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Interference with AMPA receptor endocytosis : effects on behavioral and neurochemical correlates of amphetamine… Choi, Fiona Y. 2013

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INTERFERENCE WITH AMPA RECEPTOR ENDOCYTOSIS: EFFECTS ON BEHAVIORAL AND NEUROCHEMICAL CORRELATES OF   AMPHETAMINE SENSITIZATION by Fiona Y. Choi  B.A., The University of British Columbia, 2000 M.A., California State University, San Bernardino, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE and POSTDOCTORAL STUDIES (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  November 2013  ?Fiona Y. Choi, 2013  ii Abstract  A hallmark of drug addiction is the compulsive drug taking behaviour that persists despite detrimental and adverse consequences.  The gradual sensitization to the effects of drugs has been proposed to be an underlying mechanism for increased drug use and it serves as an animal model of craving and enduring neural plasticity.  Evidence from studies conducted in rodents, non-human primates and humans have supported a role for sensitization in the development of states that promote drug taking.  Rodent studies have also demonstrated that repeated exposure to drugs producing behavioural sensitization increases dopamine (DA) levels in the nucleus accumbens (NAc) and contributes to the acquisition of drug self-administration behaviour as well as the effort level to actively acquire a drug.  The changes in neural functioning following repeated drug exposure is attributed to alterations in synaptic connections that underlie and are crucial for normal brain function as well as mechanisms of learning and memory.   There is recent evidence supporting the development of a new class of interference peptides aimed at repairing functional and structural alterations in brain regions implicated in a number of psychiatric disorders. One such interference peptide, Tat-GluA23Y, blocks long-term depression (LTD) at glutamatergic synapses and has also been demonstrated to block the expression of behavioural sensitization and significantly reduce cue-induced reinstatement of heroin self-administration. This peptide interferes with the interaction between ?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor and Brag2, a clathrin-adaptor protein which initiates the process of AMPA receptor endocytosis, leading to LTD.  Results from the studies contained in this dissertation suggest that modulating protein-protein interactions with Tat-GluA23Y can  iii influence the synaptic modifications following repeated amphetamine exposure, to effectively block the development and long-term maintenance of behavioral sensitization in a context-dependent manner.  Effects of this peptide can be applied to understanding the circuitry and mechanisms involved in the development of psychostimulant sensitization, possibly serving as a platform from which to develop a novel pharmacotherapeutic approach for treating drug addiction.    iv  Preface  A version of chapter 2 has been published: Choi, F.Y., Ahn, S., Wang, Y.T., and Phillips, A.G. (2013). Interference with AMPA Receptor Endocytosis: Effects on Behavioral and Neurochemical Correlates of Amphetamine Sensitization in Male Rats. Journal of Psychiatry and Neuroscience, 38(6):120257. [Epub ahead of print]. F.Y. Choi, S.Y. Ahn, Y.T. Wang, and A.G. Phillips designed the research; F.Y. Choi and S. Ahn performed the animal surgeries; S.Y. Ahn and F.Y. Choi collected the microdialysis data; F.Y. Choi conducted all the behavioral experiments; F.Y. Choi and A.G. Phillips wrote the manuscript; and S. Ahn and Y.T.Wang provided comments and edits on the manuscript.  Experiments described in chapter 3 were designed by F.Y. Choi under the guidance of A.G. Phillips.  All animal surgeries, behavioral work, data collection and analysis were conducted by F.Y. Choi.    Certificate of approval: The animal studies presented in this thesis were performed with ethics approval from the University of British Columbia Animal Care Committee (certificate # A09-0281 and A09-0805).  v Table of Contents Abstract ............................................................................................................................... ii Preface................................................................................................................................ iv Table of Contents ................................................................................................................ v List of Figures ................................................................................................................... vii Acknowledgements ............................................................................................................ ix Chapter One: General Introduction ..................................................................................... 1 1.1 Social factors and epidemiology of human drug addiction .................................... 1 1.2 Neurobiology of addiction ...................................................................................... 5 1.3 Animal models of addiction .................................................................................. 12 1.4   Synaptic plasticity: LTP and LTD ........................................................................ 17 1.5   Evidence linking synaptic plasticity and addiction ............................................... 18 1.6   Research objectives and hypotheses ..................................................................... 21 Chapter Two: Interference with AMPA Receptor Endocytosis: Effects on Behavioral and Neurochemical Correlates of Amphetamine ..................................................................... 23 2.1 Overview ............................................................................................................... 23 2.2 Introduction ........................................................................................................... 24 2.3   Methods................................................................................................................. 26 2.4   Results ................................................................................................................... 35 2.5   Discussion ............................................................................................................. 45 2.6   Limitations ............................................................................................................ 48 2.7   Conclusions ........................................................................................................... 49 Chapter Three: The Role of AMPA Receptor Endocytosis in Context-dependent Behavioral Sensitization ................................................................................................... 51 3.1   Overview ............................................................................................................... 51 3.2   Introduction ........................................................................................................... 52 3.3   Methods................................................................................................................. 55 3.4   Results ................................................................................................................... 60  vi 3.5   Discussion ............................................................................................................. 68 Chapter Four: General Discussion .................................................................................... 73 4.1   Overview ............................................................................................................... 73 4.2   Behavioral and neurochemical correlates of amphetamine sensitization and disruption with the interference peptide, Tat- GluA23Y ................................................ 76 4.3   Distinctive neuroadaptations within the reward circuitry are responsible for induction, maintenance, and expression of behavioral sensitization ............................ 78 4.4   AMPAR endocytosis and subunit composition is crucial in maintaining the neural plasticity underlying behavioral sensitization ............................................................... 82 4.5   Context-dependent behavioral sensitization and the pivotal role of AMPA receptor endocytosis...................................................................................................... 87 4.6   Summary ............................................................................................................... 90 4.7   Translational implications ..................................................................................... 91   vii List of Figures  Figure 1. Neurotransmitter pathways and receptor systems implicated in the acute reinforcing effect of drugs of abuse. ........................................................................... 7 Figure 2.1 Experimental protocol used to study the effects of GluA23Y on the induction and maintenance of d-AMPH-induced sensitization. ............................................... 30 Figure 2.2  Placement of microdialysis probes and microinjection cannulae................... 32 Figure 2.3  Diffusion of dansyl-tagged interference peptide. ........................................... 34 Figure 2.4 Time course and cumulative data of locomotor activity and DA efflux in the NAc on Day 1 and Day 9 of the induction of d-AMPH behavioral sensitization. ... 37 Figure 2.5 Expression of d-AMPH sensitization.  After a 14-day incubation period, all animals received a challenge dose of d-AMPH (0.5 mg/kg, IP). ............................. 40 Figure 2.6  Time course and cumulative data of locomotor activity during induction of d-AMPH behavioral sensitization following microinjection of Tat-GluA23Y or controls into VTA or NAc. ..................................................................................................... 42 Figure 2.7 Expression of behavioral sensitization to a challenge dose of d-AMPH in animals that received microinjections of Tat-GluA23Y or controls. ......................... 44 Figure 3.1 (Top) Treatment and Experiment 1 Timeline (Bottom) Panel A: Day 1 and Day 9 of Induction treatment period.  Panel B: Day 24 d-AMPH Challenge. Panel C: Second Induction treatment period, Day 24 and Day 32. Panel D: Day 47 final d-AMPH Challenge ...................................................................................................... 62 Figure 3.2 (Top) Treatment and Experiment 2 Timeline (Bottom) Panel A: Induction in Context A followed by d-AMPH Challenge in Context A & B. Panel B: d-AMPH Challenges in Context B and final Challenge in Context B ..................................... 66 Figure 3.3 (Top) Treatment and Experiment 3 Timeline (Bottom A) Induction of d-AMPH sensitization on Experimental Day 1 or 3 and Day 13 or 15. [NP-Env: Non-paired Environment; P-Env: Paired Environment]. (Bottom B) Final d-AMPH challenge on Experimental Day 30 and Day 32. Treatment Groups depict drug treatment given during the Induction phase: Vehicle + Saline;Vehicle + d-AMPH; GluA23Yscr + d-AMPH; or GluA23Y + d-AMPH.  On the Challenge days, d-AMPH is given in the treatment paired context and non-paired context on alternate days. .......................................................................................................................... 67 Figure 4.1 Schematic model of the circuitry involved in drug-induced neural plasticity underlying induction, maintenance and expression of behavioral sensitization. ...... 81 Figure 4.2  Model of drug-induced synaptic and structural plasticity in excitatory synapses onto MSNs in the NAc ............................................................................. .84  viii Figure 4.3  Model of drug-induced synaptic plasticity in excitatory synapses onto VTA DA neurons ............................................................................................................... 85   ix Acknowledgements I am extremely grateful to have studied under the mentorship of Dr. Anthony Phillips. His enthusiastic support and guidance are very much appreciated and I greatly respect his scientific curiosity and his approach to research, which will continue to be a source of inspiration to me.   I would like to thank my supervisory committee, Dr. Yu Tian Wang, Dr. Jeremy Seamans, Dr. Allen Young, and Dr. Michael Krausz, who have kindly taken the time to provide their expertise and advice to guide and advance my thesis research. There are also many other people who have helped make this work possible.  Thank you to all my friends and colleagues (both past and present) in the Kinsmen labs and the Brain Research Center, with special thanks to: Dr. Soyon Ahn, Dr. Pamela Arstikaitis, Dr. Karen Brebner, Dr. Kelly Butts, Caitlin Celinski, Dr. Ainsley Coquinco, Dr. Gemma Dalton, Dr. Carine Dias, Fred LePiane, Bonita Ma, Liya Ma, Diane Parsons, Kitty So, Giada Vacca, and all the dedicated staff of the Animal research unit.   I would also like to acknowledge the funding I received from the CIHR through a Doctoral Research Fellowship (IMPART) as well as a research assistantship through an operating grant (MOP 38069). Finally, I would especially like to thank those closest to me, my parents, Patrick and Anita, my sister, Yolanda, and my husband, Kevin, for their constant encouragement and unwavering support during my studies.  It would not have been possible without each one of you.    1 Chapter One: General Introduction  1.1 Social factors and epidemiology of human drug addiction  A hallmark of drug addiction is the compulsive drug-seeking behaviour that persists despite adverse consequences detrimental to both quality of life and health.  In particular, abuse of the psychostimulants amphetamine and its analogs, methamphetamine (known colloquially as speed, ice, crystal) and 3,4-methylenedioxy-N-methylamphetamine (MDMA) "Ecstasy", have become a concern of modern society.  In partnership with the Canadian Centre on Substance Abuse (CCSA), the Student Drug Use Surveys Working Group collected data on student drug use between 2007-2008.  Based on these surveys, 7.1% of students (grades 7, 9, 10, 12) in British Columbia reported lifetime use of ecstasy or amphetamines (CCSA, 2011).  According to the Canadian Alcohol and Drug Use Monitoring Survey (CADUMS) conducted by Health Canada in 2009, excluding cannabis, the most commonly reported drugs used during one?s lifetime are hallucinogens, used by 11.4%, and cocaine (10.6%), and then speed (6.4%) and ecstasy (4.1%).  The prevalence of use of at least one of five drugs (which include: cocaine or crack; speed; ecstasy; hallucinogens or heroin) in the past-year was 2.0%, however, the rate of use by youth at 5.5% was almost four times higher than that reported by adults at 1.3% (CADUMS, 2009).  Compared with youth who have never used, reports show that amphetamine users initiate other drug use at younger ages, use more types of drugs including injection- drugs, report more emotional distress and engage in higher-risk sexual behavior (Rotheram-Borus et al., 1999). Amphetamines have a number of pharmacological properties that account for their popularity and abuse potential.  For example, amphetamines are abused recreationally for their   2 ability to promote the rapid onset of euphoria, elevating mood and an overall sense of well-being (Hegadoren et al., 1999).  In addition, long-distance drivers, physicians, students, construction workers, and athletes, to name a few, may use amphetamines inappropriately in occupational settings for their ability to increase stamina, promote wakefulness and relieve stress (Nencini and Ahmed, 1989;Singh and Jindal, 1980).  The availability and relatively low cost of illicit amphetamines also contribute to increased abuse rates.  In contrast to cocaine, methamphetamine can be manufactured very cheaply from ingredients found in over-the-counter nasal decongestants and ingredients such as petroleum fuel, lye, and lithium from lithium-ion batteries. Therapeutic doses of amphetamines range from 5mg to 60mg and generally increase alertness, energy and a feeling of well-being, while decreasing appetite.  Physiological effects include rapid heartbeat and breathing, increased blood pressure, sweating, dilated pupils and dry mouth.  The user may become more talkative or excitable and this is sometimes coupled with the feeling of power and superiority and acts of hostility or aggressive behavior.  With larger doses, the effects transition to cardiac symptoms such as arrhythmia (irregular heartbeat) or tachycardia (very rapid heartbeat), tremors, severe paranoia and hallucinations.  Effects of amphetamine may be fatal due to heart failure, extreme high fever, or rupture of brain aneurysm leading to hemorrhagic stroke.  In some cases, violent or risky behavior can lead to accidental amphetamine-related death.  Chronic misuse of amphetamines can also lead to long-lasting impairments in brain function due to a general loss of dopaminergic terminals and transmission in the central nervous system as well as memory deficits (Cretzmeyer et al., 2003;Robinson and Becker, 1986).  In particular, repeated amphetamine administration can produce paranoia, delusions, or psychosis, a mental illness similar to paranoid schizophrenia   3 (Cretzmeyer et al., 2003;Lecomte et al., 2010;Satel et al., 1991).  Substance-induced psychosis can occur following a high dose of drug, or from prior exposure, producing an enduring form of psychosis (Barr et al., 2006), while users are more likely to have a psychotic relapse when confronted with stressful situations, even after years of cessation of drug use (Iyo et al., 1997).  In addition, family history of mental illness or substance abuse and a previous diagnosis of a mental illness are high predictors of co-occurring methamphetamine abuse and psychosis (Lecomte et al., 2010).  Those who prepare injectable mixtures from tablets or capsules are also at risk of infection from contaminated syringes and susceptible to kidney damage, while those who use inhalation methods risk damage to the lungs.  Regular use of amphetamines can produce psychological and physical dependence, compulsive use, craving and the experience of withdrawal symptoms such as extreme fatigue, disturbed sleep, depression and violent behavior (Ernst et al., 2000). Sustained high-doses of amphetamines can lead to a persistent depletion of brain dopamine (DA), which is associated with terminal degeneration and the redistribution of DA from synaptic vesicles into the cytosol, leading to cytosolic oxidative stress that may initiate amphetamine neurotoxicity (Guillot et al., 2008).  The cause of behavioural and cognitive impairments following long-term amphetamine use is still being investigated, however, positron emission tomography (PET) studies report that methamphetamine abusers have a lower level of dopamine D2 receptors in the orbitofrontal cortex and significant losses in dopamine transporters, which are structurally important to dopamine terminals (Volkow et al., 2001).  The orbitofrontal cortex is associated with compulsive behaviours, therefore, disruption in this brain region may contribute to the compulsive nature of addictive behavior (Volkow and Fowler, 2000;Volkow et al., 1999).   Additional evidence for the negative   4 impact of amphetamine exposure on brain functioning and behaviour comes from studies with adult rodents.  Repeated d-AMPH exposure induces cell death in the central nervous system and produces long-term changes in cognition and behaviour (Volkow et al., 2002;Cretzmeyer et al., 2003;Robinson and Berridge, 2001).  Given that amphetamine abuse can cause long-term changes in brain function, it is alarming that there is an increasing use of amphetamines amongst the younger population, both in Canada, as previously mentioned, and in the US, according to results from the 2012 National Survey on Drug Use and Health (NSDUH) conducted by the Substance Abuse and Mental Health Services Administration (SAMHSA, 2013).  Moreover, a large number of young children are also exposed to the amphetamine-derivative, methylphenidate, commonly known by the trade name, Ritalin, as a treatment for attention-deficit-hyperactivity-disorder (ADHD).  Although the subject of some controversy, ADHD is a behavioral condition generally with onset during childhood and is characterized by hyperactivity, impulsivity and the inability to concentrate leading to negative effects on learning and academic performance.   The pre-exposure to methylphenidate may increase the response to other psychostimulants later in life due to cross-sensitization between stimulant drugs.  Cross-sensitization has been shown in rodent studies in which chronic treatment with methylphenidate during the juvenile period led to a sensitized behavioral response to an acute exposure of amphetamine in adulthood (Yang, Swann & Dafny, 2003).  In other preclinical studies, pre-exposure to a drug has been shown to increase rates at which the drug is self-administered at a later time (Lorrain et al., 2000;Mendrek et al., 1998;Robinson et al., 1988).  The fact that prolonged exposure to stimulant drugs can induce heightened behavioral responding upon future re-exposure is one of the most striking features of amphetamine sensitization and is known formally as Behavioral Sensitization (Robinson and Becker, 1986).    5 The phenomenon of behavioral sensitization has been the subject of an intense research effort over the past two decades and will be discussed in greater detail in this thesis.  Sensitization is linked directly to the long-lasting sensation of drug craving, which may persist despite lengthy periods of abstinence, thus serving as an important contributing factor to the highly addictive property of drugs of abuse (Childress et al., 1999;Kalivas et al., 1998;Robinson and Berridge, 1993).  Indeed, the persistence of craving suggests that repeated exposure to certain drugs have the ability to produce some long-term neurobiological changes that occur during the drug-taking period and remains altered thereafter (Luscher and Malenka, 2011;Nestler, 2001;Robinson and Kolb, 1997).  A major theme of this thesis is the firm belief that further basic research holds the key to understanding the process of addiction and the subsequent development of new and more effective treatment strategies to prevent relapse in addicted individuals.    1.2 Neurobiology of addiction 1.2.1 The motive and reward circuit: A key role for dopamine The brain reward system is organized to respond appropriately to natural rewards such as food and sex, essential for health, survival, and reproduction.  This so-called ?reward system?, which employs dopamine (DA) as its main neurotransmitter, has also been referred to as the ?incentive motivation? system (Mogenson and Phillips, 1978), and is responsible for initiating movement towards salient environmental stimuli (incentives) that signal the availability of biologically significant outcomes such as food or water.  Addictive drugs also act primarily on the mesocorticolimbic DA system, thereby usurping the motivation to seek natural rewards in favour of their artificial hedonic sensations with even higher incentive value (Wise, 1996).     6 Figure 1 outlines the neurotransmitter pathways and receptor systems implicated in the acute reinforcing effects of drugs within the reward circuit.  The mesocorticolimbic DA system, a principal component of the neural reward circuitry, is comprised of DA neurons originating in the ventral tegmental area (VTA) that project to the core of the nucleus accumbens (NAc), amygdala, prefrontal cortex (PFC), and dorsolateral compartment of the ventral pallidum (VP) and other forebrain regions (Cooper et al., 1996).  The VTA receives both excitatory input through glutamatergic input and inhibitory input via ?-aminobutyric acid (GABA) interneurons (Lammel et al., 2012;Geisler et al., 2007)  Projections from the amygdala innervate the shell of the NAc, the ventromedial compartment of the VP, and the bed nucleus of the stria terminalis (BNST) (Edwards and Koob, 2010).  The VTA is also interconnected with the substantia nigra and pedunculopontine motor region.  The PFC (dorsal, prelimbic) preferentially projects to the NAc(core) (Cooper et al., 1996).  The motive circuit plays a critical role in translating environmental or pharmacological stimuli into behavioral responses.  A number of lesion studies as well as the microinjection of transmitter analogues have demonstrated that the NAc, amygdala, PFC or VP can inhibit or promote the acquisition and/or maintenance of conditioned responding to food or drug reward (Balleine and Killcross, 1994;Phillips et al., 2003;Ahn and Phillips, 2002;Fibiger and Phillips, 1988).    7   Figure 1. Neurotransmitter pathways and receptor systems implicated in the acute reinforcing effect of drugs of abuse.   Cocaine and amphetamines increase dopamine levels in the nucleus accumbens and amygdala via direct actions on dopamine terminals. The blue arrows represent the interactions within the extended amygdala system hypothesized to play a key role in psychostimulant reinforcement. The medial forebrain bundle represents ascending and descending projections between the ventral forebrain (nucleus accumbens, olfactory tubercle and septal area) and the ventral midbrain (ventral tegmental area; not shown in figure for clarity).  AC: Anterior commissure; AMG: Amygdala; ARC: Arcuate nucleus; BNST: Bed nucleus of the stria terminalis; Cer: Cerebellum; C-P: Caudate-putamen; DMT: Dorsomedial thalamus; FC: Frontal cortex; Hippo: Hippocampus; IF: Inferior colliculus; LC: Locus coeruleus; LH: Lateral hypothalamus; MFB: Medial forebrain bundle; N Acc.: Nucleus accumbens; OT: Olfactory tract; PAG: Periaqueductal gray; RPn: Reticular pontine nucleus; SC: Superior colliculus; SNr: Substantia nigra pars reticulata; VP: Ventral pallidum; VTA: Ventral tegmental area.  Reprinted with permission from (Edwards and Koob, 2010).   8 Several hypotheses have been developed to understand the function of the mesocorticolimbic dopamine system in response to drug reward.  The hedonic hypothesis proposes that mesocorticolimbic DA mediates the pleasure of reward stimuli and anhedonia during withdrawal (Koob and Le Moal, 2001;Koob and Le Moal, 2008).  Evidence is provided by reports that this system is activated by natural and drug rewards, and that blocking its activity impairs the behavioral effects of most natural and drug rewards (Guillot et al., 2008;Wise, 1996).  A competing hypothesis focuses on the role of learning, suggesting that through cellular mechanisms of associative learning, the prediction of rewards lead to learned drug-taking habits (Belin et al., 2009;Hyman et al., 2006;Koob and Le Moal, 2001).  This hypothesis is based in part on evidence that certain populations of DA neurons fire in response to cues that predict rewards rather than to rewards per se (Schultz, 2000).  The third hypothesis is the incentive-sensitization theory of motivation which suggests that neural sensitization leads to an excessive value placed on reward-associated stimuli and behaviors which increases the incentive "wanting? to seek drugs (Robinson and Berridge, 1993;Hyman and Malenka, 2001;Robinson and Berridge, 2001).  Studies show that the simple hedonic effects or ?liking? for a reward does not require mesolimbic DA, although the system is important for motivating behavor to obtain the reward (Berridge and Robinson, 2003;Berridge et al., 2009).  These different perspectives, with a focus on learning, ?wanting?, and ?liking? share common components and rather than being mutually exclusive, commonalities help in explaining the role of the mesocorticolimbic DA system in addiction.   1.2.2 Dopamine transmission Dopamine is a monoamine neurotransmitter that along with norepinephrine, constitutes the catecholamine family of neurotransmitters.  Dopamine synthesis involves the conversion of   9 the amino acid, tyrosine, to L-dihydroxyphenylalanine (L-Dopa) by the enzyme, tyrosine hydroxylase (TH) (Cooper et al., 1996).  L-Dopa is then converted to dopamine by the enzyme, dopa decarboxylase (Cooper et al., 1996).  The release of DA from the nerve terminal into the synaptic cleft is calcium-dependent and results from nerve impulse stimulation in the form of an action potential.  The effects of the released DA in the synaptic cleft are mediated by binding to DA receptors (Meador-Woodruff, 1994).  DA receptors are found both pre- and postsynaptically on both DA and non-DA cells (Meador-Woodruff, 1994) and are classified into two families, either D1-like (D1 and D5 subtypes) or D2-like (D2, D3, and D4 subtypes).  D1-like receptors are coupled to G?s/olf proteins, stimulate adenylyl cyclase activity, increase levels of the second messenger cyclic-AMP (cAMP), and activate protein kinase A (PKA), mediating the direct pathway and excitatory transmission (Dietz et al., 2009;Surmeier et al., 2007) .  On the other hand, D2-like receptors are coupled to G?i/o proteins, which inhibit adenylyl cyclase and can lead to activation of protein kinase C (PKC), IP3 activation and the mobilization of intracellular calcium stores, mediating the indirect pathway (Dietz et al., 2009;Surmeier et al., 2007).  Dopamine autoreceptors are presynaptic receptors located on the soma, dendrites, and nerve terminals of dopamine cells (Wolf and Roth, 1990).  Stimulation of autoreceptors on the soma or dendrites causes a decrease in the firing rate of the dopamine neurons whereas activation of presynaptic autoreceptors on the nerve terminals result in diminished dopamine synthesis (Starke et al., 1989;Wu et al., 2002;Wolf and Roth, 1990).  Postsynaptic DA receptors are also found on many nondopaminergic cells including GABA and acetylcholine neurons in the striatum and regions of the frontal cortex (Cooper et al., 1996).    10 Inactivation of DA after release is accomplished mainly by the dopamine transporter (DAT) in the membrane of presynaptic DA neurons (Gainetdinov et al., 1998;Giros and Caron, 1993).  DAT plays a significant role in the reuptake of extracellular dopamine, thereby regulating synaptic neurotransmitter concentration (Gainetdinov et al., 1998;Giros and Caron, 1993).  After reuptake into the nerve terminal, DA may be metabolized by monoamine oxidase (MAO) and converted to DOPAC or repackaged into synaptic vesicles and re-released (Meador-Woodruff, 1994).  Within the synaptic cleft and extracellular space, DA may be converted by catechol-O-methyltransferase (COMT) into homovanillic acid (HVA) (Cooper et al., 1996;Meador-Woodruff, 1994). 1.2.3 Amphetamine: Mechanisms of action Amphetamine is a psychostimulant that increases the synaptic release of the monoamines, dopamine, norepinephrine and serotonin (Koob et al., 1998;Self and Nestler, 1995).  Amphetamine works primarily by reversing the transport of monoamines by binding to monoamine reuptake transporters, especially DAT (Pierce and Kalivas, 1997;Weiss and Koob, 2001;Kalivas and Stewart, 1991).  In addition, amphetamine also blocks monoamine reuptake and inhibits monoamine oxidase (Genova et al., 1997;Vanderschuren and Kalivas, 2000).  The increased release of monoamines, primarily DA and its subsequent effects on mood and motivation are believed to be the reason amphetamines are pleasurable and abused by humans (Pierce and Kalivas, 1997;Robinson and Berridge, 2001;Self and Nestler, 1995).  Administration of a single acute dose of amphetamine is capable of stimulating extracellular dopamine release and causes dose-dependent elevations of dopamine in the brain (Pierce and Kalivas, 1997)  Amphetamine, at doses of 1.0, 3.0, and 9.0 mg/kg, can elevate extracellular DA levels to 700%, 800%, and 1300% of basal levels in the rat brain, respectively (Di Ciano   11 et al., 1995).  The displacement of DA from storage vesicles, inhibition of monoamine oxidase, and the blocking of reuptake transporters suggests that amphetamine is capable of producing neurotoxic effects on brain pathways (Pierce and Kalivas, 1997;Weiss and Koob, 2001).  In addition, the massive release of serotonin from presynaptic vesicles leads to an eventual depletion of serotonin in cells and further contributes to the neurotoxic effects of amphetamine.  Behavioural studies show that acute administration of low to medium doses of amphetamine to rats causes increased locomotor activity, while high doses of the psychostimulant can induce stereotypy, such as compulsive licking and gnawing (Robinson and Becker, 1986).  Acute administration of amphetamine can also lead to an enhanced sensitivity to stressful stimuli, a decreased response to natural reinforcers, and a decreased threshold for helplessness (Volkow et al., 2002). Amphetamine also causes an increase in extracellular glutamate (Wolf and Xue, 1998;Wolf and Xue, 1999).  There are several explanations for this increase in glutamate activity after systemic administration of amphetamine.  Glutamate reuptake may be affected by D1 receptor stimulation of Na+ and K+ gated channels of neuronal cells (Wolf and Xue, 1998).  Amphetamine may also increase Ca2+-independent glutamate release through ?hypoxia-induced reversal? of the glutamate reuptake transporter (Vanderschuren and Kalivas, 2000).  In addition, the reuptake of glutamate may be blocked or impeded by a number of agents such as: oxygen radicals, nitric oxide, or arachidonic acid, which remains in the extracellular space following amphetamine administration (Wolf and Xue, 1998).   12 1.3 Animal models of addiction 1.3.1 Face validity and predictive validity The behavioral symptoms that characterize human drug addiction can be modeled in animals and these models satisfy the scientific criteria of face, predictive, construct, and external validity (Epstein et al., 2006).  Animal models offer face validity in that similar classes of drugs are self-administered by both experimental animals and humans (Crombag et al., 2002;Crombag et al., 2008). Furthermore, construct validity is also supported as similar neuroanatomical structures, mainly centered on the mesocorticolimbic system, are implicated in both preclinical and clinical studies of addiction models and human addicts (Hyman, 2005).  Through the use of neuroimaging, the frontal cortical areas have been demonstrated to be involved in drug-seeking responses and this is analogous between animal and human subjects (Childress et al., 1999;Volkow et al., 2002).  Preclinical models also provide predictive validity by confirming that certain classes of drugs, including DA receptor antagonists, can block the acquisition of drug-seeking behaviour, along with craving and relapse in animal models.   This has led to the development of some, although not entirely successful, treatment options for patients due to our limited understanding of the intricate interplay of mechanisms underlying addiction.  The nature of addiction in humans is well-defined and animal models provide construct validity, particularly the model of self-administration, which can mirror the pathology, symptomology, and the etiology of the disease to a certain extent.   The use of cue-induced relapse to drug seeking behavior in rat and mouse models is well-established and translate quite closely to the human condition thereby providing further external validity as an animal model of addiction (Epstein et al., 2006).      13 1.3.2 Drug self-administration model Animal models employ two methods of drug administration: noncontingent, which is passive exposure in which the experimenter administers the drug and contingent administration, which is active seeking such as self-administration.  In humans, drug addiction is usually the result of self-initiated drug intake which in turn results in stimulus-response learning between the action of drug-taking and the experience of drug-reward.  In addition, as second class of stimulus-stimulus associations arise linking the primary reward properties of the drug with drug-related cues and contexts.  These learned stimulus-stimulus associations are now considered to be crucial for the persistence of compulsive drug-seeking, craving and relapse following periods of abstinence (Childress et al., 1999;Kalivas et al., 1998;Robinson and Berridge, 1993;Self and Nestler, 1998). Operant self-administration models provide the closest resemblance to human drug addiction by ensuring that animals have voluntary control over drug intake (Crombag et al., 2008;Epstein et al., 2006).  In a typical self-administration model, a catheter is implanted in the jugular vein through which drug is delivered when a syringe pump is activated by an operant response, such as pressing a lever or nose poking.  Drugs that have high abuse potential in humans are also readily self-administered by animals, from psychostimulants, opiates, nicotine, to ethanol (Epstein et al., 2006).  The operant task can be modified during acquisition to measure the level of motivation for drug-seeking in terms of how hard an animal is willing to ?work? for a single drug delivery.  Once well-established, extinction training, during which operant responding does not result in drug delivery, can begin.  Relapse to drug seeking can then be measured by re-exposing the animals to cues or stressors to   14 trigger the resumption of operant responding, similar to relapse in human addicts (Self and Nestler, 1998;Crombag et al., 2008;Epstein et al., 2006).   1.3.3 Conditioned place preference Conditioned place preference (CPP) is a well-established, noncontingent paradigm to study the learned association between the rewarding effects of a drug and the environmental context in which the drug is experienced (Bardo and Bevins, 2000;Carr et al., 1988;Phillips et al., 1983).  During the conditioning phase, animals receive a drug and vehicle injection in two distinct compartments that typically differ in visual, olfactory and tactile cues.  After a sequence of pairings, drug-seeking is measured by allowing the animal free access to either of the drug-paired or vehicle-paired contexts in the absence of drug.  A positive association between the drug-paired context and reward experience will result in more time spent in that particular context.  Similar to the self-administration model, an extinction phase can be implemented and drug-seeking can be observed by either a priming injection of the drug, or by exposure to a stressor (Cruz et al., 2010;Aguilar et al., 2009).  1.3.4 Behavioural sensitization As noted above, the phenomenon of behavioral sensitization represents another noncontigent paradigm related to long-lasting behavioral and neurochemical changes induced by repeated and often intermittent exposure to psychostimulant drugs.  When a psychostimulant (e.g., amphetamine, cocaine, methylphenidate, or methamphetamine) is repeatedly administered to rodents, one of two distinct enduring behavioural changes occur.  One type is a decline in the effectiveness of the psychostimulant to affect behaviour, a phenomenon referred to as tolerance (Robinson and Becker, 1986;Kalivas and Stewart, 1991).  Alternatively, repeated   15 psychostimulant treatment can cause an enhanced response known as reverse tolerance, or behavioural sensitization.  Behavioural sensitization typically occurs with repeated, intermittent exposure to a psychostimulant, especially with doses in the low to moderate range.  This enhanced behavioural response is usually progressive, and can persist for days, weeks, months, and even years after the last drug administration (Segal and Mandell, 1974;Robinson and Becker, 1986;Kalivas and Stewart, 1991).  This response persistence has been associated with the craving related to drugs of abuse, which may lead to relapse despite a prolonged period of abstinence.   Behavioral sensitization is characterized by a progressive and enduring enhancement of the drug-induced stimulatory effects of amphetamine (Robinson and Berridge, 2001;Kalivas and Stewart, 1991). Behavioural sensitization can be observed after as little as one drug exposure and can still be detected for at least one year after the last drug exposure (Robinson and Becker, 1986;Kalivas and Stewart, 1991).  Much of the interest generated by behavioural sensitization originated from its relevance as an animal analog of human psychosis (Kalivas and Stewart, 1991); subsequently this was extended to model aspects of human addiction, such as drug craving and relapse (Davidson et al., 2001;Self and Nestler, 1998).   Importantly, behavioral sensitization has been successfully modeled in humans (Leyton, 2007;Strakowski et al., 1996;Sax and Strakowski, 2001;Strakowski et al., 2001), and PET studies have found evidence for sensitization even after a single exposure to amphetamine (Boileau et al., 2006).  The process of behavioural sensitization include two stages of drug exposure referred to as ?induction? and ?expression?.  The inclusion of a drug-free period prior to re-exposure in a challenge test constitutes an important feature of the sensitization protocol.  In the   16 induction phase, development of the sensitized response is indicated by a progressive increase in behavioural responding after each repeated drug administration.  The expression of behavioural sensitization is indicated by an enhanced behavioural response to a subsequent acute drug administration (i.e., challenge injection) following a drug-free period that may last for several weeks (Pierce and Kalivas, 1997;Robinson and Becker, 1986).  Importantly, sensitized animals show increased rates of self-administration (Mendrek et al., 1998;Sorg et al., 1994;Kalivas et al., 1993). Non-pharmacological environmental factors provide a critical contribution to the effects of drugs and the process of addiction.  Exposure to drugs occurs in the presence of many environmental conditions and cues have the ability to influence initial response as well as later responsiveness upon re-exposure to the drug.  Indeed, under most circumstances, amphetamine-induced behavioural sensitization is context-specific (Stewart and Vezina, 1991;Anagnostaras and Robinson, 1996;Badiani and Robinson, 2004;Tirelli and Terry, 1998).  The common paradigm for understanding context-dependent sensitization is the Pavlovian model, in which drug administration is the unconditional stimulus (US), and is paired with placement into a distinct environmental context (CS), where contextual stimuli become associated with psychomotor drug effects (UR).  Environmental cues (CS) are then able to induce an excitatory conditioned response (CR) amplifying the sensitized response in a context where drug is anticipated.  Although a considerable body of evidence supports a pivotal role for DA in behavioral sensitization, other neurotransmitters have also been implicated.  Specifically, the enhanced behavioural effects of amphetamine can be blocked by administration of glutamate antagonists such as MK-801 (Wolf and Xue, 1998).  Moreover, repeated psychostimulant   17 administration elevates glutamate transmission in both the NAc and the VTA (Vanderschuren and Kalivas, 2000;Churchill et al., 1999).   Recent studies have shown that mechanisms of action on the glutamate system play a pivotal role in the neuroplasticity that result in drug addiction (Kalivas, 2009).   1.4   Synaptic plasticity: LTP and LTD  A synapse is able to change its strength of synaptic transmission, in a process referred to as synaptic plasticity.  This is of critical importance to both normal brain function and dysfunction.  The best characterized forms of synaptic plasticity in the brain are long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission, and these have been extensively studied as the cellular substrates of learning and memory (Bear and Malenka, 1994;Bliss and Collingridge, 1993;Collingridge et al., 2004).  Glutamate is the primary fast excitatory neurotransmitter in the brain and acts on two classes of postsynaptic ionotropic glutamate receptors, the N-methyl-D-aspartate (NMDA) receptors and the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptors.  NMDA receptors have little involvement in basal synaptic transmission due to voltage-dependent Mg2+ blockade of the channel, but are responsible for producing different forms of synaptic plasticity in AMPAR-mediated synaptic transmission, including the establishment of LTP and LTD (Collingridge et al., 2004;Wang, 2008;Bear and Malenka, 1994).  Alternatively, AMPARs are the main receptors mediating both basal synaptic transmission and also the expression of LTP and LTD (Bear and Malenka, 1994;Bliss and Collingridge, 1993).  After much debate, it is now generally accepted that the induction of LTP and LTD occur postsynaptically and are dependent on Ca2+ influx through the activated NMDA receptors.  On the contrary, the expression of LTP and LTD remain under discussion and may involve both   18 changes in presynaptic neurotransmitter release and postsynaptic changes to AMPAR expression (Bear and Malenka, 1994;Bliss and Collingridge, 1993).  Recent research suggests that there are important rapid changes in the relative expression of postsynaptic AMPARs which likely contribute to the expression of LTP and LTD (Bredt and Nicoll, 2003;Malinow and Malenka, 2002).  Of particular importance is the constitutive cycling of AMPARs between intracellular compartments and the plasma membrane via vesicle-mediated plasma membrane insertion, which is referred to as exocytosis, and the internalization, or removal, of AMPARs from the plasma membrane, referred to as endocytosis (Bear and Malenka, 1994;Malinow and Malenka, 2002;Bredt and Nicoll, 2003).  The process of exocytosis and endocytosis leads to an increase or decrease in the number of AMPARs expressed in the postsynaptic membrane density and plays an essential role in regulating the strength of the synapse.  It is now generally agreed that LTP involves facilitated insertion of AMPARs into the postsynaptic membrane while LTD involves the clathrin-mediated removal of AMPARs from the postsynaptic membrane (Bredt and Nicoll, 2003;Malinow and Malenka, 2002). 1.5   Evidence linking synaptic plasticity and addiction The acute effects of abused drugs are typically short-lived and fade away once metabolized by the body, therefore cannot help explain the development of addictive behaviors.  In order to understand addiction, it is necessary to pinpoint the specific changes that occur following exposure to the substance and the path of neurological influence.   In almost all types of adaptive experience-dependent plasticity, the neural adaptations that are responsible for behavioral changes are also involved in synaptic changes, contributing to drug-dependent synaptic plasticity.  Drug-induced synaptic plasticity is critically dependent on the molecular actions of a drug, but is also influenced by, though still poorly understood, environmental   19 stimuli that constitute the context of exposure.  It is highly likely that neural adaptations resulting from repeated exposure to drugs involve cellular substrates similar to those responsible for learning and memory, namely LTP and LTD at excitatory synapses (Hyman et al., 2006;Nestler, 2001)      Synaptic adaptations linked to drugs of abuse and mechanisms of learning have both been found to activate similar signal transduction pathways (Nestler, 2001;Hyman and Malenka, 2001;Berke and Hyman, 2000), and produce comparable morphological alterations in dendritic profiles (Robinson and Kolb, 1997;Robinson and Kolb, 1999;Robinson and Berridge, 2001).  Evidence from imaging studies also suggest that similar neuronal circuits are activated by drug-related stimuli and during processes of learning and memory (Wilson et al., 2004). Furthermore, repeated administration of amphetamine may block LTD, a persistent decrease in excitatory synaptic transmission, in the excitatory synapses of VTA neurons, and may contribute to the initiation of the sensitization process (Jones et al., 2000).  On the other hand, long-lasting expression of sensitization is linked to adaptations in the neurons of the NAc (Robinson and Kolb, 1999;Pierce and Kalivas, 1997) and there is evidence to suggest a long-lasting enhancement of LTD of glutamatergic transmission in the shell region of the NAc in mice sensitized to cocaine (Thomas et al., 2001).  Activity-dependent plasticity plays a critical role in the development and expression of behavioral sensitization (Wolf et al., 2004), and the trafficking of AMPAR is essential to neuronal plasticity (Malinow and Malenka, 2002).  Regulated endocytosis of postsynaptic AMPA receptors is required for different types of LTD, including NMDA receptor-dependent LTD, mGluR-dependent LTD, and insulin-induced LTD (Lu et al., 2001;Man et al., 2000;Malinow and Malenka, 2002).  DA receptors modulate AMPAR trafficking (Mangiavacchi and Wolf, 2004;Sun et al., 2005), providing   20 further evidence for DA-releasing psychomotor stimulants being involved in synaptic plasticity and learning and memory mechanisms.   Internalization of AMPA receptors is mediated by clathrin-dependent endocytosis (Man et al., 2000;Luscher et al., 1999;Carroll et al., 1999;Lin et al., 2000) and can be further differentiated into constitutive and regulated endocytosis.   When the regulated pathway is stimulated with NMDA activation or insulin, the surface expression of AMPA receptors is immediately diminished, leading to a lasting depression (LTD) of receptor-mediated transmission (Beattie et al., 2000;Man et al., 2000;Wang and Linden, 2000).  Among the heteromeric structural composition of AMPA receptors, those receptors consisting of the GluA2 subunit are most closely linked to regulated endocytosis (Man et al., 2000).  The CT region of the GluA2 subunit contains certain sequence motifs that account for this specificity.  This includes domains that binds to NSF and AP2 clathrin adaptor, as well as a PDZ-binding domain that is specific to certain PDZ-containing proteins such as GRIP and PICK1 (Chung et al., 2000;Lee et al., 2002).  Of particular importance is the discovery of a new tyrosine phosphorylation-dependent sequence of amino-acids containing 3 tyrosine residues (869YKEGYNVYG877) that is essential for NMDAR-mediated and insulin-stimulated AMPA receptor endocytosis (Ahmadian et al., 2004) .  Based on this sequence, a GluA23Y interference peptide was developed and found to block stimulated-LTD in hippocampal slices (Ahmadian et al., 2004).  A recent study identified that BRAG2 is the synaptic protein which interacts with the GluA2 subunit to activate Arf6 and the internalization of AMPA receptors to produce LTD.  Through blocking the GluA2 and Arf6 interaction, as well as targeted deletion of BRAG2 in hippocampal neurons, it was demonstrated that AMPA receptor endocytosis and the resulting LTD can be interrupted (Scholz et al., 2010).  Similarly, the   21 GluA23Y interference peptide may function to disrupt the regulated AMPA receptor endocytosis involved in drug-induced plasticity to possibly attenuate stages of sensitization induction, maintenance and expression.  1.6   Research objectives and hypotheses This thesis is comprised of a series of experiments utilizing an animal model of behavioral sensitization to explore the role of an aspect of synaptic plasticity in the form of LTD within GluA2-containing synapses in drug-induced changes in brain function that may give rise to persistent and enhanced behavioral and neurochemical changes related to the addictive properties of psychostimulant drugs.  The interference peptide, Tat-GluA23Y, developed by Y.T. Wang and colleagues (Ahmadian et al., 2004), was used as a tool to disrupt critical protein-protein interactions responsible for regulated AMPAR endocytosis critical for LTD.  This in turn permitted a careful and systematic assessment of the role of LTD in the VTA as distinct from the NAc in the induction and maintenance of amphetamine-induced behavioral sensitization. This series of experiments also explore neurochemical and behavioral correlates of d-AMPH-induced sensitization as well as the pivotal role that the environment plays on experience-dependent neuroplasticity.   The following chapter consists of a manuscript that summarizes the experiments investigating the neurochemical and behavioral effects of the GluA23Y-derived peptide on the development and maintenance of d-AMPH-induced behavioural sensitization.  This was achieved through in vivo microdialysis and measuring locomotor activity simultaneously.  In addition, the interference peptide was locally infused in order to better determine its site of action.   Since the first series of experiments demonstrated that Tat-GluA23Y was able to modify the neuroplasticity responsible for d-AMPH-induced behavioral sensitization, the next   22 question that arose was whether this modification would be long-lasting and persistent across changing environmental contexts.  The third chapter consists of a manuscript based on a series of experiments again utilizing the GluA23Y interference peptide to examine the role of AMPA receptor endocytosis and LTD on long-term maintenance of behavioral sensitization and a specific situation where sensitization is under direct influence of environmental context.  By varying the environmental context in which the animal is given d-AMPH while restricting exposure to the GluA23Y peptide to only one particular environment, the study provides the first evidence linking synaptic plasticity to the formation of associations between contextual stimuli and the direct psychostimulant properties of d-AMPH.  Accordingly, these data also contributed to a better understanding of how sensitization develops and is maintained through an interaction between drug-induced neuroplasticity and contextual cues.   The final chapter provides an overall discussion on the mechanisms underlying amphetamine-induced behavioral sensitization in light of the results garnered from the current series of experiments.  In addition to providing critical evidence linking synaptic plasticity to drug addiction, this thesis suggests that there is a remarkable possibility to block, or even reverse, an established drug-induced neuroadaptation of relevance to drug addiction in humans.   23 Chapter Two: Interference with AMPA Receptor Endocytosis: Effects on Behavioral and Neurochemical Correlates of Amphetamine 2.1    Overview 2.1.1 Background Behavioral sensitization is linked to drug-craving in both clinical and preclinical studies of addiction.  Increased motor activity is accompanied by enhanced dopamine (DA) release, particularly in the nucleus accumbens (NAc). The neural bases of sensitization are linked to alterations in synaptic connections that also underlie learning and memory. The present study utilizes an ?interference peptide?, Tat-GluA23Y, that blocks long-term depression (LTD) at glutamatergic synapses by disrupting the endocytosis of ?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs), to explore the role of this form of synaptic plasticity in the induction and maintenance of sensitization. 2.1.2 Methods Rats were given 5 injections of d-Amphetamine (d-AMPH, 1.0 mg/kg, i.p.) every second day.  Tat-GluA23Y, was administered by two routes of administration (intravenous (i.v.), intracranial (i.c.) to VTA, or i.c. to NAc) prior to each injection of d-AMPH.  Following a 14-day drug-free period, expression of behavioral sensitization was evoked by a challenge injection of d-AMPH (0.5 mg/kg, i.p.). DA efflux in the NAc was measured by HPLC-ED analyses of brain dialysates on Days 1, 9 and 24 of the i.v. peptide experiment. 2.1.3 Results Systemic administration of Tat-GluA23Y during the induction phase blocked maintenance of behavioral sensitization and attenuated the maintenance of neurochemical sensitization.  Intra-  24 VTA infusion of Tat-GluA23Y prior to each administration of d-AMPH did not affect induction, but inhibited maintenance and subsequent expression of sensitization, whereas intra-NAc infusion of the peptide did not affect induction or maintenance of sensitization.   2.1.4 Limitations The relevance of behavioural sensitization in rodents is related to the development of craving and does not provide direct measures of drug reinforcement.   2.1.5 Conclusions These findings confirm that drug-induced neuroplasticity is labile and may be subject to disruption at a time when long-lasting associations between drug reward and contextual stimuli are formed.  Furthermore, the unique ability of Tat-GluA23Y to block maintenance of behavioral sensitization implicates LTD in the consolidation of essential associative memories.  Tat-GluA23Y has the unique ability to disrupt functional neuroadaptations triggered by repeated psychostimulant exposure and therefore may protect against the development of craving and drug-seeking behaviors. 2.2   Introduction  Persistent thoughts and actions related to the procurement and use of illicit drugs, along with vulnerability to relapse following exposure to contextual stimuli associated with drugs of abuse, are hallmark features of drug addiction.  Behavioral sensitization, characterized by increased motor activity, is induced by repeated exposure to a variety of illicit drugs including amphetamines, and may be a key mechanism leading to compulsive drug use in both humans and animals.(Kalivas et al., 1998;Boileau et al., 2006;Leyton, 2007;Hyman and Malenka,   25 2001) Once established, behavioral sensitization is often accompanied by enhanced dopamine (DA) release in the nucleus accumbens (NAc).(Fiorino and Phillips, 1999;Robinson and Becker, 1982;Imperato et al., 1996) Whereas terminal regions of the mesocorticolimbic dopamine system are clearly implicated in the long-term expression of drug-induced sensitization (Pierce and Kalivas, 1997), its induction and initial maintenance is dependent on modulation of synaptic events in close proximity to DA-containing neurons in the ventral tegmental area (VTA) (Nestler, 2001;Pierce and Kalivas, 1997;Robinson and Berridge, 1993). The enduring neural adaptations that accompany repeated exposure to drugs of abuse have been linked to specific forms of synaptic plasticity including long-term potentiation (LTP) and long-term depression (LTD).(Grueter et al., 2012;Luscher and Malenka, 2011;Nestler, 2001;Thomas et al., 2001) This in turn has highlighted possible similarities between neural adaptations related to acquisition and maintenance of addictive behaviors and those that underlie other forms of learning and memory.(Hyman, 2005;Berke and Hyman, 2000)  Previous work has pointed to a crucial role for the endocytosis of ?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) in a psychostimulant-induced behavioral sensitization model of drug-craving, (Brebner et al., 2005;Van den Oever et al., 2008) extinction of morphine-induced place preference,(Dias et al., 2012) and cue-induced reinstatement of heroin self-administration.(Van den Oever et al., 2008)  These studies utilized a small ?interference? peptide, Tat-GluA23Y, to disrupt the regulated endocytosis of AMPARs.  This peptide has the unique ability to block LTD by inhibiting facilitated AMPAR endocytosis(Ahmadian et al., 2004;Brebner et al., 2005)  via disrupting protein-protein interaction between the GluA2 subunit of AMPAR and Brag2, a clathrin-adaptor protein which activates Arf6 to initiate regulated AMPAR endocytosis(Scholz et al., 2010).   26  In our previous study, acute intravenous administration of Tat-GluA23Y immediately prior to drug challenge test, as well as direct intracerebral injection into the NAc (but not VTA) blocked the expression of d-Amphetamine (d-AMPH)-induced sensitization of motor activity.(Brebner et al., 2005) The present study addresses several critical issues of relevance to the potential therapeutic value of Tat-GluA23Y ,including the critical question of whether this interference peptide can block the induction and maintenance of behavioral sensitization when given  prior to each of 5 injections of d-AMPH.  Given the involvement of the mesocorticolimbic DA system in both the induction and expression of behavioral sensitization (Gulley and Stanis, 2010;Lodge and Grace, 2008;Vanderschuren and Kalivas, 2000;Pierce and Kalivas, 1997) as well as the rewarding effects of psychostimulant drugs,(Koob and Le Moal, 2008;Schultz, 2000;Hyman et al., 2006;Fibiger and Phillips, 1988) the present study also employed brain microdialysis to monitor the efflux of DA in the NAc during the induction and expression of d-AMPH sensitization.   Furthermore, the induction of behavioral sensitization involves neuroplasticity in the VTA,(Churchill et al., 1999;Perugini and Vezina, 1994;Tolliver et al., 1999) therefore we also examined whether microinjection of Tat-GluA23Y into the VTA, but not the NAc, would block the induction and/or maintenance of sensitization. 2.3   Methods 2.3.1 Animals Male Sprague-Dawley rats (Charles River, Quebec) weighing 250-275 g were used for all experiments. Animals were housed in pairs in transparent cages in a temperature-controlled animal colony and acclimatized to a 12-hr reverse light-dark cycle (lights on 19:00 hours) for 7 days prior to the beginning of experiments.  They were subsequently housed individually   27 once experiments began.  Food and water were available ad libitum during all phases of the study.  All the experiments were approved by the University of British Columbia Animal Care Committee and conducted in accordance with policies outlined by the Canadian Council on Animal Care. 2.3.2 Drugs D-AMPH (USP, Rockville, MD) was diluted in 0.9% sterile saline. Tat-GluA23Y peptide was constituted of 9 amino acids (YKEGYNVYG), and was attached to an HIV-1 Tat peptide (YGRKKRRQRRR) in order to cross the blood brain barrier and permeate cells.(Ahmadian et al., 2004) The scrambled peptide, Tat-GluA23Yscr, was comprised of the same 9 amino acids placed in random sequence (VYKYGGYNE) and served as a control.  Both Tat-GluA23Y and Tat-GluA23Yscr (GL Biochem-Shanghai Ltd, Shanghai, China) were diluted in 0.9% sterile saline. 2.3.3 Surgical procedures Surgeries were performed under isoflurane anaesthesia (3% isoflurance (Baxter, Canada) in oxygen) and maintained with 1.5-2.5% isoflurane for the duration of the procedure.   As an adjunct to gaseous anaesthesia, the analgesic, ketoprofen (Anafen), was administered subcutaneously at the time of surgery to help minimize post-surgical distress.  All rats were all given a 1-week recovery period prior to experimental procedures during which their conditions were closely monitored twice daily for possible post-operative complications.  Jugular catheter implantation.  Once a surgical plane of anesthesia was achieved, hair was clipped in two areas measuring 3.5cm x 6cm on the dorsal surface and 2.5cm x 4cm on the   28 anterior right region of the ventral surface of each rat.  An iodophor scrub (Betadine) was applied to the skin surface alternated twice with 70% ethyl alcohol to disinfect the skin.  A single incision measuring 2.5cm was made using a No.10 surgical blade on the dorsal surface and an incision measuring 2cm was made on the ventral surface above the right jugular vein.  A chronic indwelling silastic catheter connected to a guide cannula (Plastics One, 22 ga) was secured to the dorsal surface of the rat with Dacron mesh. The silastic tubing was adhered to the vein by a series of sutures (4-0 silk, nonabsorbable).  Surgical wounds were closed with Polyglactin 910 (Vicryl, absorbable).   Microdialysis and intracerebral cannulation.  Animals were secured in a stereotaxic frame with the dorsal surface of the cranium oriented in a horizontal plane.  For the microdialysis experiments, rats were implanted with bilateral guide cannulae (nitric acid passivated stainless steel, 19-gauge x 15mm), positioned 1mm below dura directly above the NAc (+1.7 AP and ?1.1 ML from bregma).  For the microinjection experiments, rats were implanted with bilateral cannulae (stainless steel, 23 gauge) directed at the VTA (AP, -5.8; ML, ?0.6; DV, -7.0) or NAc (AP, +1.7; ML, ?1.1; DV, -6.8) at the border of the NAc core and shell.  Guide cannulae were secured to the skull via four stainless-steel screws and dental cement.  Sterile obdurators are placed into the guides to maintain patency. 2.3.4 Microdialysis and high pressure liquid chromatogaphy Microdialysis probes were constructed from Filtral 12 AN69HF semi-permeable hollow fibres (2mm long, 340 ?m OD, 65 kDa molecular weight cut-off; Hospal, Germany) and silica inlet-outlet lines (75/150 ?m ID/OD).  Samples were analyzed via high-pressure liquid chromatography (HPLC) with electrochemical detection.  HPLC systems were composed of   29 the following: an ESA 582 pump (Bedford, USA), a pulse damper (Scientific Systems, USA), an inert manual injector (Rheodyne, USA), a Super ODS TSK column (Tosoh Bioscience, USA) and an Intro Electrochemical detector (Antec Leyden, The Netherlands).  The mobile phase [70mM sodium acetate buffer, 40 mg/l EDTA and 5mg/l sodium dodecyl sulfate (adjustable); pH 4.0, 10% methanol] flowed through the system at 0.10 ml/min. EZChrome Elite software (Scientific Software, USA) was used to acquire and analyze chromatographic data. 2.3.5 Behavioral apparatus Eight open-field Plexiglas chambers, measuring 40cm x 40cm x 40cm were used to measure horizontal locomotion. Top-mounted cameras allowed video-capture and tracking of locomotor activity that was measured and scored by digital monitoring software, Ethovision 3.1 (Noldus, Inc).  Chambers were also fitted with a liquid-swivel (Instech 375s; Plymouth Meeting, PA, USA) required for in vivo microdialysis.   All experiments were run in a darkened room under dim red lighting between 0800 and 1800 h. 2.3.6 Experimental design  Figure 2.1 below outlines the procedural timeline from the initial day of surgery to the final day of drug challenge to measure induction and expression of d-AMPH-induced behavioral sensitization.  Prior to the start of experiments, animals were habituated to the locomotor activity testing chambers for 30min. on 2 separate occasions.  During the induction phase, animals received a total of five injections of either d-AMPH (5 x 1.0 mg/kg, i.p.) or saline on alternate days, all given in the testing chambers.  Following the 5th day of treatment (Day 9), all rats remained in the colony room for a 2-week drug-free period.  On Day 24, all groups   30 were challenged with a lower dose of d-AMPH (0.5 mg/kg, i.p.) to reduce the possible confounding effect of drug-induced stereotypy.    Figure 2.1 Experimental protocol used to study the effects of GluA23Y on the induction and maintenance of d-AMPH-induced sensitization.   Rats received a sensitizing regimen of d-AMPH (5 x 1.0 mg/kg, IP) every other day.  Tat-GluA23Y (1.5 nM/gr, IV), Tat-GluA23Yscr (1.5 nM/gr, IV), or vehicle control (0.9% saline, 1ml/kg, IV) were administered 60 min prior to each injection of d-AMPH.  Rats were then left undisturbed for a 2-week drug-free period after which all groups were challenged on Day24 with a lower dose of d-AMPH (0.5 mg/kg, IP). A 4th group, receiving 5 injections of 0.9% saline + peptide vehicle during the Induction phase provided control data on the acute effects of d-AMPH when given a single injection of 0.5 mg/kg on the final challenge trial.  DA efflux in the NAc was measured by microdialysis in each animal at 2 of 3 time-points: Day1, 9, or 24.  In the subsequent experiment, Tat-GluA23Y, Tat-GluA23Yscr, or vehicle, were administered via intracerebral (IC) microinjection into the VTA or NAc during the Induction period prior to each d-AMPH or saline injection.  Following a 14-day drug-free period, all animals were given a challenge injection of d-AMPH on Day24. Intravenous (i.v.) administration of Tat-GluA23Y peptide and microdialysis.  DA efflux in the NAc was measured by microdialysis at 3 time-points: the 1st or 5th exposure (Day 1 or 9) to d-AMPH or saline and during d-AMPH challenge on Day 24.  The day prior to microdialysis experiments on Days 1, 9 and 24, probes were flushed with artificial cerebrospinal fluid (aCSF) (10.0 mM sodium phosphate buffer with 147.0 mM NaCl, 3.0 mM KCl, 1.0 mM MgCl2 and 1.2 mM CaCl2; pH 7.4) and inserted unilaterally via the guide cannulae into the   31 NAc (dialysis membrane spanned -4.8 to -6.8 mm ventrally).  Rats remained in the locomotor activity testing chamber overnight (14-16h) with continuous perfusion of aCSF at 1?l/min.  Food and water were available ad libitum.  In the morning, food and water were removed and 15 ?l dialysis samples were collected at 15-min intervals from the NAc and immediately assayed for DA using HPLC-EC.  Baseline sampling continued until four samples showed <10% fluctuation in DA content.   Animals were then treated with Tat-GluA23Y (5 x 1.5 nM/gr, i.v.), Tat-GluA23Yscr (5 x 1.5 nM/gr, i.v.), or vehicle control (5 x 0.9% saline, i.v.), and microdialysis sampling and behavioral recording continued for 60 min., followed by injections of d-AMPH.  A fourth group received vehicle control (i.v.) and saline (i.p.) during the conditioning period.  Microdialysis samples were collected and locomotor activity was measured for a further 2 h., after which rats were returned to their home cages.  Experimental procedures on challenge Day 24 were similar except peptide treatment was not given; instead, all animals received d-AMPH injections immediately following baseline sampling. Intracerebral (i.c.) administration of Tat-GluA23Y in VTA or NAc.  In separate groups of rats, Tat-GluA23Y (15 pmol, i.c.), Tat-GluA23Yscr (15 pmol, i.c.), or vehicle control, was microinjected bilaterally into either the VTA or NAc 40 min. prior to each d-AMPH (5 x 1.0 mg/kg, i.p.) or saline injection.  Injection needles were inserted via guide cannulae and connected to an infusion pump (Harvard Apparatus, USA).  A total volume of 0.3?l/side was delivered over 1 min.  Injection needles remained in place for 2 min. to allow for diffusion.  Rats were then placed in open field chambers and locomotor activity was recorded and scored for 90 min.  At the end of each testing session, animals were returned to their home cages.  After the 5th treatment session, rats remained in the animal colony for a 2-week drug-free   32 period.  On Day 24, all animals were challenged with a lower dose of d-AMPH (0.5 mg/kg, i.p.) and locomotor activity was recorded.   2.3.7 Histology Following completion of all testing procedures, animals were given a lethal dose of sodium pentobarbitol and perfused transcardially with 0.9% saline followed by 10% formaldehyde solution.  Brains were rapidly removed and cryoprotected in 20% sucrose in 10% formaldehyde for several days for cryoprotection.  Serial 30?m coronal sections were cut on a cryostat, sections were mounted on glass slides, dried and stained with cresyl violet and cover slipped.  Placements of the microdialysis probe or guide cannulae were verified under a light microscope and located on figures adapted from the Paxinos and Watson atlas of the rat brain(Paxinos et al., 1980), presented below in Figure 2.2.  Figure 2.2  Placement of microdialysis probes and microinjection cannulae.     33 Distance from bregma is indicated to the right (mm).  (A) Black bars represent the location of microdialysis membranes.  (B) Black circles represent the location of microinjection needle tips in the NAc (left) and VTA (right).   Injection of dansyl-tagged Tat-GluA23Y peptide in brain A dansyl-lysine group was added to the Tat-peptide to serve as a fluorescent marker to estimate the diffusion of peptide from the injection sites in the VTA or NAc.  Rats were prepared with bilateral guide cannulae directed at the VTA or NAc according to the stereotaxic procedure described above.  One week following surgery, the dansyl-Tat-GluA23Y peptide was microinjected with injection needles inserted through guide cannulae connected to an infusion pump (0.3?l/side over 1 min).  Injection needles were left in place for an additional 2 min. to allow for diffusion.  Sixty min. following the microinjection, rats were deeply anesthetized and perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer for 30 min.   Brains were stored overnight in 10 % sucrose containing fixative and fixed brains were sectioned at 50 ?m on a cryostat.  Sections were then mounted on slides and examined by fluorescent microscopy, with examples presented below in Figure 2.3.   34  Figure 2.3  Diffusion of dansyl-tagged interference peptide.   Examples of the diffusion of the fluorescent dansyl-Tat-GluA23Y peptide within the NAc (A) and VTA (B) following intracranial microinjection (0.3?L total volume).  The schematic diagram on the left illustrates the location of the peptide diffusion area shown in the flourescent image on the right.     35  2.3.8 Statistical analysis All values are expressed as mean ? (standard error of the mean) SEM.  Statistical significance was assessed using one-way ANOVA or two-way ANOVA with repeated measures with time as the within-subject factor and treatment groups as the between-subjects factor.  Where appropriate, post hoc analyses were made using Tukey HSD to examine simple main effects.  Statistical significance was evaluated at p < 0.05. 2.4   Results 2.4.1 Intravenous Tat-GluA23Y administration blocks induction of behavioral sensitization and attenuates d-AMPH-induced DA efflux in the NAc  Figure 2.4 illustrates d-AMPH-induced locomotor activity and DA efflux in the NAc on Day 1 and Day 9 of induction of sensitization.  Based on the time-course of behavioral response to each d-AMPH injection (see Fig. 2.4A and Fig. 2.4C), peak locomotor activity was observed during the initial 30 min period post-injection.  Therefore, statistical analyses were performed on the cumulative data of distance traveled in the 30min following d-AMPH (Fig. 2.4E).  Repeated d-AMPH treatment (5 x 1.0 mg/kg) led to a progressive increase in locomotor activity in rats given vehicle (n=12) or Tat-GluA23Yscr (n=9) while the interference peptide, Tat-GluA23Y (n=13), blocked the induction of locomotor sensitization (Day x Treatment group: F(1,3) = 17.364, p<0.01; post hoc: vehicle + d-AMPH group, Day 1 vs. Day 9, p<0.01; Tat-GluA23Yscr + d-AMPH group, Day 1 vs. Day 9, p<0.01;  Tat-GluA23Y + d-AMPH group, Day 1 vs. Day 9, ns).    36 Consistent with previous microdialysis experiments, d-AMPH treatment on both Day 1 and Day 9 was accompanied by a significant increase in DA efflux in the NAc in rats treated with vehicle (n=9) or Tat-GluA23Yscr (n=10) (Fig. 2.4B, repeated measures ANOVA, F(8,24) = 30.75, p<0.01; Fig. 2D, repeated measures ANOVA, F(8,24) = 37.12, p<0.01).  Tat-GluA23Y peptide pretreatment (n=8) did not significantly affect levels of d-AMPH-induced DA efflux on Day 1.  However, repeated treatment with Tat-GluA23Y significantly attenuated the magnitude of d-AMPH-induced DA efflux in the NAc by the 5th d-AMPH injection (Fig. 4F, pairwise comparison, Day 1: 633% ? 170% vs. Day 9: 278% ? 53%, p<0.01).    37  Figure 2.4 Time course and cumulative data of locomotor activity and DA efflux in the NAc on Day 1 and Day 9 of the induction of d-AMPH behavioral sensitization.  Vehicle control, Tat-GluA23Yscr or Tat-GluA23Y was given 60 min. prior to d-AMPH or Saline. (A) On Day 1, Tat-GluA23Y treatment did not affect d-AMPH-stimulated locomotor activity  [Vehicle + d-AMPH (n=8), Tat-GluA23Yscr + d-AMPH (n=14), Tat-GluA23Y + d-AMPH (n=11), vehicle + saline (n=10)].  (B) Tat-GluA23Y treatment did not affect d-AMPH-induced DA efflux in NAc on Day 1 of induction [Vehicle + d-AMPH (n=11), Tat-  38 GluA23Yscr + d-AMPH (n=8), Tat-GluA23Y + d-AMPH (n=10), vehicle + saline (n=6)]. (C) 5 repeated treatments with Tat-GluA23Y blocked the development of locomotor sensitization measured on Day 9 of induction [Vehicle + d-AMPH (n=9), Tat-GluA23Yscr + d-AMPH (n=13), Tat-GluA23Y + d-AMPH (n=9), vehicle + saline (n=10)].  (D) Repeated treatment with Tat-GluA23Y significantly attenuated d-AMPH-induced DA efflux in the NAc on Day 9 of induction [Vehicle + d-AMPH (n=7), Tat-GluA23Yscr + d-AMPH (n=9), Tat-GluA23Y + d-AMPH (n=8), vehicle + saline (n=6)].  (E) Changes in cumulative distance traveled over 30min following IP injections of d-AMPH or saline between Day 1 and Day 9.  Repeated d-AMPH treatment (5 x 1.0 mg/kg) led to a progressive increase in locomotor activity in rats given vehicle (n=12) or Tat-GluA23Yscr (n=9) while the interference peptide, Tat-GluA23Y (n=13), blocked the development of locomotor sensitization (Day x Treatment group: F(1,3) = 17.364, p<0.01; post hoc: vehicle + d-AMPH group, Day 1 vs. Day 9, *p<0.01; Tat-GluA23Yscr + d-AMPH group, Day 1 vs. Day 9, *p<0.01;  Tat-GluA23Y + d-AMPH group, Day 1 vs. Day 9, ns).  (F) Comparison of change in DA efflux in NAc at 30min peak following IP injections of d-AMPH or saline between Day 1 and Day 9.  Repeated treatment with Tat-GluA23Y significantly attenuated the magnitude of d-AMPH-induced DA efflux in the NAc by the 5th d-AMPH injection (pairwise comparison, Day 1: 633% ? 170% vs. Day 9: 278% ? 53%, *p<0.01).    2.4.2 Repeated i.v. Tat-GluA23Y administration during induction phase blocks maintenance and expression of behavioral and neurochemical sensitization to d-AMPH  Following a 14-day drug-free period, rats previously given Tat-GluA23Y prior to each d-AMPH injection showed a significantly reduced locomotor response to the d-AMPH challenge compared to groups that received vehicle or Tat-GluA23Yscr prior to d-AMPH in the induction phase.  Furthermore, distance traveled for rats given repeated Tat-GluA23Y  and d-AMPH did not differ significantly from rats receiving the challenge dose of d-AMPH for the first time (Fig. 2.5C Treatment group: F(3,40) = 5.89, p<0.01; post hoc: Tat-GluA23Y + d-AMPH vs. vehicle + d-AMPH, p<0.05; Tat-GluA23Y + d-AMPH vs. Tat-GluA23Yscr + d-AMPH, p<0.05; Tat-GluA23Y + d-AMPH vs. vehicle + saline, ns).  These data indicate that Tat-GluA23Y peptide treatment blocked the induction of behavioral sensitization.   39  Clear evidence of a sensitized neurochemical effect was provided by a comparison of the magnitude of d-AMPH-induced DA efflux in the NAc on challenge tests on Day 24 in rats treated repeatedly with d-AMPH versus rats receiving the challenge dose of d-AMPH for the first time (Fig. 2.5D, Treatment group: F(3,32) = 36.285, p<0.01; post hoc: vehicle + d-AMPH vs. vehicle + saline, p<0.05; Tat-GluA23Yscr + d-AMPH vs. vehicle + saline, p<0.01).  Most importantly, repeated treatment with Tat-GluA23Y during the induction phase blocked the expression of neurochemical sensitization to a challenge injection of d-AMPH in the absence of peptide (pairwise comparison, Tat-GluA23Yscr + d-AMPH vs. Tat-GluA23Y + d-AMPH, p<0.05).    40 Figure 2.5 Expression of d-AMPH sensitization.  After a 14-day incubation period, all animals received a challenge dose of d-AMPH (0.5 mg/kg, IP).   (A) Time course of behavioral activity following d-AMPH challenge.  Group notations indicate the treatment given during the induction of sensitization  [Vehicle + d-AMPH (n=10), Tat-GluA23Yscr + d-AMPH (n=10), Tat-GluA23Y + d-AMPH (n=10), vehicle + saline (n=9)].  (B) Microdialysis in NAc measuring DA efflux in each group following d-AMPH challenge.  Again, group notations indicate treatment during induction of sensitization [Vehicle + d-AMPH (n=13), Tat-GluA23Yscr + d-AMPH (n=10), Tat-GluA23Y + d-AMPH (n=9), vehicle + saline (n=8)].  (C) Cumulative distance traveled over 30 min following d-AMPH challenge injection.   Rats treated with Tat-GluA23Y during the induction period did not express locomotor sensitization.  Treatment group: F(3,40) = 5.89, p<0.01; post hoc: vehicle + d-AMPH vs. vehicle + saline, *p<0.05; Tat-GluA23Yscr + d-AMPH vs. vehicle + saline, *p<0.05; Tat-GluA23Y + d-AMPH vs. vehicle + saline, ns). (D) Change in DA efflux at 30 min peak.  Repeated treatment with Tat-GluA23Y during the induction of sensitization blocked the expression of neurochemical sensitization to d-AMPH.  (Treatment group: F(3,32) = 36.285, p<0.01; post hoc: vehicle + d-AMPH vs. vehicle + saline, *p<0.05; Tat-GluA23Yscr + d-AMPH vs. vehicle + saline, *p<0.01; pairwise comparison, Tat-GluA23Yscr + d-AMPH vs. Tat-GluA23Y + d-AMPH, *p<0.05).   2.4.3 Microinjection of Tat-GluA23Y into the VTA or NAc has no effect on induction of d-AMPH sensitization Infusion of Tat-GluA23Y into the VTA prior to a systemic d-AMPH injection did not significantly affect d-AMPH-induced locomotion on the 1st day of exposure (Fig. 2.6A, repeated measures ANOVA, F(9,38) = 8.34, p<0.05; post hoc: Tat-GluA23Y + d-AMPH vs. Tat-GluA23Yscr + d-AMPH, ns; Tat-GluA23Y + d-AMPH vs. vehicle + d-AMPH, ns).  Furthermore, repeated d-AMPH injections led to significant increases in activity and total distance traveled on Day 9 of the induction phase in all groups, including those pretreated with Tat-GluA23Y (Fig. 2.6C and 2.6E, Day x Treatment group: F(1,5) = 5.47, p<0.01; post hoc: vehicle + d-AMPH group, Day 1 vs. Day 9, p<0.01; Tat-GluA23Yscr + d-AMPH group, Day 1 vs. Day 9, p<0.01; Tat-GluA23Y + d-AMPH group, Day 1 vs. Day 9, p<0.01).     41 Similarly, infusions of Tat-GluA23Y into the NAc prior to each  systemic d-AMPH injection did not affect d-AMPH-induced locomotion on the 1st or 9th day of the induction phase (Fig. 2.6B and 2.6D).  Repeated d-AMPH injections led to a significant increase in peak activity and total distance traveled from Day 1 to Day 9, in all groups including the one pretreated with Tat-GluA23Y (Fig. 2.6F, Day x Treatment group: F(1,5) = 19.85 p<0.01; post hoc: vehicle + d-AMPH group, Day 1 vs. Day 9, p<0.01; Tat-GluA23Yscr + d-AMPH group, Day 1 vs. Day 9, p<0.01; Tat-GluA23Y + d-AMPH group, Day 1 vs. Day 9, p<0.01).     42  Figure 2.6  Time course and cumulative data of locomotor activity during induction of d-AMPH behavioral sensitization following microinjection of Tat-GluA23Y or controls into VTA or NAc.  (A) Day 1 of induction period for rats receiving microinjections into the VTA [Vehicle + d-AMPH (n=6), Tat-GluA23Yscr + d-AMPH (n=7), Tat-GluA23Y + d-AMPH (n=7), vehicle +   43 saline (n=8), Tat-GluA23Yscr + saline (n=8), Tat-GluA23Y + saline (n=8)].  (B) Day 1 of induction period for rats receiving microinjections into the NAc [Vehicle + d-AMPH (n=9), Tat-GluA23Yscr + d-AMPH (n=6), Tat-GluA23Y + d-AMPH (n=8), vehicle + saline (n=6), Tat-GluA23Yscr + saline (n=6), Tat-GluA23Y + saline (n=6)].  Time-course of locomotor activity on Day 9 of induction period in the same animals; VTA (C), NAc (D). Comparison of cumulative distance traveled over 30min following IP injection of d-AMPH, between Day 1 and Day 9; VTA (E), NAc (F).  Repeated d-AMPH injections led to significant increases in activity and total distance traveled on Day 9 of the induction phase in all groups, including those pretreated with Tat-GluA23Y (VTA: Day x Treatment group: F(1,5) = 5.47, p<0.01; post hoc: vehicle + d-AMPH group, Day 1 vs. Day 9, *p<0.01; Tat-GluA23Yscr + d-AMPH group, Day 1 vs. Day 9, *p<0.01; Tat-GluA23Y + d-AMPH group, Day 1 vs. Day 9, *p<0.01).  (NAc: Day x Treatment group: F(1,5) = 19.85, p<0.01; post hoc: vehicle + d-AMPH group, Day 1 vs. Day 9, *p<0.01; Tat-GluA23Yscr + d-AMPH group, Day 1 vs. Day 9, *p<0.01; Tat-GluA23Y + d-AMPH group, Day 1 vs. Day 9, *p<0.01).  Note that Tat-GluA23Y injections into either structure had no effect on induction of d-AMPH sensitization.  2.4.4 Microinjection of Tat-GluA23Y into the VTA, but NOT the NAc, inhibits maintenance and expression of d-AMPH sensitization  When challenged with d-AMPH (0.5mg/kg) following a 14-day drug-free period, rats treated with microinjections Tat-GluA23Y into the VTA during the induction phase failed to express behavioral sensitization on Day 24 (Fig. 2.7A and 2.7C, Treatment group: F(5,38) = 3.25, p<0.05; post hoc: Tat-GluA23Y + d-AMPH vs. vehicle + d-AMPH, *p<0.05; Tat-GluA23Y + d-AMPH vs. Tat-GluA23Yscr + d-AMPH, *p<0.05).   In contrast, all sensitized animals, including those pretreated with microinjections of Tat-GluA23Y into the NAc, displayed an enhanced response to the d-AMPH challenge relative to control subjects receiving  d-AMPH of the first time (Fig. 7B and 7D, d-AMPH main effect: F(5,36) = 6.54, p<0.01).     44  Figure 2.7 Expression of behavioral sensitization to a challenge dose of d-AMPH in animals that received microinjections of Tat-GluA23Y or controls. Time-course of locomotor activity following challenge injection of d-AMPH in animals given microinjections of Tat-GluA23Y or controls into the VTA (A) or NAc (B) during induction.  (C) Cumulative distance traveled over 30 min following d-AMPH challenge injection.  Repeated microinjections of Tat-GluA23Y into the VTA during the induction period inhibited the maintenance and expression of d-AMPH sensitization.  (Treatment group: F(5,38) = 3.25, p<0.05; post hoc: Tat-GluA23Y + d-AMPH vs. vehicle + d-AMPH, *p<0.05; Tat-GluA23Y + d-AMPH vs. Tat-GluA23Yscr + d-AMPH, *p<0.05)  (D) Repeated microinjections of Tat-GluA23Y into the NAc during the induction period had no effect on maintenance and expression of d-AMPH sensitization.  All sensitized animals, including those pretreated with Tat-GluA23Y, expressed a greater response to the d-AMPH challenge (d-AMPH main effect: F(5,36) = 6.54, *p<0.01).   45  2.5   Discussion Consistent with the behavioral sensitization literature, locomotor activity increased significantly from Day 1 to Day 9 as a result of repeated amphetamine exposure.(Robinson and Berridge, 1993;Kalivas and Stewart, 1991)  Importantly, this effect was maintained in all control groups after a 14-day drug-free period, as evidenced by a significantly enhanced locomotor response relative to drug-na?ve controls following the challenge dose of d-AMPH (0.5 mg/kg) on Day 24.  Intravenous administration of Tat-GluA23Y did not affect the initial acute locomotor response on Day 1, however, repeated co-administration of Tat-GluA23Y with each amphetamine injection prevented the induction of behavioral sensitization normally observed after five intermittent injections of d-AMPH.  A challenge injection of d-AMPH following a 14-day drug-free period confirmed that co-administration of Tat-GluA23Y peptide during the induction phase blocked the induction and maintenance of d-AMPH-induced behavioral sensitization observed in the Tat-GluA23Yscr and vehicle-treated groups.   The microdialysis experiment confirmed that acute d-AMPH induced a significant increase in DA efflux in the NAc in the control groups on the 1st and 5th day of drug exposure. It is important to note that the induction of sensitization is not always accompanied by an increase in DA release in the NAc, especially after a short incubation period.  Paulson & Robinson (Paulson and Robinson, 1995) measured motor behavior and DA neurotransmission at different times after discontinuation of repeated d-AMPH treatment, and failed to observe a sensitized neurochemical response to a challenge dose of d-AMPH after either 3 or 7 days of withdrawal. In contrast, animals that received the same sensitization protocol displayed enhanced DA release when tested after a 28 day drug-free period.  These results indicate that   46 the development of neurochemical sensitization, expressed as an increase of DA release in the NAc, may develop more slowly than enhanced motor activity.  In the present study, when tested after a 14-day incubation period, both Tat-GluA23Yscr and vehicle -treated groups displayed significantly higher levels of evoked-DA than the d-AMPH na?ve group, thereby confirming the occurrence of neurochemical sensitization (Robinson and Berridge, 1993).   Here we also identify a distinct role for regulated endocytosis of GluA2?containing AMPARs in modulating the efflux of DA within the NAc after repeated but not acute exposure to d-AMPH.  A transient (1-5 days) synaptic potentiation of AMPAR currents in VTA DA neurons(Borgland et al., 2004) is attributed to a change from GluA2-containing to GluA2-lacking AMPARs, induced by the initial exposure to a psychostimulant drug.(Argilli et al., 2008;Brown et al., 2010)  In the present study, the significant attenuation of DA efflux observed after the 5th injection of d-AMPH, when GluA2-containing AMPAR endocytosis is blocked by co-administration of Tat-GluA23Y, clearly implicates the development of LTD in the maintenance of a robust neurochemical response to repeated psychostimulant exposure. The behavioral pharmacological response to d-AMPH, whether in the form of a conditioned response associated with repeated drug exposure or as drug-induced behavioral sensitization studied here, is highly influenced by associative processes (Robinson and Berridge, 1993;Anagnostaras and Robinson, 1996), which in turn are encoded as enduring memories of drug-related environmental stimuli. We hypothesize that formation of such memories is dependent on an LTD-like process that may well involve the replacement of GluA2-containing by GluA2-lacking AMPARs.  When AMPAR endocytosis is disrupted by the interference peptide Tat-GluA23Y, the inhibition of LTD results in the absence of both   47 neurochemical and behavioral sensitization in rats subsequently exposed to a challenge dose of d-AMPH 14 days later.  The microinjection studies targeting the NAc and VTA revealed important differences in the neuroplasticity involved in the induction of sensitization as distinct from those processes related to the maintenance of behavioral sensitization.  Notably, microinjection of Tat-GluA23Y into either the VTA or the NAc had no effect on the initial increase in drug-induced locomotion from drug injection 1 to 5.  In contrast, disruption of GluA2 endocytosis in the VTA during the induction phase did have a marked effect on d-AMPH-induced locomotion in response to the final challenge dose of this drug on day 24.  A direct comparison of the VTA microinjection data to results obtained with systemic administration of Tat-GluA23Y suggests that the blockade of sensitization observed in the latter condition involves actions of Tat-GluA23Y at sites beyond the VTA, which remain to be identified.  Accordingly, there is a distinction between processes responsible for the immediate increase in motor activity during repeated injections of a psychostimulant drug and those neuroadaptations related to the encoding and augmentation of behavioral sensitization over a drug-free period prior to the expression of sensitization (Anagnostaras and Robinson, 1996).  Importantly, our findings are consistent with the literature linking neural activity within the VTA to neuroadaptations that establish and maintain sensitization (Ungless et al., 2001;Kalivas and Duffy, 1993;Vezina and Stewart, 1990;Wolf and Xue, 1999), as distinct from other forms of adaptation in the NAc responsible for the expression of behavioral sensitization (Robinson and Kolb, 1997;Kalivas and Duffy, 1993).  The imposition of a prolonged drug-free period following repeated non-contingent treatments with cocaine is related to enhanced AMPAR function in MSNs in the NAc shell, which is linked to increased   48 surface expression of GluA1 and GluA2 subunits.(Boudreau et al., 2007;Boudreau et al., 2009) This state may be permissive for the expression of behavioral sensitization, as both enhanced AMPAR currents and surface expression receptor subunits are reversed immediately following a challenge dose of cocaine.(Boudreau et al., 2007;Kourrich et al., 2007;Thomas et al., 2001)   A recent discussion of synaptic plasticity induced by drugs of abuse and its contributions to the neuropathology of addiction highlights the many and varied processes involved (Van den Oever et al., 2012).  Initial findings emphasize the induction of LTP by acute exposure to drugs of abuse that was subsequently linked to an increase in AMPAR trafficking(Wolf et al., 2004).  Of particular importance to the present discussion is the finding that the prominence of GluA2-containing AMPARs in a drug-na?ve state is replaced by increased expression of the GluA2-lacking variant of AMPARs following exposure to a psychostimulant drug.(Argilli et al., 2008;Brown et al., 2010)  Together, these findings suggest that increased Ca2+ permeability and conductance of GluA2-lacking AMPA receptors are essential factors in the sensitized response to a psychostimulant drug challenge.  If this change in AMPAR subtype is a critical step in the modification of glutamatergic synapses on VTA-DA neurons required for the encoding of associations between contextual stimuli and drug reward, it follows that interference with regulated endocytosis of GluA2-containing AMPARs by Tat-GluA23Y, is an ideal tool to prevent the ascendency of drug-related memories.  2.6   Limitations Behavioral sensitization in rodents, as a model of addiction, does not provide direct measures of drug reinforcement. One may also question the relevance of experimenter-administered   49 drug treatment to an understanding of processes related to human addiction.  Critics of non-contingent drug administration methods favour the use of drug self-administration protocols, however, it is also important to employ procedures that lead to behavioral, psychological or neurobiological outcomes that parallel those seen in clinical addiction.(Robinson and Berridge, 2008) Animals that develop behavioral sensitization acquire self-administration more readily, indicating increased motivation for drug reward,(Vezina, 2004) they also exhibit impairments in tests of cognition,(Schoenbaum and Shaham, 2008) display prolonged neurochemical alterations such as enhanced glutamate release(Pierce et al., 1996) or drug-induced DA efflux(Paulson and Robinson, 1995) as observed in the present study.  The intermittent treatment and relatively high dose of drug per injection, may contribute to the utility of sensitization in mimicking the initial period of irregular drug use and experimentation, while the maintenance of a heightened response seen after a prolonged drug-free period may effectively model drug craving. Although we have not observed adverse side effects in rats treated repeatedly with the Tat-GluA23Y peptide, formal toxicology is required before it may be considered as a candidate for use in human clinical trials. 2.7   Conclusions The interplay between glutamatergic synapses and dopaminergic projections onto medium spiny neurons (MSNs) within the NAc core has long been recognized as a critical factor in the expression of behavioral sensitization, as well as different forms of relapse to drug-seeking behavior. In a similar manner, AMPARs in the VTA have also been implicated in the induction of behavioral sensitization (Luscher and Malenka, 2011).  The present study provides compelling evidence linking AMPAR endocytosis within the VTA to the induction and maintenance of behavioral sensitization.  In this context, inhibition of the endocytosis of   50 GluA2-containing AMPA receptors by Tat-GluA23Y interference peptide may serve to maintain or restore the balance of GluA2-containing to GluA2-lacking AMPARs.  Furthermore, these effects may reflect modulation of DA transmission within the NAc.  The current findings complement our previous observation that disruption of GluA2 AMPAR endocytosis by direct microinjection of Tat-GluA23Y peptide into the NAc can prevent the expression of behavioral sensitization to d-AMPH.(Brebner et al., 2005)   By preventing the induction of LTD, the Tat-GluA23Y interference peptide may retain the mesocorticolimbic DA system and related glutamatergic modulation in a drug-na?ve state.  An immediate consequence of this mode of action would be to block the encoding of critical drug ? contextual stimuli associations essential for the maintenance and expression of behavioral sensitization.  Given the arguments that support a close relationship between behavioral sensitization and drug-craving (Vezina et al., 1999;Lorrain et al., 2000) and its relevance to clinical addiction in humans, (Boileau et al., 2006) one may contemplate the use of the Tat-GluA23Y interference peptide to protect against the development of craving and drug-seeking behaviours.   51 Chapter Three: The Role of AMPA Receptor Endocytosis in Context-dependent Behavioral Sensitization   3.1   Overview  In humans, drug addiction is usually characterized by a strong association between the experience of drug-reward and drug-related environmental contexts.  These learned associations are now considered crucial to the persistence of compulsive drug-seeking and craving during periods of abstinence, and when presented, can often trigger relapse.  In a series of 3 experiments, we show in a rat model of behavioral sensitization that blocking regulated AMPAR endocytosis has lasting effects on d-AMPH-induced long-term sensitization, context-dependent sensitization, and to a certain extent, dual-context sensitization. Our results demonstrate that intravenous injections of a peptide inhibiting GluA2 endocytosis attenuated d-AMPH-induced locomotion, inhibited the expression of sensitization to a d-AMPH challenge, and blocked the further development of sensitization to subsequent d-AMPH injections in the absence of peptide.  Using dual conditioning contexts, our results support the phenomenon of context-dependent sensitization and reveal the role of ?occasion-setting?, in which there is an active inhibitory process that can block the expression of sensitization in contexts where the drug is not expected, while amplifying the sensitized response in a context where the drug is expected.  When sensitization was induced concurrently in two separate environments, we observed an enhanced motor stimulant effect of d-AMPH in a second environment in which the control peptide was given.  We interpret this effect as evidence of a role for AMPAR trafficking in inhibitory occasion-setting.  Environmental cues are a major contributing factor to triggering craving, therefore identifying the involvement of GluA2 endocytosis as one of the mechanisms underlying context-  52 dependent drug responses can inform the development of new therapeutic approaches for the treatment of drug addiction. 3.2   Introduction Two of the primary barriers to treating addiction are the persistent nature of drug craving maintained over long periods of abstinence and the powerful ability of drug-associated environmental stimuli to trigger this craving and subsequent drug-seeking behavior (Anagnostaras and Robinson, 1996;Crombag et al., 2008;Everitt and Robbins, 2005;Hyman and Malenka, 2001;Phillips and Fibiger, 1990).  The learned associations between the effects of drugs and the drug exposure environment may in turn share neural mechanisms with other aspects of Pavlovian conditioned associative learning including processes related to synaptic plasticity.  In particular, neural adaptations in the mesocorticolimbic regions of the brain, including a terminal region in the NAc that receives dopaminergic projections from the VTA and excitatory glutamatergic inputs from the PFC has been implicated in many drug-related learned behaviors, including context-specific behavioral sensitization(Dias et al., 2012;Vezina and Leyton, 2009;Badiani and Robinson, 2004).  As noted repeatedly, behavioral sensitization, characterized by the progressive and long-lasting increase of locomotor response after repeated drug exposure, is an animal model of drug-craving (Robinson and Becker, 1986;Vanderschuren and Kalivas, 2000).  As discussed in Chapter 2, behavioral sensitization is related to LTD at glutamatergic synapses in the VTA for maintenance and additional synaptic adaptations in the NAc is involved in long-term expression.  LTD in the brain is proposed to be a cellular substrate for learning and memory, and more importantly, LTD in the NAc can be blocked by a GluA2-derived ?interference peptide? (Tat-GluA23Y), by disrupting regulated a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor   53 (AMPAR) endocytosis (Ahmadian et al., 2004).  Recent findings using Tat-GluA23Y  in models of addiction show that this interference peptide blocks the expression of d-AMPH-induced behavioral sensitization (Brebner et al., 2005), extinction of morphine-induced place preference (Dias et al., 2012), and cue-induced reinstatement of heroin self-administration (Van den Oever et al., 2008).   AMPARs receptors are comprised of heteromeric subunits and those consisting of the GluA2 subunit in particular are linked to regulated endocytosis (Man et al., 2000).  The CT region of the GluA2 subunit contains sequence motifs that account for this specificity, consisting of domains that bind to NSF and AP2 clathrin adaptor, as well as a PDZ-binding domain that is specific to certain PDZ-containing proteins such as GRIP and PICK1 (Chung et al., 2000;Lee et al., 2002).  Of particular importance is the discovery of a new tyrosine phosphorylation-dependent sequence of amino-acids containing 3 tyrosine residues (869YKEGYNVYG877) that are essential for NMDAR-mediated and insulin-stimulated AMPA receptor endocytosis.  Based on this sequence, a Tat-GluA23Y interference peptide was developed and found to block stimulated-LTD in hippocampal slices (Ahmadian et al., 2004).  A recent study identified that BRAG2 is the synaptic protein which interacts with the GluA2 subunit to activate Arf6 and the internalization of AMPA receptors to produce LTD.  Through blocking the GluA2 and Arf6 interaction, as well as targeted deletion of BRAG2 in hippocampal neurons, it was demonstrated that AMPA receptor endocytosis and the resulting LTD can be interrupted (Scholz et al., 2010).   Chapter 2 describes studies in which Tat-GluA23Y blocked the development and expression of behavioral and neurochemical sensitization to repeated, intermittent d-AMPH injections.   The present study begins by extending our analysis of the role of the interference   54 peptide, Tat-GluA23Y, in behavioral sensitization.  First, we aimed to replicate and extend the previous findings that Tat-GluA23Y given prior to each d-AMPH treatment has the ability to block the expression of behavioral sensitization upon subsequent drug challenge.  This observation raises the possibility of  more permanent blocking-effect of Tat-GluA23Y therefore animals were given a second induction series of d-AMPH injections in the absence of Tat-GluA23Y and then tested again for expression of sensitization following a second drug-free incubation period. The phenomenon of LTD has been linked to novel environment stimuli as indicated by the observation that administration of low-frequency stimulation during exploration of a novel (and not a familiar) environment results in the expression of LTD (Manahan-Vaughan and Braunewell, 1999).  Furthermore, formation of associative spatial memories can also be initiated by hippocampal LTD-like processes (Etkin et al., 2006).  Environmental context also plays a critical role in the development of behavioral sensitization (Stewart and Vezina, 1991;Anagnostaras and Robinson, 1996;Badiani and Robinson, 2004;Tirelli and Terry, 1998), raising the possibility that unique associations are formed between the processing of novel environmental stimuli and the direct stimulant properties of drugs such as amphetamines and cocaine.  Given the data linking LTD to processing of novel stimuli, we tested the hypothesis that this form of synaptic plasticity may exert its influence on behavioural sensitization by blocking associative learning linking the contextual stimuli to the rewarding effects of psychostimulant drugs.   We first demonstrated the context specific effect of d-AMPH in the present experimental setup by first pairing 4 injections of the psychostimulant drug with a unique context (Context A) and then confirmed a sensitized locomotor response in Context A but not   55 in a novel environment (Context B).  Co-administration of the interference peptide, Tat-GluA23Y, with each injection of d-AMPH in Context A prevented the development of context-specificity.  Importantly, when the rats treated previously with Tat-GluA23Y and d-AMPH in Context A were given d-AMPH alone in Context B, normal sensitization was observed.   Finally, we examined the effects of exposing individual animals to two separate and distinct drug environments on consecutive days, during which the Tat-GluA23Y peptide was co-administered with d-AMPH in only one of the two environments.  Two possible outcomes were anticipated, with the first being the restriction of a sensitized response to the environmental context in which Tat-GluA23Y and d-AMPH were not paired.  This result would imply that the role of LTD is in forming an association between the contextual stimuli and the unconditioned drug-reward.  Alternatively, by permitting two separate occasions for the formation of contextual associations between the stimulant effects of d-AMPH and specific contexts (A vs. B), this offers the possibility of establishing competing associations (occasion-setting) which in turn limit the amount of locomotor behaviour expressed in a given environment.  If active inhibitory processes are involved, it is conceivable that such inhibition may be mediated by LTD.  Accordingly, we hypothesized that rats receiving co-administration of Tat-GluA23Y and d-AMPH in one context would display a significantly enhanced locomotor response when challenged with d-AMPH in the second environment.    3.3   Methods 3.3.1 Animals Male Sprague-Dawley rats weighing approximately 250 g were housed in pairs in transparent cages in a temperature-controlled animal colony. Animals were acclimatized to a 12-hr   56 reverse light dark cycle (lights on 1900 hrs) for 7 days prior to the beginning of experiments, and were individually housed once experiments begin.  Animals were given ad libitum access to food and water during all phases of the study.  All the experiments have received approval from the Institutional Animal Care and Use Committee and met the International Animal Guide for the Care and Use of Laboratory Animals. 3.3.2 Drugs D-AMPH (USP, Rockville, MD) was diluted in 0.9% sterile saline. Tat-GluA23Y peptide consisting of 9 amino acids (YKEGYNVYG), and was attached to an HIV-1 Tat peptide (YGRKKRRQRRR) to facilitate transport across the blood brain barrier and to permeate cells.(Ahmadian et al., 2004) The scrambled peptide, Tat-GluA23Yscr, was comprised of the same 9 amino acids placed in random sequence (VYKYGGYNE) and served as a control.  Both Tat-GluA23Y and Tat-GluA23Yscr (GL Biochem-Shanghai Ltd, Shanghai, China) were diluted in 0.9% sterile saline. 3.3.3 Behavioral apparatus Eight open-field Plexiglas chambers, measuring 40cm x 40cm x 40cm were used to measure horizontal locomotion.  For experiments 2 and 3, two distinct environmental contexts were created, differentiated by color, texture, and odor cues.  Context A consisted of black chamber walls and soft newspaper bedding (CareFresh) on the floor of the chambers.  Context B consisted of white chamber walls and wire mesh flooring with a combination of pine and cedar bedding beneath the wire mesh to provide an odor cue that was distinct from Context A.  Top-mounted cameras allowed video-capture and tracking of locomotor activity that was   57 measured and scored by digital monitoring software, Ethovision 3.1 (Noldus, Inc).  All experiments were run in a darkened room under dim red lighting between 0800 and 1800 h.   3.3.4 Surgical procedures Animals were anaesthetized with isoflurane (induction at 3%, maintenance at 2-2.5%, in oxygen). As an adjunct to gaseous anaesthesia, the analgesic, ketoprofen (Anafen), was administered subcutaneously at the time of surgery to help minimize post-surgical distress.  Once a surgical plane of anesthesia was achieved, hair was clipped in two areas measuring 3.5cm x 6cm on the dorsal surface and 2.5cm x 4cm on the anterior right region of the ventral surface of each rat.  An iodophor scrub (Betadine) was applied to the skin surface alternated twice with 70% ethyl alcohol to disinfect the skin.  A single incision measuring 2.5cm was made using a No.10 surgical blade on the dorsal surface and an incision measuring 2cm was made on the ventral surface above the right jugular vein.  A chronic indwelling silastic catheter connected to a guide cannula (Plastics One, 22 ga) was secured to the dorsal surface of the rat with Dacron mesh. The silastic tubing was adhered to the vein by a series of sutures (4-0 silk, nonabsorbable).  Surgical wounds were closed with Polyglactin 910 (Vicryl, absorbable).  After surgery, the cannulae were regularly flushed with heparinized saline to ensure patency.  All Rats were given a 1-week recovery period prior to experimental procedures during which their conditions were closely monitored twice daily for possible post-operative complications.   58 3.3.5 Experiment 1: Induction, expression and long-term maintenance of behavioral sensitization.   A total of 6 treatment groups (n=8) were involved in the first experiment.  2 groups received 5 infusions of Tat-GluA23Y peptide (1.5 nM/gr, i.v.) 60 min preceding each of 5 injections of d-AMPH (1.0 mg/kg, i.p.) or saline (0.9% NaCl) given every other day, on Experimental Days (ED) 1, 3, 5, 7, and 9.  Groups 3 and 4 received the inactive form of the peptide, Tat-GluA23Yscr (1.5 nM/gr, i.v.), 60 min preceding injection of d-AMPH (1.0 mg/kg, i.p.) or saline (0.9% NaCl) every other day.  Groups 5 and 6 received vehicle saline (0.9% NaCl, i.v.), 60 min preceding injection of d-AMPH (1.0 mg/kg, i.p.) or saline (0.9% NaCl) every other day.  Following injections, rats were placed in open-field test chambers where locomotor activity was measured and recorded for 90 min. All rats were returned to their home cages at the end of the 90 min. session. After the 5th injection, rats underwent a 14-day drug-free period.  Beginning on ED 24, all rats were exposed to 5 challenge doses of d-AMPH (0.5 mg/kg i.p.) every other day over 10 days (ED 24, 26, 28, 30, and 32) and behavioral analysis was conducted as previously described.  Following the 5 challenge injections, animals underwent a second 14-day drug-free period and were challenged with a final dose of d-AMPH (0.5 mg/kg i.p.) on ED 47.   3.3.6 Experiment 2: Induction and expression of context-dependent behavioral sensitization.   Animals were divided into 4 treatment groups (n=8).  3 groups were given Tat-GluA23Y peptide (4 x 1.5 nM/gr, i.v.) or Tat-GluA23Yscr peptide (4 x 1.5 nM/gr, i.v.) or vehicle control (4 x 0.9% saline, i.v.) 60 min prior to d-AMPH (4 x 1.0 mg/kg, i.p.) on ED 1, 3, 5 and 7 in   59 Context A.  A control group received vehicle control paired with saline injections on all 4 treatment days.  After the 4th injection, rats underwent a 14-day drug-free period.  On the 15th day post-induction (ED 22), all rats were re-exposed to d-AMPH (0.5 mg/kg i.p.) in the drug-paired Context A. 2 days later (ED 24), rats were given a second challenge test of d-AMPH (0.5 mg/kg i.p.) in a new environment, Context B, to determine the presence of context-dependent sensitization.   3 additional injections of d-AMPH (0.5 mg/kg i.p.) were administered in Context B in the absence of Tat-GluA23Y to determine if sensitization could be induced in a new context.  Animals underwent a second 14-day drug-free period and were challenged with d-AMPH (0.5 mg/kg i.p.) on ED 45 in Context B. 3.3.7 Experiment 3: Concurrent induction and expression of behavioral sensitization in two separate environmental contexts.   For the third experiment, animals were assigned to 6 treatment groups (n=8).  2 groups were given four infusions of Tat-GluA23Y (1.5 nM/gr, i.v.) or Tat-GluA23Yscr  (1.5 nM/gr, i.v.) 60 min preceding d-AMPH (1.0 mg/kg, i.p.) paired with Context A, then on alternate days given infusions of vehicle (0.9% NaCl, i.v.) 60 min preceding d-AMPH (1.0 mg/kg, i.p.) paired with Context B.  Another 2 groups were given four infusions of Tat-GluA23Y (1.5 nM/gr, i.v.) or Tat-GluA23Yscr  (1.5 nM/gr, i.v.) 60 min preceding d-AMPH (1.0 mg/kg, i.p.) paired with Context B, then on alternate days were given infusions of vehicle (0.9% NaCl, i.v.) 60 min preceding d-AMPH (1.0 mg/kg, i.p.) paired with Context A. One control group (d-AMPH control) was given vehicle (0.9% NaCl, i.v.) 60 min preceding d-AMPH (1.0 mg/kg, i.p.) paired with Context A then on alternate days received the same d-AMPH treatment paired with Context B.  The final group (saline control) was given vehicle (0.9% NaCl, i.v.) 60 min preceding injections of saline (0.9% NaCl, i.p.) paired with Context A then on alternate days   60 received the same control treatment paired with Context B.  Behavioral testing procedures were identical to those employed in Experiment 1.  Following the 8th injection (ED 16), rats underwent a 14-day drug-free period after which challenge injections of d-AMPH (0.5 mg/kg i.p.) were paired with re-exposure to Context A, followed 2 days later in Context B, or vice versa to counter-balance for effects of previous treatment. 3.3.8 Data analysis All values are expressed as mean ? (standard error of the mean) SEM.  Statistical significance was assessed using one-way ANOVA or two-way ANOVA with repeated measures with time as the within-subject factor and treatment groups as the between-subjects factor.  Where appropriate, post hoc analyses were performed using Tukey HSD to examine simple main effects.  Statistical significance was set at p < 0.05. 3.4   Results 3.4.1 Inhibition of GluA2 endocytosis blocks long-term maintenance of d-AMPH sensitization. Once established, behavioral sensitization to d-AMPH is long-lasting and can be expressed following extended drug-free periods (Robinson & Becker, 1986).  As indicated by the findings presented in Chapter 2, the development of sensitization appears to involve neuroadaptations that require GluA2 endocytosis.  Tat-GluA23Y interference peptide provides a highly selective means to block GluA2 endocytosis and thereby to examine the role of LTD mechanisms in synaptic neuroplasticity related to learned associations required for the long-term maintenance of behavioral sensitization.  In the first experiment, rats were exposed to   61 two phases of induction in which Tat-GluA23Y was paired with d-AMPH only during the first series.  The expression of long-term sensitization was tested twice, each time following a 2-week drug free period. The experimental timeline and resulting data are represented below in Figure 3.1.  While repeated injections of d-AMPH (1.0 mg/kg) led to a significant increase in locomotor activity from Day 1 to Day 9, intravenous infusions of Tat-GluA23Y prior to each of the initial injections of d-AMPH significantly attenuated the expression of sensitization following a drug-free period.     62  Figure 3.1 (Top) Treatment and Experiment 1 Timeline (Bottom) Panel A: Day 1 and Day 9 of Induction treatment period.  Panel B: Day 24 d-AMPH Challenge. Panel C: Second Induction treatment period, Day 24 and Day 32. Panel D: Day 47 final d-AMPH Challenge On induction day 1, treatment with d-AMPH produced significantly greater locomotor activity compared to treatment with saline, as expected (Fig.3.1A: Treatment group: F(5,47) = 6.66, p<0.05, post hoc: vehicle + d-AMPH vs. vehicle + saline, p<0.05; Tat-GluA23Yscr + d-  63 AMPH vs. Tat-GluA23Yscr + saline, p<0.05).  Locomotor activity of rats receiving co-administration of interference peptide and d-AMPH did not differ significantly from locomotor activity in the saline-control group (Tat-GluA23Y + d-AMPH vs. Tat-GluA23Y + saline, ns).  Subsequent repeated d-AMPH treatments led to a significant increase in locomotor response in all d-AMPH groups, indicative of behavioral sensitization (Day x Treatment group F(5, 43) = 9.12, p<0.05: pairwise comparison: vehicle + d-AMPH Day 1 vs. Day 9, p< 0.05; Tat-GluA23Yscr  + d-AMPH Day 1 vs. Day 9, p< 0.05; Tat-GluA23Y + d-AMPH Day 1 vs. Day 9, p<0.05).  Repeated saline treatments led to a gradual, though not significant decrease in locomotor response in all saline groups.  Following a 14-day drug-free period, all groups were given a challenge injection of d-AMPH, and sensitized groups (d-AMPH pretreated) were compared to their respective acute exposure control groups (saline pretreated).  Both vehicle + d-AMPH and Tat-GluA23Yscr + d-AMPH groups displayed a sensitized response to drug challenge (Fig. 3.1B: Treatment group: F(5,47) = 4.21, p<0.05, post hoc: vehicle + d-AMPH vs. vehicle + saline, p<0.05; Tat-GluA23Yscr  + d-AMPH vs. Tat-GluA23Yscr  + saline, p<0.05).  Importantly, although repeated d-AMPH treatments led to a significant increase in locomotor activity from Day 1 to Day 9 in Tat-GluA23Y treated rats, when challenged with d-AMPH after a drug-free period, Tat-GluA23Y + d-AMPH rats failed to express a sensitized response when compared to acute challenge rats (Tat-GluA23Y + d-AMPH vs. Tat-GluA23Y + saline, ns).   3.4.2 Prior treatment with GluA23Y peptide + d-AMPH prevents subsequent induction of d-AMPH sensitization Our results indicate that disruption of regulated GluA2 endocytosis with the Tat-GluA23Y peptide can have both short- and long-term effects on amphetamine-induced behavioral   64 sensitization.  Animals treated initially with saline during the induction phase subsequently developed behavioral sensitization when given five injections of d-AMPH (0.5 mg/kg).  Importantly, animals treated initially with Tat-GluA23Y prior to d-AMPH injections did not develop behavioral sensitization when exposed to a second sensitizing regimen of the lower dose of d-AMPH 2-weeks later.  Specifically, repeated treatments with the lower challenge dose of d-AMPH (0.5 mg/kg) led to a significant increase in locomotor activity in all groups regardless of pretreatment condition except for rats that received co-administration of Tat-GluA23Y + d-AMPH during the initial phase of sensitization (Fig. 3.1C: Day x Treatment group F(5,39) =1.09, p<0.05, pairwise comparison: vehicle + saline Day 24 vs. Day 32, p<0.05; Tat-GluA23Yscr + saline Day 24 vs. Day 32, p<0.05; Tat-GluA23Y + saline Day 24 vs. Day 32, p<0.05; vehicle + d-AMPH Day 24 vs. Day 32, p<0.05; Tat-GluA23Yscr + d-AMPH Day 24 vs. Day 32, p<0.05; Tat-GluA23Y + d-AMPH Day 24 vs. Day 32, ns).  The final d-AMPH challenge on Day 47 revealed that in addition to suppressing the expression of sensitization on Day 24, treatment with Tat-GluA23Y + d-AMPH during the first phase of induction blocked the future development of sensitization to subsequent d-AMPH injections (Fig 3.1D: Treatment group: F(2,18) = 3.40, p<0.05, post hoc: vehicle + d-AMPH vs. Tat-GluA23Y + d-AMPH, p<0.05; Tat-GluA23Yscr + d-AMPH vs. Tat-GluA23Y + d-AMPH, p<0.05).    3.4.3 Inhibition of GluA2 endocytosis prevents induction of context-dependent sensitization  As predicted from the initial experiments, in Experiment 2, 4 d-AMPH treatments were sufficient to induce a significant increase in locomotor activity.  Data and experimental timeline are represented in Figure 3.2 (Fig. 3.2A: Day x Treatment group F(3,30)=7.212,   65 p<0.05, pairwise comparison: vehicle + d-AMPH Day 1 vs. Day 7, p< 0.05; Tat-GluA23Yscr  + d-AMPH Day 1 vs. Day 7, p< 0.05; Tat-GluA23Y + d-AMPH Day 1 vs. Day 7, p<0.05).  After the 14-day drug-free incubation period, challenge injections of d-AMPH given in Context A were accompanied by sensitized locomotor responses only in the groups treated with vehicle control or Tat-GluA23Yscr.  The acute challenge group treated with saline as well as the experimental group treated with Tat-GluA23Y + d-AMPH had significantly lower locomotor activity scores (Treatment group: F(3,30)=4.06, p<0.05, post hoc: vehicle + d-AMPH vs. vehicle + saline, p<0.05; vehicle + d-AMPH vs. Tat-GluA23Y + d-AMPH, p<0.05; Tat-GluA23Yscr + d-AMPH vs. vehicle + saline, p<0.05; Tat-GluA23Yscr + d-AMPH vs. Tat-GluA23Y + d-AMPH, p<0.05).   When d-AMPH was given in Context B (the non-paired environment) sensitized locomotor responses were no longer evident in the vehicle + d-AMPH or Tat-GluA23Yscr + d-AMPH groups, indicating that the previously observed expression of sensitization was context-dependent (Treatment group: F(3,30)=10.48, ns).   In contrast with the results observed in the first experiment, repeated d-AMPH injections in a new environment (Context B) were now able to induce sensitization and the expression of this newly acquired sensitization was demonstrated after a final d-AMPH challenge on Day 45.  This effect was observed in all groups, including rats previously treated with Tat-GluA23Y, which had failed to display sensitization in Context A  (Fig. 3.2B: Day x Treatment group F(3,30)=2.49, p<0.05, pairwise comparison: saline + d-AMPH Day 24 vs. Day 45; vehicle + d-AMPH Day 24 vs. Day 45, p< 0.05; Tat-GluA23Yscr  + d-AMPH Day 24 vs. Day 45, p< 0.05; Tat-GluA23Y + d-AMPH Day 24 vs. Day 45, p<0.05).   66  Figure 3.2 (Top) Treatment and Experiment 2 Timeline (Bottom) Panel A: Induction in Context A followed by d-AMPH Challenge in Context A & B. Panel B: d-AMPH Challenges in Context B and final Challenge in Context B  3.4.4 Selective effect of GluA23Y on context-dependent sensitization reveals the influence of occasion setting in behavioral sensitization. In order to gain further understanding of the influence of contextual cues on the development of sensitization, we exposed rats to a sensitizing regimen of d-AMPH in two separate environments on consecutive days.  Tat-GluA23Y or control treatments were administered in only 1 of the 2 environments.  Experimental protocol and resulting data are represented below in Figure 3.3.     67  Figure 3.3 (Top) Treatment and Experiment 3 Timeline (Bottom A) Induction of d-AMPH sensitization on Experimental Day 1 or 3 and Day 13 or 15. [NP-Env: Non-paired Environment; P-Env: Paired Environment]. (Bottom B) Final d-AMPH challenge on Experimental Day 30 and Day 32. Treatment Groups depict drug treatment given during the Induction phase: Vehicle + Saline;Vehicle + d-AMPH; GluA23Yscr + d-AMPH; or GluA23Y + d-AMPH.  On the Challenge days, d-AMPH is given in the treatment paired context and non-paired context on alternate days.  Following 8 treatment sessions, the 4 pairings of Tat-GluA23Y + d-AMPH significantly attenuated the development of locomotor sensitization in the paired environment only, while sensitization developed for animals treated with Tat-GluA23Y + d-AMPH in the non-paired environment, and also for those treated with vehicle + d-AMPH or Tat-GluA23Yscr + d-AMPH in both paired and non-paired environments (Fig.3.3A. Treatment group: F(5,47)=4.91,   68 p<0.05, post hoc: Tat-GluA23Y + d-AMPH (P-Env) vs. Tat-GluA23Y + d-AMPH (NP-Env), p<0.05; Tat-GluA23Y + d-AMPH (P-Env) vs. vehicle + d-AMPH, p<0.05; Tat-GluA23Y + d-AMPH (P-Env) vs. Tat-GluA23Yscr + d-AMPH (P-Env), p<0.05; Tat-GluA23Y + d-AMPH (P-Env) vs. Tat-GluA23Yscr + d-AMPH (NP-Env).   When the d-AMPH challenge was given in the initial environmental context, all d-AMPH treated groups that had received 8 pairings (4 in each of the 2 environments) displayed sensitized locomotor responses compared to control rats (given 4 saline injections in both environments) (Fig.3.3B. Treatment group: F(5,47)=3.58, p<0.05, post hoc: vehicle + saline vs. vehicle + d-AMPH, p<0.05; vehicle + saline vs. Tat-GluA23Y + d-AMPH peptide-paired, p<0.05; vehicle + saline vs. Tat-GluA23Y + d-AMPH vehicle-paired, p<0.05; vehicle + saline vs. Tat-GluA23Yscr + d-AMPH peptide-paired p<0.05; vehicle + saline vs. Tat-GluA23Yscr + d-AMPH vehicle-paired, p<0.05).  Importantly, when rats treated with Tat-GluA23Y + d-AMPH were given the challenge injection in the non-paired environment, the locomotor activity response was significantly greater than was observed following the d-AMPH challenge in the peptide-paired environment (Tat-GluA23Y + d-AMPH group: non-paired vs. peptide-paired, p<0.05).  3.5   Discussion These findings indicate that sensitization can occur simultaneously in two distinct contexts and although Tat-GluA23Y treatment did not block sensitization in the environment in which it is paired, it appeared to have blocked an inhibitory effect on the associative memory of the environmental cues paired with d-AMPH in the second context.  The striking increase in locomotion observed when rats were challenged in the context where d-AMPH was administered in the absence of Tat- GluA23Y supports the notion that occasion setting plays an   69 important role in the learned associations between drug reward and the contextual cues that predict the presence of drug reward.  Specifically, when regulated GluA2 endocytosis is blocked with Tat- GluA23Y, it would appear that the learned associations of occasion setting are interrupted in that particular context (GluA23Y -paired), leading to a stronger encoding of drug reward and environmental stimuli in the non-paired context.   The development of more effective treatments for drug addiction may benefit from a better understanding of the neurobiological mechanisms underlying craving and the remarkable ability of contextual cues to trigger drug craving.  Here we replicate the finding that although repeated injections of d-AMPH (1.0 mg/kg) cause a significant increase in locomotor activity from Day 1 to Day 9, intravenous infusions of Tat-GluA23Y prior to each of the initial injections of d-AMPH can attenuate the expression of sensitization following a drug-free period.  Animals treated initially with saline during the induction phase subsequently developed behavioral sensitization when given five injections of d-AMPH (0.5 mg/kg).  Importantly, animals treated initially with Tat-GluA23Y prior to d-AMPH injections did not develop behavioral sensitization when given a second sensitizing regimen with a lower dose of d-AMPH 2-weeks later.  These results indicate that disruption of regulated AMPAR endocytosis with the Tat-GluA23Y peptide can have both short- and long-term effects on amphetamine-induced behavioral sensitization.  Rats initially exposed to a sensitizing regimen of d-AMPH on alternate days developed sensitization in the original environment (Context A).  Expression of behavioral sensitization was confirmed in control groups with a challenge injection of 0.5 mg/kg d-AMPH 14 days later.  Subsequent injections of d-AMPH in the absence of Tat-GluA23Y in a distinctly different context (Context B) verified context-dependent sensitization as rats that previously   70 expressed sensitization in Context A failed to exhibit heightened locomotor activity to a d-AMPH injection.  When rats were subsequently exposed to a sensitizing regimen of d-AMPH in Context B in the absence of the Tat-GluA23Y interference peptide, sensitization developed in all groups.  This important finding indicates that the inability to develop sensitization after repeated challenge injections of d-AMPH in rats previously treated with Tat-GluA23Y in Context A, was due specifically to the blockade of conditioned associations responsible for contextual conditioning and not to a general impairment of sensitization per se.  Pairing of the environmental cues with stimulatory drug effects appear to involve a strong learning component, that in these studies are revealed to require regulated AMPAR endocytosis and the critical protein-protein interactions that, once impaired by the Tat-GluA23Y interference peptide, can eliminate any further development of sensitization.  This context-specific sensitization involves Pavlovian associative learning, in which drug administration is the unconditional stimulus (US), and is paired with a distinct environmental context (CS), where contextual stimuli become associated with psychomotor drug effects (UR).  Environmental cues (CS) can also become cues for ?occasion-setting? and are then able to induce an excitatory conditioned response (CR) amplifying the sensitized response in the presence of conditioned cues in a context where drug is anticipated.   Contexts may reinstate drug-seeking indirectly by functioning as an occasion-setter (Holland, 1991).  According to this view, contexts function as retrieval cues, and in the dual-context sensitization paradigm used in the present study these cues may have been ambiguous, because certain cues such as those accompanying i.p. drug injections are paired with both environments.  The contextual cues are able to retrieve the non-drug or the drug experience during d-AMPH challenge.     71 The learned association between drug and the environmental context of administration following context-specific behavioral sensitization may be mediated by the activation of a small number of sparsely distributed neurons (Mattson et al., 2008).  Histochemical studies utilizing immediate-early gene markers to indicate neuronal activation have revealed that following repeated exposure to cocaine in a specific environmental context, only a subset of neuronal ensembles are selectively activated by cocaine in the drug-paired environment but not in a non-drug-paired environment (Mattson et al., 2007;Crombag et al., 2002).  This explains our observations that the blocking effect of the Tat-GluA23Y interference peptide is sustained when animals are tested in the original conditioning environment, even when Tat-GluA23Y is no longer administered.  It is possible that the synaptic modifications and recruitment of AMPARs to strengthen the excitatory synapse on the subset of dopaminergic neurons responsible for sensitization are permanently inhibited, but when the drug administration environment changes to a new context, with novel contextual cues, previously non-activated neurons in surrounding ensembles may mediate the robust sensitized response we observed.  As discussed previously, in rodents and humans, psychomotor sensitization is quite consistently found after limited drug experience and develops quickly after the first few drug exposures and is often maintained after long periods of abstinence.  This sensitization is associated with enhanced rewarding and reinforcing effects of drugs.  Sensitized animals also show increased motivation for natural reinforcers (Fiorino and Phillips, 1999), suggesting that incentive sensitization represents a general increase in the activity of neural systems that regulate motivated behavior.  Incentive sensitization is hypothesized to be an important initial step in the addiction process (Robinson and Berridge, 1993) such that the first few occasions   72 of drug use can enhance the attractiveness of the drug and promote further use.  During these drug exposures, the association of environmental stimuli with drug effects are established and these drug-associated cues play an essential role in predicting and controlling future behavior and response to drugs (Everitt and Robbins, 2005).  The combination of the environmental context and drug effects increases the likelihood of further drug use, possibly escalating into greater drug intake and the eventual loss of control over drug intake as drug self-administration escalates into habitual behavior.  Once established, these habits persist during long periods of abstinence until triggered by drug-associated cues or contexts.  As the neural changes underlying sensitization are extremely long-lasting (Paulson et al., 1991) and persist after a drug-free period, it follows that a better understanding of the mechanisms underlying sensitization and the development of procedures leading to their reversal or inhibition would be promising approaches to improve the treatment of addiction.  The Tat-GluA23Y interference peptide utilized in the present thesis provides precisely such a novel target and is now undergoing formal assessment as a therapeutic compound in studies sponsored by both NIDA and BC Genome.   73 Chapter Four: General Discussion 4.1   Overview Drug addiction is a persistent and pervasive disorder for which we still lack quick and effective therapies.  As described in detail in the Introduction, this may be due, in part, to long-term changes in brain structure and or function, resulting in lasting alterations in both physiological and psychological responses to the drug or associated cues which in turn may trigger memories of a prior drug experience (Leyton, 2007;Robinson and Becker, 1986).  A progressive augmented behavioral hyperactivity to repeated administration of psychostimulants was first documented in the 1930s, (Downs and Eddy, 1932) whereas clinical effects of sensitization were reported in humans in the 1950s (Angrist and Sudilovsky, 1978;Connell, 1958).  Recent research has led to significant advancements in understanding the neurobiology of addiction by linking drug-evoked synaptic plasticity to the neural plasticity underlying processes of learning and memory (Hyman, 2005;Nestler, 2001;Brebner et al., 2006;Wang, 2008).   Behavioral sensitization has been an effective tool used to understand aspects of addiction, particularly the craving that occurs following repeated exposure to a drug (Kalivas et al., 1998;Self and Nestler, 1998).  However, based on past studies and results contained in the chapters of this thesis, it is prudent to recognize that studying behavioral sensitization requires a clear definition of three main factors.  The first of which is the time point at which sensitization is being investigated: induction, maintenance or expression.  The second is the involvement of distinct brain areas in which specific changes are occurring during each time period of sensitization.  The third aspect essential to using behavioral sensitization as a model is the recognition of the pivotal role that contextual cues play in the entire process.     74 In order for sensitization to be successfully induced, the dosage of drug and number of repeated exposures are important points of consideration (Bjijou et al., 2002;Cador et al., 1995).  Interrupting the induction of both behavioral and neurochemical sensitization is possible when the interference peptide, Tat-GluA23Y was co-administered with d-AMPH in the series of studies described in Chapter 2.  This demonstrates that blocking endocytosis of GluA2-containing AMPARs impedes the neuroplasticity contributing to the induction phase, typically observed as a gradual increasing behavioral response during repeated intermittent drug exposure.  The maintenance period that follows is highlighted in the series of studies contained in both Chapters 2 and 3 of this thesis.  Important modifications are occurring during this phase, determining whether the changes that happened during induction are preserved or sustained for an extended period of time.  Whether induction manifests into the expression of sensitization at a later time depends on processes and length of time given to the maintenance phase before a challenge drug is given to test for expression.  Expression to re-exposure typically requires a longer period of incubation.  If given after 1-3 days, a similar level of responsiveness or even tolerance may be expected (Kuczenski and Segal, 1997;Robinson and Becker, 1982).  However, following 7 days, or in the current studies, an incubation period of 14 days, a challenge injection of a lower dose of the drug produces a robust expression of sensitization.  Previous studies show evidence for sensitization even after 28 days of incubation (Paulson and Robinson, 1995).  It is important to uncover the mechanisms that are occurring during this period of incubation and to understand the specific factors that are changing.  Results from studies included in this thesis suggest that endocytosis of AMPARs, specifically, the removal of GluA2-containing and possibly the insertion of GluA2-lacking AMPARs contribute greatly to this drug-induced neuroplasticity.     75 The aims of this thesis were threefold.  The first aim was to extended previous studies by the Phillips and Wang groups focused on the role of LTD in the induction and maintenance of  d-AMPH-induced behavioral sensitization in rats.  The interference peptide, Tat-GluA23Y, was used as a pharmacological tool to block regulated GluA2 endocytosis critical for induction of LTD.  As the reader will recall, this short-sequence interference peptide contains three tyrosine residues identical to those that must undergo phosphorylation to ensure GluA2 CT-dependent AMPA receptor endocytosis and the expression of LTD (Ahmadian et al., 2004;Wang and Linden, 2000).   Tat-GluA23Y has the unique ability to interfere with regulated AMPA receptor endocytosis leading to a disruption in the relative expression of GluA2-containing (Ca2+-impermeable) and GluA2-lacking (Ca2+-permeable) AMPARs (Sun et al., 2005;Van den Oever et al., 2012).  This leads to the impairment of LTD.  The second aim focused on drug-induced neuroadaptations in different regions of the mesolimbic circuitry, specifically, the involvement of the VTA as distinct from the NAc during different stages of behavioral sensitization.  The third and final aim of the thesis addressed the possible role of LTD in mediating the learned association between the rewarding effects of d-AMPH and the contextual stimuli experience at the time of drug treatment, which exert influence on the persistence of behavioral sensitization.  We will attempt to integrate these findings into a coherent explanation of the role of AMPAR endocytosis and LTD in the induction and maintenance of behavioural sensitization as a prelude to sharing ideas about the potential for the findings from this thesis to advance therapeutic developments in the treatment of addiction.    76 4.2   Behavioral and neurochemical correlates of amphetamine sensitization and disruption with the interference peptide, Tat- GluA23Y The second chapter of this thesis describes a series of experiments in which one of the objectives was to correlate behavior with DA efflux in the NAc in response to repeated d-AMPH and then to examine the effect of the Tat-GluA23Y interference peptide at different sensitization stages.  Augmentation of motor stimulant effects following repeated intermittent administration of d-AMPH, cocaine or morphine are well documented, as is the accompanying increase in extracellular DA, as measured by in-vivo microdialysis (Kalivas and Duffy, 1990;Robinson et al., 1988;Akimoto et al., 1989).  As discussed in the results section of chapter 2, this specific microdialysis experiment confirmed a significant increase in DA efflux in the NAc following a single acute dose of d-AMPH that was also evident following the final repeated drug exposure.  Following the challenge injection of d-AMPH 14 days later, the drug-treated groups exhibited significantly higher levels of evoked-DA efflux than the d-AMPH na?ve group, thereby confirming previous reports of neurochemical sensitization (Robinson and Berridge, 1993;Robinson and Becker, 1982).   Of particular interest was our observation of a unique role for regulated endocytosis of GluA2?containing AMPARs in modulating the efflux of DA within the NAc after repeated but not acute exposure to d-AMPH.  Co-administration of Tat-GluA23Y with d-AMPH during the induction period did not affect levels of d-AMPH-induced DA efflux on Day 1.  However, repeated treatment with Tat-GluA23Y significantly attenuated the magnitude of d-AMPH-induced DA efflux in the NAc by the 5th d-AMPH injection.  This can be explained by other results showing that a transient (1-5 days) synaptic potentiation of AMPAR currents in VTA DA neurons(Borgland et al., 2004) is related to a change from GluA2-containing to GluA2-  77 lacking AMPARs, induced by the initial exposure to a psychostimulant drug (Argilli et al., 2008;Brown et al., 2010).  The co-administration of Tat-GluA23Y in our study would block endocystosis of GluA2-containing AMPARs, thereby preventing the trafficking of GluA2-lacking AMPARs.  The resulting disruption of the transient potentiation of AMPA currents in the VTA may therefore account for the significant attenuation of DA efflux observed after the 5th injection of d-AMPH.   At the behavioral level, repeated d-AMPH treatment in rats led to a progressive increase in locomotor activity from the 1st to the 5th injection, as is well-documented in literature (Robinson et al., 1988;Kalivas and Stewart, 1991).  Repeated co-administration of Tat-GluA23Y blocked this progressive increase in behavioral activity, which unlike the neurochemical correlates, remained stable across repeated exposures to Tat-GluA23Y.  When a d-AMPH challenge was given in the absence of Tat-GluA23Y after a drug-free period, the profiles of behavioral and neurochemical responses were similar and animals showed neither locomotor nor neurochemical sensitization.  This introduces an important observation, namely that the progressive increase in behavioral activity leading to sensitization may not be immediately reflected in measurable neurochemical correlates.  However, during the test for expression of sensitization, behavioral and neurochemical responses seem to be a direct reflection of one another.  This suggests that further investigation is needed to more fully explore the mechanisms and neurocircuitry underlying the manifestation of behaviour and its neurochemical correlates.   Taken together, these findings implicate AMPAR endocystosis and potentially the development of LTD in the maintenance of a robust behavioral and neurochemical response to a sensitizing regimen of psychostimulant exposure. The behavioral pharmacological responses   78 to d-AMPH, whether in the form of a conditioned motor response associated with repeated drug exposure or as drug-induced behavioral sensitization studied here, are highly influenced by associative processes outlined in learning and addiction literature (Robinson and Berridge, 1993;Anagnostaras and Robinson, 1996), which in turn are encoded as enduring memories of drug-related environmental stimuli.  The formation of such contextual memories may be dependent on an LTD-like process, since disruption by the interference peptide Tat-GluA23Y results in the absence of both neurochemical and behavioral sensitization when rats are re-exposed to a challenge dose of d-AMPH.  The role of AMPAR endocystosis in the associative process and the formation of contextual learning will be further discussed in section 4.5 of this general discussion. 4.3   Distinctive neuroadaptations within the reward circuitry are responsible for induction, maintenance, and expression of behavioral sensitization Microinjection studies targeting the NAc and VTA revealed interesting differentiations between the neuroplasticity involved in the induction of sensitization and processes related to the maintenance and final expression of sensitization.  Microinjection of Tat-GluA23Y into either the VTA or the NAc had no effect on the gradual increase of locomotor activity in response to each repeated injection of d-AMPH.  This seems to suggest that the initial induction of sensitization is not affected by site-specific blockade of AMPAR endocytosis.  However, when animals were given a d-AMPH challenge injection following an extended drug-free period, effects of the intra-VTA microinjections of Tat-GluA23Y were clearly manifested and animals did not express behavioral sensitization.  In contrast, animals given intra-NAc microinjections of Tat-GluA23Y during the induction phase maintained and expressed behavioral sensitization when challenged with d-AMPH.  This pattern of   79 responding differs from that observed when Tat-GluA23Y was given intravenously.  In this series of experiments, systemic peptide treatment significantly blocked the induction of behavioral sensitization and a d-AMPH challenge injection after a period of incubation did not evoke the expression of sensitization.  Taken together, our results suggest that the AMPAR endocytosis-mediated effects on initial induction of d-AMPH sensitization may also occur at sites beyond the VTA, while the maintenance of sensitization directly involves processes dependent upon AMPAR endocytosis within the VTA.   This distinction between mechanisms responsible for the initial increase in motor activity following repeated injections of d-AMPH and neuroadaptations related to the encoding and augmentation of behavior during a drug-free period leading to the expression of sensitization compliments previous conclusions (Anagnostaras and Robinson, 1996).   As previously mentioned, our findings are consistent with the literature linking neural activity within the VTA to the initial neuroadaptations that establish sensitization, (Perugini and Vezina, 1994;Kalivas and Duffy, 1993;Vezina and Stewart, 1990;Wolf and Xue, 1999) as distinct from a related set of adaptations in the NAc that are engaged during the expression of behavioral sensitization (Robinson and Kolb, 1997;Kalivas and Duffy, 1993).   More importantly, our results highlight a critical differentiation between the increase in behavioral activity during induction of sensitization and the plasticity that underlies the long-term maintenance of sensitization once established.  We propose that the VTA is involved in both the induction and maintenance of sensitization, whereas mechanisms within the cortical region involving a major glutamatergic input to the NAc most likely contribute to the maintenance of sensitization.  The mPFC integrates information from numerous brain regions including those related to the contextual learning component of drug-associated cues   80 underlying behavioral sensitization (Gipson et al., 2013a;Gipson et al., 2013b) .  The following schematic drawing (Figure 4.1) represents a summary of the proposed circuitry involved in induction, maintenance and expression of behavioral sensitization.  Projections of VTA dopamine neurons innervate NAc medium spiny neurons (MSNs) and mPFC neurons directly, as well as other projections to amygdala and hippocampal neurons are not included in this schematic for simplicity.  GABAergic afferents (some direct, some indirect) from the NAc to the VTA provide feedback to VTA dopamine neurons.  Glutamatergic afferents project to the NAc from mPFC, amygdala, and hippocampus, integrating information encoding contextual learning of conditioned drug cues (Gipson et al., 2013a;Luscher and Malenka, 2011).     81      Figure 4.1 Schematic model of the circuitry involved in drug-induced neural plasticity underlying induction, maintenance and expression of behavioral sensitization.       82 4.4   AMPAR endocytosis and subunit composition is crucial in maintaining the neural plasticity underlying behavioral sensitization  Results from our current microdialysis studies indicate that repeated intravenous Tat-GluA23Y attenuated d-AMPH-induced DA efflux in the NAc during induction and eliminated the sensitized response to a drug challenge.  This suggests that regulated endocytosis of GluA2?containing AMPARs is involved in the maintenance of a robust neurochemical response to repeated d-AMPH exposure as measured in the NAc.  Medium spiny neurons (MSNs), the main cell type within the NAc, receive input from limbic and cortical areas and regulate behavioral output through projections to the motor region.  Recent findings by Wolf and colleagues have identified several distinct mechanisms that work in conjunction to mediate drug-induced adaptations in the NAc (Boudreau et al., 2007;Wolf et al., 2004).   First, there is strong evidence for increased excitatory synaptic strength at MSNs of the NAc receiving glutamatergic input related to increased surface expression of CP-AMPARs and increased AMPA/NMDA ratios (Wolf, 2010).  The interference peptide, Tat-GluA23Y, could inhibit the cycling of CP-AMPARs to the surface, thereby limiting excitatory synaptic strength and blocking sensitization.  After withdrawal from a sensitizing regimen of cocaine, the intrinsic membrane excitability of MSNs is decreased due to decreased Na+ and Ca2+ conductance and increased K+ conductance (Mu et al., 2010;Hu et al., 2004).  Finally, repeated cocaine exposure decreases extracellular non-synaptic glutamate levels in the NAc, including a decrease in the activity of the cystine-glutamate exchanger which functions to exchange extracellular cystine for intracellular glutamate (Baker et al., 2002;Madayag et al., 2007).  This in turn provides glutamate tone on extrasynaptic group II mGluRs that exert inhibitory control over glutamate neurotransmission (Baker et al., 2002;Moran et al., 2005).  As a result,   83 there is a disinhibition of synaptic glutamate release, enabling the expression of behavioral sensitization to a drug challenge (Wolf, 2010).   The shift in the overall expression of different AMPAR subtypes at excitatory synapses on dopaminergic neurons play a key role in modifying the drug-induced adaptive response.  The prominence of GluA2-containing AMPARs in a drug-na?ve state is replaced by the GluA2-lacking variant of AMPARs following exposure to a psychostimulant drug (Van den Oever et al., 2012;Luscher and Malenka, 2011).  GluA2-lacking AMPARs are Ca2+-permeable and have increased conductance, ultimately changing the synaptic properties at these sites and are essential factors in the sensitized response to a psychostimulant drug challenge.  The synaptic changes occur in a time-dependent manner and there is a transient reorganization of AMPA and NMDA receptors at NAc MSN synapses which may precede or work independently from structural changes that occur in the spine head of NAc MSNs (Gipson et al., 2013a;Luscher and Malenka, 2011;Russo et al., 2010;Wolf, 2010).  The following figures provide summaries of the proposed model of drug-induced synaptic and structural plasticity in excitatory synapses onto MSNs in the NAc (Figure 4.2), and onto DA neurons in the VTA (Figure 4.3).    84  Figure 4.2 Model of drug-induced synaptic and structural plasticity in excitatory synapses onto MSNs in the NAc.   85  Figure 4.3 Model of drug-induced synaptic plasticity in excitatory synapses onto VTA DA neurons.    86   In drug-na?ve animals, excitatory synapses in both VTA DA neurons and NAc MSNs express NMDARs and GluA2-containing AMPARs (Fig.4.2A and 4.3A).  Repeated d-AMPH exposure induces endocytosis of AMPARs on MSNs in the NAc, resulting in a higher proportion of NMDA receptors and long-term depression after a short withdrawal period (0-1 day following repeated drug exposure) (Fig.4.2B).  Following a longer incubation period (2-4 weeks or more), GluA2-lacking AMPARs appear, leading to synaptic potentiation.  This increased surface expression of GluA2-lacking AMPARs may also be accompanied by the development of mushroom-shaped spines (Fig.4.2C).  These changes quickly revert back to the pre-incubation state upon exposure to a drug challenge, restructuring the spine and resulting in a depression of synaptic strength (Fig.4.2D) (Luscher and Malenka, 2011;Russo et al., 2010;Wolf, 2010).  In excitatory synapses of VTA DA neurons, evidence have shown that one dose of cocaine can cause a proportion of GluA2-containing AMPARs to be exchanged for GluA2-lacking AMPARs through endo- and exocytosis (Ungless et al., 2001) (Fig.4.3B).  This results in the reduction of surface expression of GluA2-containing AMPARs while increasing the expression of CP-GluA2-lacking AMPARs and a transient enhancement of excitatory synaptic strength, though the total number of AMPARs may remain the same (Ungless et al., 2001;Saal et al., 2003) (Fig.4.3C).  Following repeated drug injections, the overall balance of AMPAR subtypes is restored as GluA2 is synthesized from internal stores of mRNA through mGluR1 activation and mTOR signaling (Luscher and Malenka, 2011;Bellone and Luscher, 2005;Mameli et al., 2007).   From the perspective of studies described in this thesis, systemic co-administration of Tat-GluA23Y with d-AMPH would block the endocytosis of GluA2-containing AMPARs,   87 normally induced by repeated drug exposure.  As a result of the actions of Tat-GluA23Y, the AMPA/NMDA ratio would remain stable thereby preventing the progression of change through the stages of drug-induced synaptic and structural plasticity related to sensitization.  Normally, during the drug-free incubation period, prior repeated drug treatment would have caused exocytosis of GluA2-lacking AMPARs resulting in synaptic potentiation and then a subsequent drug challenge would result in a heightened response and expression of behavioral sensitization.  The finding that Tat-GluA23Y peptide-treated animals in our studies do not express behavioral sensitization despite receiving repeated intermittent injections of d-AMPH, is consistent with the hypothesis that VTA synapses are maintained in the original drug-na?ve state, thereby preventing the proposed drug-related synaptic and structural plasticity.  4.5   Context-dependent behavioral sensitization and the pivotal role of AMPA receptor endocytosis  Synaptic plasticity underlying the development of sensitization has been proposed as a key factor in associative learning processes by which environmental cues paired with drug experiences gain the capacity to elicit a behavioral response even in the absence of a drug.  As discussed previously in the introduction, in both rodents and humans, psychostimulant sensitization is observed consistently after limited drug experience and is often maintained over long periods of drug-free status.  This sensitization is associated with enhanced rewarding and reinforcing effects of drugs.  Sensitized animals show increased incentive motivation for natural reinforcers, suggesting a general increase in the sensitivity of neural systems that regulate motivated behavior.  Incentive sensitization is an important initial step in the addiction process, while the first few occasions of drug use can enhance the attractiveness of drugs and promote further use.  During these drug exposures, the association of   88 environmental stimuli with drug effects is established and these drug-associated cues play an essential role in predicting and controlling future behavior and response to drugs (Everitt and Robbins, 2005).  Since the neural changes underlying sensitization are extremely long-lasting (Paulson et al., 1991) and are primarily observed after a drug-free period (Vanderschuren and Kalivas, 2000;Pierce and Kalivas, 1997), understanding the mechanisms underlying sensitization and if possible, the reversal or inhibition of such mechanisms would contribute greatly to the development of improved treatment approaches.  As previously demonstrated by different laboratories, sensitization often is very weak and may not occur at all if a drug is paired with contextual cues of the home environment (Badiani and Robinson, 2004;Crombag et al., 2008;Tirelli and Terry, 1998)  Salient novel cues serve essential roles in triggering behavioral responses to re-exposure in the novel environment and play an important role in mediating neuroadaptations.  As demonstrated in experiments described in Chapter 3, when the interference peptide blocked development of sensitization in the original context, a second sensitizing regimen of drug treatment failed to produce sensitization in Tat-GluA23Y peptide-treated animals.  However, if animals were given the second sensitizing treatment of d-AMPH in a different environment, sensitization readily develops.  This highlights the fact that the mechanisms blocked by the interference peptide are not involved simply in basic physiological processes, but instead appear to be essential for forming learned associations between  contextual cues and the unconditioned effects of the drug such as enhanced motor activation.produce sensitization. According to associative learning theory, the context in which a drug is given acquires associatively activated representations and thus establishes a situation referred to as ?occasion-setting? in   89 which stimulatory effects become expected (Badiani and Robinson, 2004;Robinson and Berridge, 2001).  A role for ?occasion setting? is supported by our study in which d-AMPH was paired concurrently on separate days with two distinct environments.  In the experimental group, the interference peptide was paired with one of the two environments , while control subjects received a scrambled version of the peptide.   In the control groups, a similar level of sensitizated locomotor activity was observed in each environment. In the experimental group, a greatly enhanced response to d-AMPH challenge was seen in context paired with the Tat-GluA23Y peptide-paired context, while activity in the non-paired context was comparable to that observed in control groups.  The concept of ?occasion setting? provides for the creation of an active association between specific contextual cues and activation of a specific pattern of behavior. Importantly, the theory also proposes the active inhibition of competing associations thereby assuring the dominance of one set of associations. We propose that this inhibitory process involves AMPAR-mediated endocytosis and subsequent induction of LTD within a network that represents the competing environmental context. By blocking with Tat-GluA23Y peptide, the inhibitory effect that Context A would normally exert on the Context B network is impeded and the locomotor effect in Context B develops in a unrestricted manner as revealed by a subsequent challenge dose of d-AMPH in Context B. The highly specific nature of this context-dependent effect is highlighted further by the finding that rats treated previously with Tat-GluA23Y peptide in a specific context still retain the ability to develop new contextual associations in a second novel environment.   90 4.6   Summary  These findings reveal a novel aspect of the conditions under which behavioral sensitization develops and highlight the contributions of AMPAR endocytosis in the formation of the learned associations underlying context-dependent behavioral sensitization. Pairing of the environment with stimulatory drug effects appear to involve a strong learning component, that in these studies are revealed to require regulated AMPAR endocytosis and the critical protein-protein interactions that, once impaired by the Tat-GluA23Y interference peptide, can eliminate any further development of sensitization.  Recent studies suggest that stimuli in the drug environment activate specific neuronal ensembles within striatal neurons and that repeated activation of these same ensembles result in context-specific sensitization (Hope et al., 2006;Koya et al., 2009;Mattson et al., 2007;Mattson et al., 2008).   A new technique termed ?Daun02 inactivation method?, permits the selective inactivation of only those neurons activated by cocaine in an environment repeatedly paired with drug injections was made possible (Koya et al., 2009).   This study found that only a small subset (2-3%) of  NAc neurons are selectively activated by cocaine in a specific environment and are responsible for the expression of context-specific sensitization (Koya et al., 2009).   In relation to the current thesis, it is highly possible that the interference peptide blocks the activation of a subset of neuronal ensembles hence blocking the induction and maintenance of sensitization.  Even when a 2nd series of drug is administered without the interference peptide, sensitization does not develop possibly because d-AMPH is triggering the same neuronal ensembles that have been previously activated and modified by the interference peptide.  However, when animals are exposed to the 2nd round of sensitization in   91 a different novel context, a new set of neuronal ensembles would be activated and therefore sensitization can once again be induced.    4.7   Translational implications  To date, there are few pharmacological therapies that are approved to treat psychostimulant addiction.  Many drugs have been tested, but none have shown conclusive efficacy with tolerable side effects in humans (Kampman et al., 2005).   These potential treatment drugs have included DA-receptor ligands such as DA receptor agonists, partial agonists, and antagonists, as well as DA-reuptake inhibitors (Amato et al., 2011;Pani et al., 2011).   There has also been particular interest in the antipsychotic medication, aripiprazole, a partial D2-like receptor agonist, which is currently approved for the treatment of schizophrenia, depression, and bipolar disorder and potentially proposed to reduce cocaine craving and use (Meini et al., 2011a;Meini et al., 2011b).   Another targeted area for drug development is dysregulation of glutamatergic signaling found in addiction and this led to tests of N-acetylcysteine, a derivative of the amino acid cysteine, with the ability to normalize extracellular levels of glutamate following cocaine administration (Baker et al., 2003;Kalivas et al., 2003).  Clinical trials report that N-acetylcystine treatment attenuates cocaine craving and use and normalizes glutamate levels in the brain (Larowe et al., 2013;Olive et al., 2012;Mardikian et al., 2007).  Some recent studies have found that the glutamate release-inhibiting properties of the antidepressant buproprion and the anticonvulsant, topiramate, are effective in reducing methamphetamine use and craving (Elkashef et al., 2012;Elkashef et al., 2008).  Several other compounds have been investigated, with little success, such as ?-adrenergic antagonists, opiod-receptor antagonists, 5-HT3-receptor antagonists, antidepressants, and anticonvulsants (Kreek et al., 2004;Kreek et   92 al., 2002;Karila et al., 2008;Vocci and Ling, 2005;van den Brink and van Ree, 2003).  Before the interference peptide Tat-GluA23Y can become a target of drug discovery, an extensive amount of further research will be required, including toxicology and side effects.   As mentioned in the introduction of this thesis, Tat-GluA23Y has only been applied to a handful of studies examining animal models of addiction (Brebner et al., 2005;Dias et al., 2012;Van den Oever et al., 2008).  However, Tat-GluA23Y has been used in several studies to interfere with AMPAR trafficking in order to probe the roles of LTD in other learning and memory-related behaviours.  When applied to spatial learning performance on a Morris water maze, the interference peptide blocks the expression of hippocampal LTD and prevents the acute stress-impairment of memory retrieval (Wong et al., 2007).  In a Pavlovian fear conditioning model, blocking LTD with Tat-GluA23Y during an initial extinction training session interrupted both the expression and recall of extinction learning, while having no effect if given during the conditioning training, indicating that interfering with AMPAR endocytosis impairs the selective modification of a learned association without impacting the ability to form new memories (Dalton et al., 2008).  These various applications of the Tat-GluA23Y interference peptide in learning and memory models have important implications for its possible use in the treatment of drug addiction.  Obviously, a compound that has broad effects on memory processes would have limited application.  Fortunately, the available data, including that reported in our original report (Brebner et al., 2005) indicate selective effects that do not affect operant behavior for natural food rewards.  We are also encouraged by the current results, that blocking AMPAR endocytosis and the resulting formation of LTD leads to the very selective blockade of contextual control of sensitization ONLY in the environment in which Tat-GluA23Y interference peptide was co-administered with d-AMPH.     93 Obviously, introduction of a pharmacological agent such as Tat-GluA23Y to interfere with neuroadaptations in the brain, whether aberrant, as in the case of drug addiction or stress-induced impairment of memory retrieval, or functional, such as behavioral flexibility in facilitating extinction of fear conditioning, must be approached with caution.  Nevertheless, the urgent need for effective pharmacotherapies is underscored by the occurrence of sensitization in other therapeutic treatments including the use of stimulants for the treatment of attention deficit hyperactivity disorder (ADHD); opiates for the treatment of pain; and antidepressants for the treatment of mood disorders, to name a few (Kupfer et al., 1992;Baldessarini, 1995).  Broadening our understanding of the processes involved in the development of sensitization and identifying specific mechanisms that can be modulated in order to inhibit sensitization, may give rise to new drug development strategies such as the use of interference peptides advocated here.     94 Reference List  Aguilar MA, Rodriguez-Arias M, Minarro J (2009) Neurobiological mechanisms of the reinstatement of drug-conditioned place preference. Brain Res Rev 59:253-277. 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