-AAV (n=5) or GFP-AAV (n=7) (F(11,121)=1.04, p=0.95). There was a significant effect of Time on infusions during self-administration (F(11,121)=4.83, p=0.000).

35

Figure 3-11. Lever presses on the previously active lever during extinction training for rats that were analyzed for the western blot data. There was no effect of Group x Time detected for presses on the previously active lever during extinction training for rats that were analyzed for the western blot data and had received GLT-1-AAV (n=5) or GFP-AAV (n=7) (F(11,121)=1.05, p=0.40). There was a significant effect of Time detected during extinction training (F(11,121)=15.85, p=0.000).

36

Figure 3-12. Western blot data of GLT-1 expression for each virus group and cocaine naïve controls. A one-way ANOVA omnibus revealed an overall significant main effect (F(2,15)=4.25, p=0.03). A Bonferroni Post-Hoc test revealed a significant difference in total GLT-1 protein amount between Cocaine Naïve Control (n=6) and rats that had received GFP-AAV (n=7), (t(1,11)=2.84, p<0.05). A Bonferroni Post-Hoc test revealed no difference for GLT-1 protein expression in cocaine naïve control rats and rats that had received GLT-AAV (n=5) (t(1,10)=0.82, p>0.05).

Figure 3-13. Western blot data of xCT expression for each virus group. An independent samples T-test revealed no significant difference in xCT protein expression for rats that had received GLT-1-AAV (n=5) and GFP-AAV (n=7) (t(1,10)=1.59, p=0.144).

*

37

CHAPTER 4 DISCUSSION

Chronic cocaine use alters glutamate systems in the nucleus accumbens that are

associated with addiction and relapse. Ceftriaxone prevents relapse by upregulating the

cystine-glutamate exchange, GLT-1 and restores glutamate homeostasis in the NAc

(Knackstedt et al., 2010). The current study upregulated GLT-1 using an adeno-

associated virus which did not altered the protein expression of xCT. Although

upregulation of GLT-1 is necessary for relapse prevention (Reissner et al., 2014), our

findings indicate upregulation of GLT-1 alone is not sufficient in preventing relapse (Fig.

3-5). Upregulation of GLT-1 is expected to increase removal of synaptically-released

extracellular glutamate therefore reducing the amount of glutamate binding to the post-

synaptic receptors during reinstatement. Here we found that the upregulation of GLT-1

in the NAc did not attenuate reinstatement, although it did restore levels of GLT-1a

expression following cocaine use (Fig. 3-11).

GLT-1 transporters are predominately located on astrocytes (Rimmel &

Rosenburg, 2016), and thus the decrease of colocalization between neurons and

astrocytes could be contributing to the ineffectiveness of upregulation of GLT-1 to

prevent relapse as the transporters may not be readily available to uptake glutamate

efficiently. Following cocaine self-administration and extinction, Scofield et al. (2016)

found a decrease in co-localization of neurons and glial cells in the nucleus accumbens

and a decrease in astrocyte size. Ceftriaxone reverses the colocalization of glial cells

and neurons therefore this could be an important factor in its ability to prevent relapse

(Scofield et al., 2016). Here, we upregulate only GLT-1a in glial cells. The upregulation

of transporters would not alter the colocalization of glial cells and neurons. Therefore,

38

the colocalization of the glial cells and neurons could be a vital alteration in preventing

reinstatement of cocaine-seeking.

Following chronic cocaine use, there is downregulation of cystine-glutamate

exchange, and therefore decreased tone on mGluR2/3 (Baker et al., 2002; Baker et al.,

2003). Stimulation of mGluR2/3 reduces synaptically released glutamate (Cartmell,

Schoepp, & Lilly, 2000; Smith et al., 2017). Repeated exposure to cocaine decreases

mGluR2/3 function (Xi et al., 2002) and results in an increase of synaptically released

glutamate (Moran et al., 2005). The upregulation of GLT-1 did not alter the expression

of cystine-glutamate exchange (Fig. 3-6); therefore, it is unlikely it altered basal

glutamate levels or mGluR2/3 tone. With the increased release of synaptically released

glutamate, the upregulation of GLT-1 may not be enough to prevent glutamate

postsynaptic binding, leading to downstream activation. Future research should quantify

glutamate levels during reinstatement after GLT-1 have been upregulated with the GLT-

1 AAV.

Ceftriaxone’s therapeutic effects also could be due to other alterations in the

brain. Ceftriaxone is administered through intraperitoneal injections and therefore affect

many areas in the brain and body. The upregulation of GLT-1 is observed in the PFC

following ceftriaxone treatment at higher doses (Dasa et al., 2015; Sari et al., 2009).

GLT-1 expression in various other brain regions have not be quantified but may be

altered by ceftriaxone treatment. For example, ceftriaxone increases the density of GLT-

1a in glial cells, as well as presynapticcally (Omrani et al., 2009). Also, GLT-1b

expression in the inferior colliculus is altered by ceftriaxone (Jhala, Wang & Hazell,

2011). The effects of GLT-1b upregulation, as well as other possible alterations by

39

ceftriaxone, have not been well characterized. Other glutamate transporters that may be

targeted by ceftriaxone, but not in the current study, could also be playing a role in

relapse prevention.

It should be noted that the AAV-GFAP-GLT-1a virus spread did not fill the

entirety of the NAc (Fig. 3-2), and thus reinstatement may have occurred due to the

presence of glutamate efflux in areas where there was no overexpression. In order to

investigate this possibility, in the future we plan to collect microdialysis samples during

reinstatement test with the probe directly in the virus spread. This will give us a clear

understanding of glutamate levels in the presence of upregulation of GLT-1a. However,

there are other reports of viral spread not filling the entire NAc and reinstatement of

cocaine-seeking was reduced (Knackstedt et al., 2010).

Although the majority of GLT-1 are located on glial cells (Danbolt, 2001; Murphy-

Royal et al., 2017), there is evidence that a portion of GLT-1 transporters are located on

hippocampus and cortical terminals (Chaudhry et al., 1995). The AAV used here

contained GFAP promoters, which allowed to specific targeting of glial cells, but would

not promote expression of the virus in neurons. Thus, presynaptic expression of GLT-1a

was not altered and may account for the lack of change in reinstatement behaviors. As

described above, the glia may have retracted and thus the GLT-1a may not be as

readily available to uptake glutamate as prior to cocaine use.

AAV upregulation of GLT-1 allowed us to observe the relationship between xCT

and GLT-1 expression in a unique way. Previous research has put emphasis on the co-

regulation of these two glutamate transport systems (Pendyam, Mohan, Kalivas & Nair,

2009). Upregulation of xCT by N-acetylcystein normalizes glutamate homeostasis and

40

restores tone on transporters and receptors (Baker et al., 2003). Here we found that

AAV-mediated over-expression of GLT-1a did not prodcue similar increases in xCT.

Although an increase in synaptically released glutamate occurs during cue-

primed reinstatement to drug-seeking (Smith et al., 2017), an increase of dopamine in

the nucleus accumbens core also occurs during cue-primed reinstatement (Ito et al.,

2000). Our focus on glutamate uptake did not alter dopamine release or uptake, and

thus the increase in synaptically released dopamine during the reinstatement test could

be contributing to the reinstatement to drug-seeking observed. The role of dopamine

binding in the NAc during cue-primed reinstatement hass not fully been defined and

could be playing a large role in the reinstatement of drug-seeking.

The present results suggest that, although the downregulation of GLT-1 plays a

vital role in reinstatement to drug-seeking and upregulation of the GLT-1 is necessary

for the prevention of reinstatement behaviors, the upregulation of GLT-1 is not the sole

alteration from ceftriaxone contributing to the prevention of reinstatement to drug

seeking. Upreglulation of GLT-1a did not alter xCT expression, which yeilds a better

understanding of the relationship between xCT and GLT-1a. Restoration of GLT-1a

expression to levels of cocaine naïve controls, while not altering xCT expression, is not

a sufficent means to prevent reinstatement of cocaine-seeking. This information will

aide in pharmacological treatments for relapse prevention in the future.

41

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BIOGRAPHICAL SKETCH

Carly Logan was born in Sharon Pennsylvania and attended Hubbard High

School, graduating in 2010. She then began her attendance at Kent State University. In

spring 2015, Carly graduated magna cum laude with a Bachelor of Science degree in

Psychology. In fall 2015, she began a PhD in behavioral and cognitive neuroscience

area within the Psychology Department at the University of Florida, with a research

focus on the neurobiology of addiction. Carly is awarded her Master of Science degree

in the psychology department in the behavioral and cognitive neuroscience area. Carly

is continuing her PhD in psychology at the University of Florida continuing her research

on the neurobiology of addiction.