AAV MEDI ATED U PREG ULATION OF GLT 1 DOE S NOT AT T ENUATE THE REINSTAT EMENT OF COCAINE SEE KING By CARLY N. LOGAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMEN TS 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 Ja ckson Brianna Parlette, Brianna Yaffe for their skills and dedication I thank Dr. Lori Knackstedt for guidance an d suppo rt 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 Pr evention 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 We stern Blotting ................................ ................................ ................................ ..... 22 Statistical Analysis ................................ ................................ ................................ .. 23 3 RESULTS ................................ ................................ ................................ ............... 25 Self admi nistration and Extinction ................................ ................................ ........... 25 Cue Primed Reinstatement ................................ ................................ ..................... 26 AAV GFAP GLT 1 Increases GLT 1 Protein Expression to Levels of Cocaine Nave 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 number s ................................ .................. 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 pre sses during self administration ................................ ................... 29 3 5 I nfusions attai ned during self administration ................................ ...................... 30 3 6 L ever presses on the previously active lever during the first twe lve days of extinction training ................................ ................................ ................................ 30 3 7 C ue primed reinstatement test ................................ ................................ ............ 31 3 8 Rats analyzed in the western blot data inactive lever presses during s elf administration ................................ ................................ ................................ ..... 32 3 9 The rats analyzed in the we stern data active lever pre sses during s elf administration ................................ ................................ ................................ ..... 33 3 10 Rats analyzed in the western blot data infusions during self administration ....... 34 3 11 Leve r p resses 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 an d cocaine nave controls ................................ ................................ ................................ .... 36 3 13 Western blot data of xCT expression for each virus group ................................ 36
8 LIST OF ABBREVIATIONS AAV Adeno associated virus CN S 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 Requir ements for the Degree of Master of Science AAV MEDI ATED UPREG ULATION OF GLT 1 DOE S NOT AT T ENUATE THE REINSTAT EMENT OF COCAINE SEE KING By Carly N. Logan May 2017 Cha ir: 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 administrati on extinction re instatement paradigm are useful tools to study relapse prevention. In th is model the operant response required for cocaine delivery is established, then extinguished and reinstated with cues previous ly 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 down regulation of the cystine glutamate exchange r and its catalytic subunit xCT and down regulation of GLT 1 glut a mate transporters. The antibiotic c eftriaxone has been shown to upregulate the cystine glutamate exchange r (xCT), and GLT 1 expression after cocaine use, ultimately p reventing reinstatement of drug seeking behaviors. T o determine which neurob iological 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, r eceiving injections of either AAV GFAP GLT 1 a 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 overexp ression occurred) before undergoing a cue primed reinstatement test. Rats that had received the AAV GFAP GLT 1 a reinstated cocaine se eking in a similar manner as the rats that had received AAV GFAP e GFP. These results indicate that the upregulation of GLT 1 transporters alone is not sufficient to prevent the reinstatement of cocaine seeking behaviors.
11 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, 200 2) Treatment and rehabilitation plans for cocaine addiction are complicated by h ig h rates of relapse even after long periods of abstinence (O'Brien, 2001). Current r esear ch is striving t o better understand the neurobiological mechanisms involved in addic tion to produce pharmacological therapies and more effective treatments for addiction and relapse prevention Operant S elf administration E xtinction reinstatement Animal M odel Animal models of substance abuse have been developed to exam ine the cellular an d molecular processes involved in acquisition, maintenance, and relapse to cocaine seeking The operant self administration extinction re instatement paradigm is considered a valid animal method for studying the relapse of drug seeking (Epstein et al., 2006 ) This paradigm includes a period of sel f adminis tration during which the animal perform s an operant behavior (such as pressing a lever) to receive an infusion of a drug. The d rug infusion is paired with a conditioned stimulus, such as illumination of a l ight above the lever and a tone. A n inactive lever is present that results in no drug infusion or conditioned stimuli presentation when pressed. This inactive lever preference for the active lever and serves as a locomotor contro l Once a pre set criterion of drug intak e is met animals enter a period of extinction training in the operant chamber during which both levers are extended but
12 presses on n either 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 (ligh t 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 significan tly more during the reinstatement test than during the previous days of extinction training (Katz & Higgins, 2005) Neurobiology U nderlying Cue primed Reinstatement of Cocaine s eeking The neurocircuitry of relapse has been thoroughly studied for decades and is well u nderstood. 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). Cu e primed reinstatement is blocked by pharmacological ( McLaughlin and See, 2003) and optical (Stefanik, Kupchik & Kalivas 2016) inactivation o f 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 glutamater gic activation of the basolateral amygdala and dopaminergic projections to the NAc (Everitt & Wolf, 2002 ; Kalivas & McFarland, 2003 ) Pharmacological i nactivation of the rostral basolateral amygdala (Kantak et al., 2002) and optical inactivation of the baso lateral amygdala (Stefanik et
13 al., 2013) blocks cue primed reinstatement. Inhibiting glutamatergic binding in the ventral tegmental a rea, which projects to the nucleus accumbens, basolateral amygdala and the prefrontal cortex ( Kalivas & McFarland, 2003 ) b locks 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 pr ojections from the prefrontal cortex, basolateral amygdala and ventral tegmental ar ea 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 bindi ng in the NAc is necessary for reinstatement as reinstat ement behaviors were blocked with an intra accumbens infusion of AMPA/kainate receptor antagonist CNQX whereas NMDA an tagonists did not attenuate reinstatement to cocaine seeking (Backstro m & Hyytia, 2007) There is an increase of AMPA receptors in the NAc after one month of cocaine withdrawal (Wolf & Tseng, 2012). Administration of m GluR5 antagonists ( Wang, Moussawi, Kna ckstedt, Shen & Kalivas, 2012) or infusion of negative allosteric modulators into the nucleus accumbens core attenuate cue primed reinstatement (Kum aresan, 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 i s necessary for cue primed reinstatement.
14 With consideration of certain receptor antagonist preventing reinstatement, it can be postulated that glutamate plays an important role in reinstatement to drug seeking. T here 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 increase d synaptically released gluta mate and decrease of glutamate uptake, stimulates mGluR5 producing n itric oxide which contributes to cue primed re instatement (Smith et al., 2017). These findings have narrowed relapse prevent ion research to glutamate rgic alter ations in the nucleus accumbens for possible pharmacological treatments Glutamate H omeostasis Glutamate is the major excitatory neurotransmit ter 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 exchange r (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 exchange r 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 pr imarily located in glial cells
15 and is a substrate not energy dependent system (McB ean & Flynn, 200 1 ; 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 grou p 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 b oth 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 glutama te 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 avoida nce 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 play s 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 G LT 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
16 responsible for removing 90% of synaptically rele ased 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 expr ess ion 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 termin al 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 expressio n 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 H omeostasis Glutamate regulatory systems within the nucleus accumbens are altered from chronic coc aine 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 f ollowing 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) Glutamater gic Alterations Associated with P revention of Reinstatement to D rug seeking Following cocaine use, there is a down regulation of xCT and GLT 1, and a decrease in basal extracellular glutamate. 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 c eftriaxone for six days fo llowing cocaine self administration increases protein expression of GLT 1 and xCT in the nucleus accumb ens ( Knackstedt et al., 2010; LaCrosse et al., 2016 ; Lew erenz et al., 2009 ) Ceftriaxone avoids the increase of extracellular synaptically released glutam ate 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 prime s ( Knackstedt et al., 2010) Ceftriaxone treatmen t attenuated reinstatement to cocaine seeking for weeks after administration has ceased (Sondheimer & Knacksted t, 2011). S ystemic injection s of N acetylcysteine, a cysteine pro d ru g, delivers large amounts of cysteine to the brain, and elevates extrace llul ar glutamate by increasing cystine glutamate exchange and thereby increasing glutamate export into the extrasynaptic space (Ba ke r al., 2003). N acetylcysteine pr events cocaine primed
18 increases in synaptically released glutamate and prevents rei nstatement t o 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., 201 4 ) 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 1 a expression using an adeno associated virus and examine cue primed of reinstatement of cocaine seeking We hypothesized upregulation of GLT 1 a would increase the re uptake of glu tamate, thus preventing the increase of synaptically released glutamate that has been observ ed during reinstatement of cocaine seeking, and therefore prevent reinstatement
19 CHAPTER 2 MATERIALS AND METHODS Subjects Twenty five a dult m ale Sprague Dawley rats (Charles River, 300 350 g; Raleigh, NC, USA ) w ere 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 o f standard lab chow daily and water ad libitum Surgical P rocedures Ketamine (87.5 mg/ kg, i.p.) and xylazine (5 mg/kg, i.p.) were administered to anesthetize the anim als prior to surgery. Keterolac was administered following surgery to provide analgesia. Catheters (SILASTIC silicon tubing, ID 0.51 mm, OD 0.94 m m, 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 cannul a (Plastics One, Roanoke VA, USA) embedded in a rubber harness (In stech, Plymouth Meeting, PA, USA ) worn by the rat for th e duration of the self a dministration. Immediately following catheter implantation, guide cannulas (Plastics One, Roanoke, VA, USA) w ere 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 l ) was administered for three days after surgery as an antibiotic. Catheters were fl ushed with heparinized saline ( 1 l 100 U/ml) prior to and following self administration sessions
20 Viral V ectors We employed two a deno associated virus es (AAV) constructed with an adeno helper pla s mid (pF6), and AAV helper encoding serotype 8, and the AAV packaging vector containing the GFAP promoter s to permit the spec ific 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 1 a plasmid ( Li et al. 2014) and was a k ind gift from David Poulson (SUNY Buffalo ) and was amplified and packaged into an AAV 8 by the UNC Viral Vector Core E ither A AV GFAP GLT 1 a or AAV GFAP eGFP was injected into the nucleus accumbens core using Harvard Apparatus pump at 0 .25 L/min. Injectors extended 1mm below the cannula into the NAc Cocaine Self administration E xtinction t raining, and R e instatement 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 chamber s under a FR1 schedule during two hour sessions. When the active lever was pressed intravenous infusion cocaine ( 0.35 mg/infusion ) was delivered Infusion s were paired sim ultaneously 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 i nactive lever were not reinforced but the lever presses were recorded The c riteria for self administration was ten or more infusions for twelve days Immediately following the conclusion of the 12 day self administration period, r ats began extinction training, d uring which time both levers were presented but presses on neither le ver resulted in programmed consequence s Rats received bilateral intra
21 NAc AAV GFAP GLT 1 (n=14 ) or the control AAV GFAP eGFP (n=16 ) immediately following the first se ssion of extinction training. Rats in both groups were either subjected to a reinstatement test (n=18) or were killed for weste r n blotting ( n=12). Following three week s of extinction training, a 1 hr cue primed reinstatement test was conducted. During thi s 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 t est were compared to the average number of presses during the final two days of extinction training. Histology and T issue P reparation 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 ) B rains 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. Brain s 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 Imm unohistochemistry to detect the his tagged AAV GFAP GLT 1 virus was c ompleted. Brain s lices were washed in PBST (PBS and 0.2% Triton) for three washes. The slices were then blocked in 10% Normal Goat Ser um in PBS before being probed with rabbit a nti his tag antibody overnight at 4 C The slices were then wash ed 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 bein g placed on slides and coverslipped using citifluor mounting solutions See T able 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 contai n ing potassium ( K PBS ) The slices were then blocked in a solution of 2% Normal Goat Serum in PBS a nd subsequently incubated in rabbit anti GFP at 4 C overnight. The slices we re then washed in KPBS (3 x 10in) before being in cubated i n go a t anti rabbit secondary anti body Th e slices are then washed three additional times in KPBS before being transferred to slides and coverslipped. Images were acquired on a fl uorescent microscope to confirm that viral overexpression was confined to the NAc Rats were eliminated from the behav ioral analysis if th ere was improper spread (e.g. up the cannula track) or improper placement ( not within the nucleus accumbens core). Western B lotting A separate group of rats (n=1 8 ) underwent surgery, self administration, extinction training and AAV infusion as described above but were not tested for reinstatement. I mmediately following the final day of extinction training, brains were extracted and NAc region was dissected on ice C ocaine nave rats were included also. These rats underwent surgeries, and remained in their home cages th rough out the experiment until their brains were extracted Following the brain extraction t he tissue was homogenized and frozen at minus 80. Western b lotting w as performed for GLT 1 a and xCT expression. Proteins were sepa rated 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 25m M, NaCl 0.13M, KCl 0.0027M, 1ml Tween20; 4 x 10min) For GLT 1 a protein quantification, t he 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 secondar y antibody was incubated for two hours at room temperature. After being washed in TBST (4 x 10min), t he membrane was exposed to Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Limited, Little Chalfont, United Kingdom ) and were develope d 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 A nalysis Behavior al 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 p ost hoc tests were used when necessary Repeated measure (RM) analy ses of variance ( ANOVAs) were used to compare the number of lever p resses 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 Imm unohistochemist ry 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 E xtinction For rats later tested for reinstatement, r epeated measures ANOVA of the inactive lever presses (Fig. 3 3 ) during self administration revealed no si gnificant 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 diffe rence for rats later assigned to received GLT 1 A AV 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(3.5,6 2. 9)=3.42 p=.018). There wa s no significant effect of Group (F(1,1 6 )=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 A AV or GFP AAV ( F(4.6,16)=.935 p=.458) A si gnificant 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 sig nificant effect of Group (F(1,16 )=1.08, p=.312). There was no significant effect of G roup 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 e xtinction training for t he 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 R einstatement The animals were tested for cue primed reinstatement to dru g seeking behaviors during a one hour tes t during which the a ctive 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=.0 06), 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 I ncr eases GLT 1 Protein E xpression to L evels of Cocaine Nave C ontrol Protein expression was measured in a se parate 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 Ti me (F(3.9,46.9)=0.49, p=0.73) or Ti me (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(11,12 1)=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 1 1 ) (F(11,121)=15.85, p=.000) and no significant effect of Group (F(1,16)=6.95, p=0.595) T here 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 coca ine nave control rats (Fig. 3 12 ) revealed a significant overall effect (F( 2,15)=4.25, p=.03). Bonferroni post ho c test s 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 s ignificantly less GLT 1 protein than cocaine nave 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 nave control animal. AAV GFAP GLT 1 Expression does not Alter xCT Protein A mount 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, 1 0)=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. Fig ure 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 can nula 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 comm issure A.C.
29 Figure 3 3 Inactive lever presses during self administration Inactive lever presses during self administrati on 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 A ctive 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 Th e 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 ) ] F igure 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 signifi cantly 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 f amilywise error inflation (therefore = 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 reinstatemen t test compared to lever presses during extinction (F(1,16)=28.24, p= 0 .000). Extinction Cu e test
32 Fig ure 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 effe ct 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 administratio n (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 no t have a significant effect of Group x Time on active lever presses during s elf 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 A AV (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 Fig ure 3 11 Lever presses on the previously active lever during extinction training for ra ts that were analyzed for the western blot data There was no effect of Group x Time detected for presses on the previously active lever duri ng extinction training for rats that were analyzed for the western blot data and ha d received GLT 1 AAV (n=5) or GF P 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 1 2 Western blot data of GLT 1 expression for each virus group and cocaine nave 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 Nave 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 nave control rats and rats that had received GLT AAV (n=5) (t(1,10)=0.82, p>0.05). Figure 3 1 3 Western blot data of xCT expression for each virus group An independent samples T test revealed no significant difference in x CT 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 system s in the nucleus accumbens that are associated with addiction and relapse. Ceftriaxone prevents relapse by upregulating the cystine glutamate exchange, GLT 1 and restores g lutamate homeostasis in the NAc (Knackstedt et al., 2010). The current study upregul ated GLT 1 u sing an adeno ass ociated virus which did not altered the protein expression of xCT Although upregulation of GLT 1 is necessary f or relapse prevention (Reissner et al. 2 014 ) our findings indicate upregulation of GLT 1 alone is not sufficient in pre venting relapse (Fig. 3 5) Upregulation of GLT 1 is expected to increase removal of synaptically released extr acellular glutamate therefore reducing the amount of glutamate binding to the post synaptic receptors during reinstatement Here we found that th e 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, S cofield et al. (2016) foun d 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 impor tant factor in its ability to prevent relapse (Sco field et al., 2016) Here we up regulate only GLT 1 a in glial cells. The upregulation of transporters would not alter t he colocalization of glial cells a nd neurons. Theref ore,
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 cyst ine glutamate exchange, and therefore decreased tone on mGluR2/3 (Baker et al., 2002 ; Baker et al., 2003 ). S timulation of mGluR2/3 reduces synap tically released glutamate (Cartmell, Schoepp, & Lilly, 2000 ; Smith e t a l., 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 increase d release of synapt ically released glutamate, the upregulation of GL T 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 to o ther alterations in the brain. Ceftriaxone is administered through i ntraperitoneal injections and therefore a ffect many areas in the brain and body T he upregulation of GLT 1 is observed in the PFC following ceftriaxone treatment at higher do ses ( Dasa et al. 2015 ; Sari et al., 2009 ) GLT 1 expres sion 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 glia l 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 characteriz ed. 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 t he 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 w as reduced ( Knackstedt et al., 2010) Alt hough the majority of GLT 1 are located on glial cells ( Danbolt, 2001; Mur phy Royal et al. 2017) there is evidence that a portion of GLT 1 tran sporters 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 desc ribed 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. Pre vious research has put emphasis on the co regulation of these two glutamate transport systems (Pendyam, Mohan, Kalivas & Nair 2009) Upregulation of xCT by N acetylcystei n normalizes glutamate homeostasis and
40 restores tone on transporters and receptors (B aker 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 re instatement test could be contributing to the reinstatement to drug seeking observed. The role of dopamine binding in the NAc during cue primed reinstateme nt has s not fully been defined and could be playing a large role in the reinstatement of drug seekin g. The present results suggest that a lthough the downregulation of GLT 1 play s a vital role in reins tatement 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 so le alteration from c eftriaxone contributing to the prevention of reinstatement to drug seeking. U preglulation of GLT 1a did not alter xCT expression, which yeilds a better understanding of the relationship between xCT and GLT 1a. Restoration of GLT 1 a expression to levels of cocaine nave controls, while not altering xCT expression, is not a sufficent m eans to prevent reinstatement of cocaine seeking This information will aide in pharmacological treatments for relapse prevention in the future.
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48 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 S cience degree in Psychology. In fall 2015, she began a PhD in behavioral and cognitive neuroscience area within the Psychology Department at the Universit y 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.