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Age-related differences in cocaine place conditioning and cocaine-induced dopamine

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Age-related differences in cocaine place conditioning and cocaine-induced dopamine
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Badanich, Kimberly A
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University of South Florida
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Ontogeny
Adolescent rat
Nucleus accumbens
Addiction
Quantitiative in vivo microdialysis
Dissertations, Academic -- Psychology -- Masters -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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ABSTRACT: In humans, adolescent exposure to illicit drugs predicts the onset of adult drug abuse and suggests that early drug use potentiates adolescent vulnerability to drug addiction. In experiment 1, it was hypothesized that adolescent rats would show a CPP for a low cocaine dose if in fact adolescents are more vulnerable to cocaine's rewarding effects. Place preferences were measured in early adolescent postnatal day (PND) 35, late adolescent (PND 45) and young adult (PND 60) rats by injecting either 0, 5 or 20 mg/kg cocaine and conditioning them to environmental cues in a 2-chamber place conditioning apparatus. Significant cocaine preferences were found for all ages at the high dose. Interestingly, PND 35's were the only age group to have a CPP at the low dose suggesting that PND 35 rats are more sensitive than late adolescent and young adult rats to cocaine's rewarding effects.In Experiment 2, it was hypothesized that age-related differences in cocaine CPP may be mediated by differences in the mesolimbic dopaminergic (DA) system throughout development. Extracellular DA levels in the nucleus accumbens septi (NAcc) of early adolescent, late adolescent and adult rats were measured via quantitative microdialysis. PND 35, PND 45 and PND 60 rats were injected daily with either 5 mg/kg/ip or saline for 4 days, surgically implanted with a microdialysis probe aimed at the NAcc. Rats were perfused with either 0, 1, 10 or 40 nM DA and the extracellular DA concentration was measured. Our results show that adolescents differ from adults in basal DA with PND 35 rats having low basal DA (0.4 nM), PND 45 rats having high basal DA (1.8 nM) and PND 60 rats having intermediate basal DA (1.3 nM). PND 45 cocaine treated rats showed a 58% decrease in basal DA. All cocaine treated rats, regardless of age, showed a significant increase in DA over baseline in response to a cocaine challenge.Additionally, there were age-related differences in the extraction fraction (Ed), an indirect measure of DA reuptake, with PND 45 and PND 60's showing a decrease in basal Ed, an effect absent in PND 35's. Together these findings suggest that there are substantial ontogenetic differences in extracellular DA and DA reuptake and that these differences may provide an explanation for adolescent vulnerability to addiction. Future research should investigate DA supply and degradation processes in naïve and cocaine treated adolescent rats and vulnerability to addiction.
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Thesis (M.A.)--University of South Florida, 2005.
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Includes bibliographical references.
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by Kimberly A. Badanich.
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Age-Related Differences in Cocaine Place Conditioning and Cocaine-Induced Dopamine By Kimberly A. Badanich A thesis submitted in partial fulfillment of the requirements for the degree of Master of Arts Department of Psychology College of Arts and Sciences University of South Florida Major Professor: Cheryl L. Kirstein, Ph.D. Member: Toru Shimizu, Ph.D. Member: James Willott, PhD. Member: Mark Goldman, Ph.D. Date of Approval: November 7, 2005 Keywords: ontogeny, adolescent rat, nucleus accumbens, addiction, quantitiative in vivo microdialysis Copyright 2005, Kimberly A. Badanich

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i Table of Contents List of Figures iii Abstract iv Chapter One: Introduction 1 Theories of Drug Addiction 2 Drugs of Abuse and the Mesolimbic Dopa mine system 4 Expectancy and Drugs of Abuse 5 Cocaine and the Mesolimbic Dopamine System 6 Drugs of Abuse and Adolescence 7 Chapter Two: Early Adolescents are More Sensitive to the Rewarding 9 Effects of Cocaine than Late Adol escent and Young Adult Rats Abstract 9 Introduction 10 Method and Materials 12 Subjects 12 Apparatus 13 Procedure 13 Design and Analyses 14 Results 15 Discussion 15 Chapter Three: Basal and Cocaine-Induced Ex tracellular Dopamine 20 In the Nucleus Accumbens Septi During Adolescence and Young Adulthood Abstract 20 Introduction 22 Method and Materials 26 Subjects 26 Pre-exposure and Surgical Procedures 26 Quantitative Microdialysis Procedures & Neurochemical Analyses 27 Design and Analyses 28 Results 29 Basal Dopamine 29 Cocaine-Induced Dopamine 29 Extraction Fraction 30 Comparison of Conventional and Quantita tive Microdialysis 31 Discussion 32 Age-Related Differences in Basal Dopamine 32 Ontogenetic Differences in Cocaine-Induc ed Dopamine 33 Extraction Fraction and Dopamine Reuptake 35 Conventional vs. Quantitative Microdialysis 35

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ii Chapter Four: Concluding Remarks and Implications 37 References 39 Appendices 48 Appendix A: Cocaine Place Conditioning-Figure 49 Appendix B: Normal Dopamine Concentra tion Gradient-Figure 50 Appendix C: Gradual Dopamine Concentra tion Gradient-Figure 51 Appendix D: Steep Dopamine Concentra tion Gradient-Figure 52 Appendix E: Basal Dopamine Levels-Figure 53 Appendix F: Dopamine and Repeated Cocai ne Pretreatment 54 for PND 35-Figure Appendix G: Dopamine and Repeated Co caine Pretreatment 55 for PND 45-Figure Appendix H: Dopamine and Repeated Cocai ne Pretreatment 56 for PND 60-Figure Appendix I: Percent Change in Dopami ne for PND 35-Figure 57 Appendix J: Percent Change in Dopami ne for PND 45-Figure 58 Appendix K: Percent Change in Dopami ne for PND 60-Figure 59 Appendix L: Age-Related Differences in Cocaine-Induced 60 Dopamine-Figure Appendix M: Basal Extraction Fraction-Figure 61 Appendix N: Cocaine-Induced Extracti on Fraction-Figure 62 Appendix O: Extraction Fraction and Dopa mine for PND 35-Figure 63 Appendix P: Extraction Fraction and Dopa mine for PND 45-Figure 64 Appendix R: Extraction Fraction and Dopa mine for PND 60-Figure 65 Appendix S: Conventional and Quantitati ve Microdialysis 66 for PND 35-Figure Appendix Q: Conventional and Quantitati ve Microdialysis 67 for PND 45-Figure Appendix T: Conventional and Quantitati ve Microdialysis 68 for PND 60-Figure

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iii List of Figures Figure 1. Cocaine Place Conditioning 46 Figure 2. Normal Dopamine Concentration Gradient 47 Figure 3. Gradual Dopamine Concentration Gradient 48 Figure 4. Steep Dopamine Concentration Gradient 49 Figure 5. Basal Dopamine Levels 50 Figure 6. Dopamine and Repeated Cocaine Pretreatment for PND 35 51 Figure 7. Dopamine and Repeated Cocaine Pretreatment for PND 45 52 Figure 8. Dopamine and Repeated Cocaine Pretreatment for PND 60 53 Figure 9. Percent Change in Dopamine for PND 35 54 Figure 10. Percent Change in Dopamine for PND 45 55 Figure 11. Percent Change in Dopamine for PND 60 56 Figure 12. Age-Related Differences in Co caine-Induced Dopamine 57 Figure 13. Basal Extraction Fraction 58 Figure 14. Cocaine-Induced Extracti on Fraction 59 Figure 15. Extraction Fraction and Dopamine for PND 35 60 Figure 16. Extraction Fraction and Dopamine for PND 45 61 Figure 17. Extraction Fraction and Dopamine for PND 60 62 Figure 18. Conventional and Quantitative Mi crodialysis for PND 35 63 Figure 19. Conventional and Quantitative Micr odialysis for PND 45 64 Figure 20. Conventional and Quantitative Mi crodialysis for PND 60 65

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iv Age-Related Differences in Cocaine Place Conditioning and Cocaine-Induced Dopamine Kimberly A. Badanich ABSTRACT In humans, adolescent exposure to illicit drugs predicts the onset of adult drug abuse and suggests that early drug use poten tiates adolescent vul nerability to drug addiction. In experiment 1, it was hypothesized that adolescent rats would show a CPP for a low cocaine dose if in fact adolescents are more vulnerable to cocaine’s rewarding effects. Place preferences were measured in early adolescent [postnatal day (PND) 35], late adolescent (PND 45) a nd young adult (PND 60) rats by in jecting either 0, 5 or 20 mg/kg cocaine and conditioning them to e nvironmental cues in a 2-chamber place conditioning apparatus. Signi ficant cocaine preferences were found for all ages at the high dose. Interestingly, PND 35’s were th e only age group to have a CPP at the low dose suggesting that PND 35 rats are more sensitive than late adolescent and young adult rats to cocaine’s rewarding effects. In E xperiment 2, it was hypothesi zed that age-related differences in cocaine CPP may be medi ated by differences in the mesolimbic dopaminergic (DA) system throughout devel opment. Extracellular DA levels in the nucleus accumbens septi (NAcc) of early adol escent, late adolescent and adult rats were measured via quantitative microdialysis PND 35, PND 45 and PND 60 rats were injected daily with either 5 mg/kg/ip or sa line for 4 days, surgically implanted with a microdialysis probe aimed at the NAcc. Rats were perfused with either 0, 1, 10 or 40 nM DA and the extracellular DA concentration was measured. Our results show that adolescents differ from adults in basal DA with PND 35 rats having low basal DA (0.4

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v nM), PND 45 rats having high basal DA (1.8 nM) and PND 60 rats having intermediate basal DA (1.3 nM). PND 45 cocaine treated ra ts showed a 58% decrease in basal DA. All cocaine treated rats, regardless of age, showed a significant increase in DA over baseline in response to a cocaine challe nge. Additionally, there were age-related differences in the extraction fraction (Ed), an indirect measure of DA reuptake, with PND 45 and PND 60’s showing a decrease in basa l Ed, an effect absent in PND 35’s. Together these findings suggest that there are substantial ontoge netic differences in extracellular DA and DA reuptake and that th ese differences may pr ovide an explanation for adolescent vulnerability to addiction. Future research should investigate DA supply and degradation processes in nave and cocai ne treated adolescent rats and vulnerability to addiction.

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1 Chapter One Introduction For decades, scientists have searched for a biological mediator(s) of drug dependency. A substantial amount of this re search has focused on one particular pathway in the brain, the mesolimbic DA system. This system originates in the ventral tegmental area (VTA) and projects to several brain structures including the NAcc, amygdala, hippocampus, septum, olfactory bulb, bed nuc leus of the stria terminalis and the prefrontal cortex (Dahlstrom and Fuxe, 1964). Although substance abuse research has focused on the mesolimbic system, a consider able amount of eviden ce indicates a much broader range of stimuli affects this pathwa y. In general, the mesolimbic system is activated by motivationally significant stimuli (Blackburn et al., 1992). Any motivationally significant stimulus, whether it is positive/hedonic or negative/anhedonic, are capable of activating the mesolimbic system (Blackburn et al., 1992). Specifically, increases in DA in the NAcc of a rodent, as measured by in vivo microdialysis, have been reported in response to many motivationally si gnificant stimuli such as food (Martel and Fantino, 1996), sexual activity (M eisel et al., 1993), novelty (R ebec et al., 1997), aversive stimuli such as shock (Morrow et al., 1995) as well as drugs of abuse (Moghaddam and Bunney, 1989; Koob and Weiss, 1992). Thus, drugs of abuse are only one type of stimuli that can activate the mesolimbic system. Mo re research should be done to clarify behavioral responses regulated by the meso limbic system. Once these have been clarified, the involvement of the mesolimbic system in substance abuse and dependency may more likely be understood.

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2 An important issue in studies involving the mesolimbic system research has been the functional and structural heterogeneity of the NAcc. The NAcc is anatomically divided into two distinct compartments: core and shell. The core has projections to the dorsolateral ventral pallidal s ubterritory, an area similar in anatomy to the striatopallidal subregion of the caudate putamen while the shell has projections to the ventromedial portion of the ventral pallidum (Zahm and Heimer, 1990). Increases in extracellular DA levels in the shell of the NA cc have been found in response to motivationally significant stimuli such as drugs of abuse (Pontieri et al., 1995), food (Tanda and Di Chiara, 1998), aversive stimuli (Kalivas and Duffy, 1995) and novel stimuli (Rebec et al., 1997). Motivation in these studies have been define d by increases in the number of lever presses for a reinforcing stimulus, increases in the amount of time spent in the environment paired with a reinforcing stim ulus ( i.e., CPP), and increases in the amount of stimulus intake (i.e., greater food intake, greater wate r intake, greater drug se lf-administration). Together, these findings indica te that the mesolimbic system and specifically the NAcc shell are activated by motivationally significant stimuli. Theories of drug addiction. In the quest to identify the mediator(s) of addiction, several theories have been proposed. One of the earliest theories of addiction focused on the rewarding effects of drugs (Crow, 1970; Rolls et al., 1974; Fibiger and Phillips, 1974). According to the reward theory of addiction, enhanced DA activity in the mesolimbic system is pleasurable and rewardi ng. The majority of research during this era found rats will voluntarily electrically stimulate the mesolimbic system and will further change their response rates for intr acrainial self-stimulation (ICSS) with the administration of dopaminergic agonists/antagonists suggestin g that DA is important in

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3 reward. However, the reward theory of a ddiction has weaknesses in that it only provides an explanation for the early stages of dr ug use and poorly addre sses the occurrence of chronic drug use. With certain drugs, like cocaine, initial administrations are the most pleasurable while later uses have a diminish ed rewarding effect. Additionally, addicts repeatedly take drugs and use higher drug doses to achieve the same subjective state as in the first use. Therefore reward could not be the sole factor driv ing drug addiction. An alternative theory, the anhe donia hypothesis (Salamone et al, 1997) provides a better explanation of chronic drug use. This theory suggests that addicts chronically use drugs to avoid the negative affect associated w ith drug use. Although drug use enhances dopaminergic activity of the mesolimbic syst em, over time drug use induces changes in synaptic transmission causing either sensiti zation (cocaine, amphetamine) or habituation (alcohol) of the system dependi ng on the type of drug administ ered. It is suggested by the anhedonia hypothesis that the addict uses drugs to avoid these physiological and psychological effects, providing a better explan ation for chronic drug use than the reward hypothesis. A limitation of the anhedonia hypothesis is that it poorly explains the early stages of drug use. The incompleteness of these two theories led to the development of current theories of drug use. Robinson and Berridge (1993) proposed the incentive salience theory of addiction. Incentive salience is describe d as “a psychological process that transforms the perception of stimuli, imbuing them with salience, making them attractive, 'wanted', incentive stimuli.” Im portantly, Robinson and Berridge stated “the mesolimbic system’s function is to attribute 'incentive salience' to the perception and mental representation of events associated wi th activation of the system.” Moreover, the function of the mesolimbic system is not si mply reward or aver sion, but also includes

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4 perception of salient stimuli. The incentive sa lience theory can be applied to many of the motivationally arousing stimuli mentioned a bove (food, sex, novelty, aversive stimuli and drugs of abuse). Di Chiara (1999) proposed that drug addiction is manifested by a generalized facilitation of associ ative learning. In this theor y, behavioral c ontrol is lost and drug-related cues acquire a motivationally relevant valence th at reliably induces drug-seeking behavior. Both the incentive sa lience and associative learning theories of drug addiction imply that the environment and dr ug-related cues play an integral role in repeated drug use and drug-seeking behavior s. Possible mechanisms underlying chronic drug use and the negative effects of withdrawal are discussed in the allostasis theory of addiction (Koob and Le Moal, 2001). The allostas is model of addiction states that during chronic drug use, homeostatic processes are dysregulated and fail to return to a normal range. This allostatic state drives subse quent drug use and in turn causes a downward spiral of dysregulation. In response to the pr oposal of these newer th eories of addiction, much research has focused on the ability of salient stimuli to induce dopaminergic activity in the mesolimbic system and specifically the NAcc. Drugs of abuse and the meso limbic dopamine system. Drugs of abuse have several similarities in common with the salient stimuli discussed above. Rats exhibit place conditioning in response to ethanol (Ris inger et al., 2001) heroin (Hand et al., 1989), morphine (Higgins et al., 1992) amphetamine (Lett, 1989; Meyer et al., 2002), methylphenidate (Meririnne et al., 2001) and cocaine (Shippenbe rg and Heidbreder, 1995; Horan et al., 2000). Rats will also self-administer drugs of abuse such as heroin (Higgins et al., 1994) cocaine (Pudiak and Bozarth, 2002) and ethanol (Tomkins et al., 2002). Drug use is mediated by the mesolimb ic system and specifically the NAcc.

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5 Lesioning DA neurons in the mesolimbic system with 6-hydroxydopamine (6-OHDA) attenuates drug-induced behavior. For exampl e, rats trained to self-administer nicotine were found to no longer lever press for the drug after infusion of 6-OHDA into the NAcc (Corrigall et al., 1992). Lesioning mesolimbi c DA neurons results in attenuation of heroin self-administra tion behavior (Gerrits and Van Ree, 1996). Drug-induced behavior can also be affected by administration of DA agonists/antagonists. Place Preference studies reported CPP after administration of SKF38393, a DA D1 agonist (White et al., 1991). However, administration of haloperi dol, a DA antagonist significantly decreased CPP scores relative to control rats (Adams et al., 2001). Drugs (amphetamine, alcohol, cocaine) also increase DA in the NAcc as measured by in vivo microdialysis (Moghaddam et al., 1989). Expectancy and drugs of abuse. Another topic of interest has been the effect of expectancies on drug use. Expectancy is defi ned as the anticipation or predictability of an event (Goldman, 2002; Schultz et al., 1997). Prediction of future events facilitates both humans and animals in their adaptation to the environment. Anticipation of dangerous future events can increase the surv ival rate of an animal by allowing it more preparatory time. In addition, humans can be nefit from expectancies. The knowledge of how and when an event takes place can greatly improve the choices a person makes in the event of adaptive situations. Expectancies ar e even evident in the CPP paradigm. As an animal is placed in a CPP apparatus, the animal typically spends more time in the chamber it ‘expects’ to induce a drug effect Even DA neurons in the VTA have been reported to predict cocaine administration. Rodents previously tr eated with repeated cocaine were shown to have increased DA neur on firing in the VTA just prior to cocaine

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6 administration (Carrelli and Ijames, 2000). This preceding neuronal firing suggests an expectancy or anticipatory effect. Our lab has shown an expectancy effect of DA in the NAcc in response to th e anticipation of both cocaine and alcohol. In this expectancy condition, rats were repeatedly administered a drug and during dialysis administered saline. DA levels post-saline injection s how an elevation and then a subsequent depression (Philpot and Kirstein, 1998; 1999). Cocaine and the mesolimbic dopamine system. Cocaine, like other drugs of abuse, acts on the mesolimbic system and sp ecifically the NAcc to produce its associated rewarding properties. Cocaine disrup ts normal DA transmission by altering DA degradation processes such as DA reuptake. In normal functioning DA reuptake, excess extracellular DA in the NAcc binds to the DA transporter (DAT) to be transported back into the presynaptic terminal (Hitri et al., 1994). However, cocaine binds to the DAT in place of DA causing the transporter to be ine ffective. Thus, excess extracellular DA is unable to bind to DAT’s, reuptake is inhibite d and excess DA remains in the extracellular fluid of the NAcc. Inhibition of DA reuptak e enhances basal accumbal DA and produces the rewarding effects associated with cocaine admi nistration. The rewarding properties of cocaine can be measured through place conditioning. Cocaine CPP is most effectively established fo r 20 mg/kg in 4 conditioning trials with the number of trials and dos es ranging from 2-6 and 5-30 mg/kg cocaine respectively (Tzschentke, 1998). A CPP effect can even be produced with one administration of cocaine; however most CPP experiments em ploy repeated cocaine administration to better mimic human patterns of abuse. Ra ts will self-administer cocaine, however microinjections of DA antagonists into the NA cc attenuates self-adm inistration behavior

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7 (Koob, 1992). Cocaine has also been shown to elevate basal accumbal DA as measured by in vivo microdialysis to approx imately 300% of baseline (Chefer and Shippenberg, 2002; Thompson et al., 2000; Camp et al., 1994; Maisonneuve and Kreek, 1994; Parsons and Justice, 1993; Kalivas and Duffy, 1993; Wei ss et al., 1992; Hurd et al., 1989). Doses of cocaine ranged from 2-20 mg/kg depending on the method of administration. Hence, the mesolimbic system is the primary pa thway involved in e xpressing cocaine’s rewarding properties. Drugs of abuse and adolescence. In humans, repeated e xposure to drugs during adolescence predicts the onset and severity of substance abus e. Cocaine use during early adolescence is associated with rapid escal ation from casual to daily substance abuse (Estroff et al., 1989). Adolescents who repeated ly use drugs also show greater substance consumption as an adult (Taioli and Wynde r, 1991; Chen and Millar, 1998). A higher rate of substance dependence is found in those initiating use during adolescence. For example, adolescents exhibit a rapid escalation from initiation to dependence (Anthony and Petronis, 1995; Clark et al. 1998) and mo re difficulty quitting (Khuder et al., 1999; Chen and Millar, 1998). Even when substance consumption is controlled for, adolescents show a higher prevalence of dependence than adults (Kandel and Chen, 2000) suggesting that the increased risk of adolescent substan ce abuse is not a consequence of greater total consumption for early users; but relates to accelerated progression of dependence. As a result of these findings, clini cal and experimental research has focused on identifying the behavioral and neurochemical mechanisms underlying adolescent vulnerability to addiction. Possibly the combination of adolescent neural developmental and drug-

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8 induced synaptic change may likely potenti ate the onset and seve rity of adult drug dependency.

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9 Chapter Two Experiment One Early Adolescents are More Sensitive to the Rewarding Effects of Cocaine than Late Adolescent and Young Adult Rats In humans, adolescent exposure to illicit drugs predicts the onset of adult drug abuse and suggests that repeated drug use pot entiates adolescent vulnerability to drug addiction. It was hypothesized that adolescent rats would s how a CPP for a low cocaine dose if in fact adolescents are more vulnera ble to cocaine’s rewarding effects. Place preferences were measured in early adolescent (PND) 35, late adolescent (PND 45) and young adult (PND 60) rats by injecting either 0, 5 or 20 mg/kg cocai ne and conditioning them to environmental cues in a 2-chambe r place conditioning apparatus. Significant cocaine preferences were found for all ages at the high dos e. Interestingly, PND 35’s were the only age group to have a CPP at the low dose. Together these findings suggest PND 35 rats are more sensitive than late adolescent and young adult rats to cocaine’s rewarding effects. Future research shoul d investigate ontogenetic differences in extracellular DA and how they relate to the development of addiction. Introduction In humans, adolescent exposure to illicit drugs predicts the onset of adult drug abuse and suggests that repeated drug use pot entiates adolescent vulnerability to drug addiction (Estroff et al., 1989; Anthony a nd Petronis, 1995; Clark et al. 1998). Adolescent vulnerability to drug addiction may be mediated by age-related differences in the behavioral response to drugs of abuse. To test this hypothesis, animal models of

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10 adolescent drug use have been developed to i nvestigate the impact of drugs on adolescent behavior and brain functioning. Specifically, the place cond itioning paradigm provides a measure of drug reward by assessing an animal ’s ability to associat e drug-induced effects with environmental cues. The amount of time spent in an environment is measured both before and after drug conditioning and a CPP is demonstrated if the rat spends a greater amount of time in the drug-paired envir onment. Given that there is increased experimentation with drugs and potentiate d vulnerability to drug addiction during adolescence (Khuder et al., 1999; Chen and Millar, 1998; Kandel and Chen, 2000), it has been hypothesized that adolescents would find drugs more rewarding than younger and older rats and that the expr ession of cocaine place conditi oning would be greatest in adolescents In adults, a place preference is most e ffectively established for a relatively high dose, 20 mg/kg cocaine, in 4 conditioning trials (Spyraki et al., 1982; Bardo et al., 1986; Calcagnetti and Schechter, 1993; Hem by et al., 1994; Durazzo et al., 1994; Kaddis et al., 1995). Whether adolescents express facilitated, inhi bited or similar cocaine CPP to adults is not as clear. Cocaine CPP has been shown in pre-adolescent (Laviola et al., 1992; Pruitt et al., 1995) and early ad olescent rodents (Laviola et al., 1992); however, adult comparisons were not included in these e xperiments. Additionally, the later study showed that early adolescents did not show a CPP at 5 mg/kg cocaine with one drugpairing. Interestingly, a CPP wa s expressed at this age af ter increasing the number of drug-pairings to 4, suggesting that adolescen t CPP models will produce variable results that are directly influenced by the type of experimental de sign employed. Others have found comparable CPP’s in adolescent and adults rats (Campbe ll et al, 2000) but

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11 neglected to include a baseline comparis on which is particularly important for developmental experiments given age-related differences in novelty -induced exploration (Stansfield and Kirstein, 2005). Although several studies have investigat ed place conditioning in adolescence, these findings are difficult to interpret due to inconsistency across experiments in the inclusion of an adult compar ison, choice of statistical desi gn, species (Schramm-Sapyta et al., 2004), age and drug dose. In view of the fact that adolescents are more vulnerable to developing drug addictions, it may not be that adolescents expre ss the greatest CPP at a particular drug dose, but that they are mo re physiologically sensit ive to the rewarding effects of cocaine. If in fact adolescents ar e more sensitive to co caine, then it would be expected that adolescents woul d demonstrate a CPP for a low cocaine dose (5 mg/kg) that which is typically not rewarding for adults. Therefore the adolescent CPP literature is in need of an experiment that incorporates adult comparisons, a baseline test, a late adolescent age group, and both low/high cocain e doses. The aim of the present study was to investigate cocaine CPP in early adoles cent (PND 35), late adolescent (PND 45) and young adults (PND 60). Our results suggest ther e are age-related differences in cocaine place conditioning with PND 35’s expressing a greater sensitiv ity to cocaine’s rewarding effects. Method and Materials Subjects. Ninety male Sprague-Dawley rats, o ffspring of breeding pairs (Harlan Laboratories, IN), were used in the presen t study. The day of birth was designated as PND 0 and litters were sexed and culled to 10 pups per litter on PND 1. Pups remained housed with their respective da ms in a temperature and humidity-controlled vivarium on

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12 a 12:12-hr light/dark cycle (lights on from 0700 h and 1900 h). On PND 21, pups were weaned and housed in groups of three. As in humans, the adolescent period for rodents begins with sexual maturation (Odell, 1990). Although the exact age range of adolescence is still contr oversial (Odell 1990; Spear, 2000; Tirelli et al., 2003), for the purposes of the present study adolescence was operationalized as PND 34-46. Rats were trained and tested at three separate ages: PND 30-35 (early adolescent), PND 40-45 (late adolescent) or PND 55-60 (young adults). To eliminate the potential confound of litter effects, no more than one pup per litter was used for any given condition and remaining pups were used for other ongoing lab experime nts. In all respects, maintenance and treatment of the rats were within the guidelines for animal care as approved by the University of South Florida’s Instituti onal Animal Care and Use Committee and the National Institutes of Health. Apparatus. The conditioning apparatus was a si ngle runway comprised of black Plexiglas (Rohm and Haas Company, Philadelphi a, Pennsylvania) that was divided into two equal sized sections: each (21 x 24.5 x 20.5 cm) with visual and tactile cues of either black and white horizontal striped (1 inch thick) walls with a gr ey sandpaper floor or black and white vertical striped walls (1 inch thick) with a wire-mesh floor. The chambers were separated by a removable Plex iglas door. A 2-chamber apparatus, rather than a 3-chamber, was used to eliminate age-related differences in novelty-induced exploration (Stansfield and Ki rstein, 2005) that may likely be induced by a less familiar central choice chamber which is typically inco rporated in the 3-chamber CPP paradigm. Procedure. The procedure consisted of four pha ses: handling (days one and two), baseline (day three), drug conditioning (days f our-seven) and a CPP test (day eight). On

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13 days one and two, rats were wheeled on a cart into the room where conditioning would take place and gently handled for 3 minutes. Handling occurred twice a day so that the rats would become used to experimenter handling (Maldonado and Kirstein, 2005). On day three, a biased design was used to dete rmine baseline chamber preferences. Naverats were placed in the center of the two CPP chambers in a dimly lit room and given free access to the entire apparatus for fifteen minut es. Time (sec) spent in each chamber was recorded. A camera was suspended above the CPP apparatus to record behavior. The camera signal was digitized and sent to a com puter (Dell OptiPlex GX110) for analysis. Once data were received, movement was anal yzed by distinguishing the tracked object (e.g., Sprague-Dawley rat) from the black background (Ethovision video tracking system, Noldus, Netherlands). The chamber in whic h each animal spent th e least amount of time was designated as the least pref erred (LP) chamber. Starti ng on the morning of day four, rats were injected with saline intraperitoneal ly and confined to the preferred (P) chamber for fifteen minutes. At least four hours afte r the morning injection, rats were injected with 5 or 20 mg/kg/ip cocaine and confined to the LP chamber for fifteen minutes. Control rats received saline injections in both chambers. Conditioning occurred twice a day over four consecutive days for a total of four LP and four P chamber exposures. The apparatus was cleaned with Quatricide (Pha rmacal Research Laboratories Incorporated) and ethanol prior to each trial to remove odors. On day eight, the conditioned effects of cocaine were tested. Rats were tested drugfree in the same manner as at baseline (day three). Design and analyses. Given that others have shown similar CPP responses between adolescents and adults (Campbell et al, 2000; Schramm-Sapyta et al., 2004), but

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14 human data has suggested adoles cent vulnerability to the re warding properties of drugs (Estroff et al., 1989; Taioli and Wynder, 1991; Chen and Millar, 1998; Anthony and Petronis, 1995; Clark et al., 1998), it was our aim to inves tigate whether there are agerelated differences in place conditioning for a low dose of cocaine. Planned comparisons (Bonferroni) were used to test for age a nd drug effects (Keppel, 1991). An adjusted alpha value, based on the number of compar isons, was used to control for familywise error. Comparisons were determined significan t at the 0.01 alpha level. Baseline and test measures were compared for each condition and the difference between the 2 measures provided a place conditioning score (sec in th e LP chamber at test – sec in the LP chamber at baseline) and was used as the de pendent measure. A CPP was defined as a drug group spending significantly more time in the least preferred chamber than agematched saline controls. Results Appendix A illustrates cocaine CPP varied with Dose and Age. All three ages demonstrated a CPP for 20 mg/kg cocaine [PND 35: t(3.91), p < 0.01; PND 45: t(3.14), p < 0.01; PND 60: t(4.06), p < 0.01]. These findi ngs are consistent with previous adult CPP findings in that adult rats generally ex press a CPP for 20 mg/kg cocaine (Spyraki et al., 1982; Bardo et al., 1986; Calcagnetti and Schechter, 1993; Hemby et al., 1994; Durazzo et al., 1994; Kaddis et al., 1995). Appendix A also illustrates that PND 35’s show a unique sensitivity to cocaine’s rewa rding effects. PND 35 was the only age to have a CPP for 5 mg/kg cocaine [t(3. 94), p < 0.01]. Additionally, PND 35’s demonstrated greater CPP than PND 45 [t( 2.69), p < .01] and PND 60 [t(2.98), p < .01].

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15 There were no differences between PND 45 and PND 60 for the 5 mg/kg condition [t(0.11), p > .01]. These results show that PND 35’s demonstrate a CPP for a low dose of cocaine and that early adolescents are more se nsitive than late adolescent and adult rats to the rewarding propert ies of cocaine. Discussion These results are the first to show a significant age difference between adolescent and adult rats for a low dose of cocaine us ing the CPP paradigm. There are several physiological and behavioral mechanisms that may induce greater cocaine sensitivity for PND 35 rats. For example, cocaine produces its rewarding effects by blocking the DAT in the NAcc and consequently increasing extrace llular levels of DA (C ooper et al., 2003). It is known that adolescents and adults have similar DAT densities in the NAcc (Tarazi et al., 1998b); however it is not known if the f unction of the DAT differs between age groups. It may be that DA reuptake rates differ between PND 35, PND 45 and PND 60 resulting in variable extr acellular DA levels as a func tion of age after cocaine administration. Another possibility is th at nave PND 35’s differ in their basal extracellular DA levels. If young adolescent rats have a differe nt tonic level of DA in the NAcc, these rats may be hyperesponsive to rewa rding stimuli. It is known that changes in extracellular DA in the NAcc are associated with drug rewa rd (Pontieri et al, 1995). Together, these results suggest that the greater cocaine sensitivity of PND 35 rats may be mediated by ontogenetic differen ces in extracellular DA. Age-related differences in cocaine CPP may reflect divergent stimulus associations causing the rewarding properties of cocaine to be expre ssed differently as a function of age. For example, it has been s uggested that drug addi ction is manifested by

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16 a generalized facilitation of associativ e learning (Di Chiara, 1999). PND 35’s may demonstrate facilitated associative learning a nd as a consequence, easily relate cocaine’s rewarding effects to drug-rela ted cues present in the CPP apparatus. Additionally, the salience of drug-related cues ma y be expressed differently as a function of age. Robinson and Berridge (1993) discussed the role of the mesolimbic system and specifically the NAcc in the salience of drug-re lated cues. Possibly the tran sitioning of the mesolimbic system from preadolescence to adulthood resu lts in aberrations in the salience of contextual cues thus mediating PND 35’s enha nced vulnerability to the rewarding effects of cocaine. Another possibility is that PND 35’s develop stronger co caine expectancies than older rats. CPP paradigms measure the rewardin g value associated with a drug as well as drug expectancy. After repeated pairings of cocaine with specific contextual cues, rats that spend more time in the drug-paired chamber can be viewed as anticipating or expecting cocaine administration. Essentially these rats are thought to be seeking out the environment associated with the administrati on of cocaine. Therefore, the results of the present experiment should be extended to s uggest that PND 35’s have greater cocaine expectancies and can easily associate contextual cues with cocaine’s rewarding effects. It would be interesting to determine if the pres ent results extend to natural reinforcers such as food, water, sex and even aversive stim uli such as shock or predatory odors. There are some caveats that should be a ddressed. Other adolescent behavioral studies have demonstrated hyposensitivity (A driani and Laviola, 2003; Laviola et al, 1995; Spear and Brick, 1979) to psychostimul ants. The present study does not provide evidence for adolescent hyposensitivity; however there are several methodological

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17 differences in the present study that should be noted. The majority of the adolescent hyporesponsivity findings used locomotor activit y as a measure of drug sensitivity while the present study used the CPP paradi gm. Although both locomotor and place conditioning paradigms are valid measures of drug sensitivity, it is known that these two behavioral measures are associ ated with the activity of two separate DA pathways, the nigrostriatal and mesolimbic DA systems. Albeit psychostimulants act on both of these pathways, drug-induced behavior is associated with differe nt dopaminergic responses in the terminal areas of these 2 pathways (C adoni and Di Chiara, 1999). Further, druginduced locomotor activity does not predic t expression of CPP (Martin-Iverson and Reimer, 1996; Hemby et al, 1992). Therefor e differences in psychostimulant-induced behavior would be expected when compari ng locomotor activity to place conditioning. Others have shown a hyposensitivity to amphetamine place conditioning (Adriani and Laviola, 2003); however care s hould be taken when comparing amphetamine and cocaine behavioral results given that these 2 drugs work via different neurochemical mechanisms with amphetamine facilitating DA release and cocaine blocking DA reuptake. Additionally, the amphetamine CPP findings ar e in response to one drug-pairing while the present study incorporated 4 drug-pairings suggesting that ontogenetic differences to the rewarding properties of psychostimulant s may vary depending on the extent of drugpretreatment. The number of drug injecti ons is a likely factor producing conflicting results between the amphetamine CPP and pres ent findings since it ha s been previously shown that four drug-pairing produced a cocai ne CPP in early adol escent mice while one drug-pairing failed to have the same effect (L aviola et al., 1992). Finally, the present CPP paradigm may be more sensitive th an others (Campbell et al, 2000; Schramm-

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18 Sapyta et al, 2004) in reveali ng ontogenetic differences in cocaine-place conditioning in that a baseline comparison was included to control for any age-related differences in novelty-induced exploration (Stansfield and Ki rstein, 2005) upon first exposure to the CPP apparatus. In summary, our results demonstrate that there are age-rela ted differences in cocaine place conditioning with PND 35’s e xpressing unique sensi tivity to cocaine’s rewarding effects. This increased sensitivity may be mediated by cocaine’s effects on the developing mesolimbic system. These neurobe havioral factors appe ar to vary as a function of age and likely potentiate adolescent vulnerability to drug addiction. Further research should focus on the ontogeny of re ward-related associative learning and the involvement of DA in these processes.

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19 Chapter Three Experiment Two Basal and Cocaine-Induced Extracellular Dopamine in the Nucleus Accumbens Septi During Adolescence and Young Adulthood In humans, adolescent exposure to illicit drugs predicts the onset of adult drug abuse and suggests that repeated drug use pot entiates adolescent vulnerability to drug addiction. Our lab has previously shown th at adolescents are more sensitive to the rewarding properties of cocaine with PND 35 rats demonstrating a CPP for a low dose of cocaine (5 mg/kg). It was hypothesized that age-related di fferences in cocaine CPP may be mediated by differences in the meso limbic dopaminergic (DA) system throughout development. Extracellular DA levels in the NAcc of both adolescent and adult rats were measured via quantitative microdialysis. Early adolescent (PND 35), late adolescent (PND 45) and adult (PND 60) rats were injected daily with either 5 mg/kg/ip or saline for 4 days and surgically implanted with a micr odialysis probe aimed at the NAcc. Rats were perfused with 0, 1, 10 or 40 nM DA and the extracellular DA concentration was measured. Results show that adolescents di ffer from adults in basal DA with PND 35 rats having low (0.4 nM), PND 45 rats ha ving high (1.8 nM) and PND 60 rats having intermediate (1.3 nM) basal DA. PND 45 cocaine treated rats showed a 58% decrease in basal DA. All cocaine treated rats, regardless of age, showed a significant increase in DA over baseline in response to a cocaine chal lenge. Additionally, th ere were age-related differences in the extraction fraction (Ed), an indirect measure of DA reuptake, with PND 45 and PND 60’s showing a decrease in basal E d, an effect absent for PND 35. Together these findings demonstrate ontogenetic differe nces in extracellular DA and DA reuptake.

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20 Future research should investigate how these differences impact DA-dependent processes, such as addicti on, attention, and learning, in adolescent and adult rats. Introduction Current neurochemical research in a dolescence has focused on the development of the mesolimbic system, a dopaminergic (DA) pathway that originates in the VTA and terminates in the NAcc (Dahlstrom and F uxe, 1967). Widespread physiological changes occur throughout adolescence including he ightened neuronal ac tivity for humans (Chugani et al., 1987), and overproduction and pruning of several receptors in monkeys and rats (Lidow et al., 1991) including ch anges in DA receptors (Teicher et al., 1995; Tarazi et al., 1998a; Tarazi et al., 1999). In male rats, DA receptors peak in density at PND 40 followed by receptor pruning into young adulthood (PND 60). Studies using gamma-butyrolactone autoreceptor models de monstrate that DA autoreceptors in the NAcc decrease in sensitivity with maturation (Andersen et al., 1997). DAT density in the NAcc rapidly increases until PND 35 (Tarazi et al., 1998b); but stabilizes thereafter. Interestingly, it has been suggested that there are age-relate d differences in DA degradation processes such as a lack of DAT upregulation in the striatum after repeated cocaine treatment (Collins and Izenwasser, 2002 ). Additionally, age related differences in enzymatic degradation of DA have been shown in the striatum (Nakano and Mizuno, 1996) and the NAcc (Philpot and Kirstein, 2004). Together, these findings suggest that the mesolimbic system undergoes marked change in DA synthesis, metabolism and receptors during adolescence and drug use during this stage of development may likely alter subsequent normal DA functioning. To our knowledge, no one has quantified basal

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21 extracellular DA levels in the adolescent rat. It would be interesting to determine if adolescents had a hypoor hypersensitive DA system to aid in the understanding of adolescent neurocircuitry and associated DA –dependent behaviors. Conventional in vivo microdialysis has b een used to measure DA concentrations in various brain regions. However, recove ry of DA by the microdialysis probe, or any analyte of interest, is influenced by degrada tion processes occurring in the brain such as reuptake and metabolism (Shi ppenberg and Thompson, 1997; Justice, 1993; Smith and Justice, 1994). Analyte degradation processes, primar ily reuptake, directly influence the rate at which an analyte is recovered by changing analyte concentration gradients (Justice, 1993; Shippenberg and Thompson, 1997; Olson and Justice, 1993; Smith and Justice, 1994). For example, cocaine bloc kade of DA reuptake decreases DA recovery while facilitation of DA reuptake increases DA recovery (Smith and Justice, 1994). The effects of DA reuptake on DA recovery by th e probe during conventional microdialysis are illustrated in Appendices B-D. A ppendix B illustrates how DA concentration gradients in the NAcc are affected by the pr esence of a microdialys is probe. The NAcc contains DAT’s located on mesolimbic DA term inals. Extracellular DA is reuptaken by the DAT (1) and recycled into the terminal button. The probe mimics the DAT by recovering extracellular DA via the process of diffusion (2). After DA is recovered, the area immediately surrounding the probe is devoid of DA and must be replenished. By the processes of diffusion, DA from undisturbed ar eas in the NAcc diffuses into the depleted sampling region (3). This flux of DA from surrounding areas demonstrates the extracellular DA concentration gradient in the NAcc of a dialysis rat. Appendix C illustrates the changes in DA recovery when DA reuptake is inhibited. When cocaine is

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22 present, it blocks the DAT, inhibits DA re uptake (1) and excess DA remains in the extracellular fluid (2). As DA is recovere d by the probe (3) and the sampling region is depleted of DA, excess DA in the extracellu lar fluid replenishes the sampling region (4) thus decreasing the need for DA to diffuse from surrounding areas. This decreased flux of DA in the extracellular fl uid produces a gradual concentr ation gradient (5) and less movement of DA to the probe. Decreased re uptake and gradual concentration gradients limit the amount of DA available to be recovered by the probe and as a result underestimate true extracellular DA (see step 3; Olson and Justice, 1993). On the other hand, facilitated reuptake overestimates true extracellular DA. Appendix D illustrates the changes in DA recovery when DA reuptake is facilitated. If DA re uptake is facilitated either by increased density of DAT’s or by faster reuptake rates (1), DA is rapidly removed from the extracellular fluid (2). As DA is recovered by the probe (3) and the sampling region is depleted of DA, DA from un disturbed areas diffuses into the depleted sampling region (4). However, enough DA mu st diffuse to replenish both the sampling region around the probe in addition to areas de void of DA from facilitated reuptake. As a consequence, an increased flux of DA in the extracellular fluid produces a steep concentration gradient (5) and facilitated movement of DA to the probe. Increased reuptake and steep concentration gradients enhance the amount of DA available to be recovered by the probe and as a result overes timate true extracellular DA (see step 3; Justice, 1993; Shippenberg and Thompson, 1997) The fact that DA degradation and DA recovery are dependent on each other suggest s that any change in DA degradation across experimental groups will confound analyses of basal extracellular DA levels. Therefore, conventional microdialysis should only be used to determine analyt e concentrations if

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23 there are no suspected differences in degrad ation processes across manipulation groups. As noted above, it has previously been shown that there are age-rela ted differences in DA degradation. Therefore it is imperative that a measure of DA recovery is included when measuring basal extracellular DA levels in adolescent rats. Quantitative microdialysis in adults has been used to simultaneously measure extracellular DA and DA recovery in the NA cc (Parsons et al., 1991a; Parsons et al, 1991b; Olson and Justice, 1993, Smith and Ju stice, 1994; Shippenberg and Thompson, 1997). In quantitative microdialysis, various concentrations of DA are perfused through the probe in order to measure the point of “no net flux” for DA. When the DA concentration in the perfusate is greater than the DA concentration in the brain, DA diffuses from the probe and into the brain. On the other hand, when the DA concentration in the brain is greater than the DA concentration in the probe, DA diffuses from the brain and into the probe. Therefore, when in vivo DA concentrations are allowed to equilibrate, the brain and probe DA concentrations are equal and there is no net flux across the dialysis membrane. Any change in the tissue concentration of DA thereafter, for example when cocaine is admi nistered, would be a di rect consequence of cocaine and not influenced by the presence of the probe. In quant itative microdialysis, extracellular DA levels are calculated by th e equation (DAin – DAout) where DAin is the DA concentration in the perfus ate and DAout is the DA concentration in the sample. The net difference is averaged and plotted agains t DAin and a linear regression line is formed for each sample. The x-intercept represents the extracellular DA concentration while the slope of the regression line represents a measure of DA recovery, or the extraction fraction (Ed). Ed has also been used as an indirect measure of DA reuptake because the

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24 Ed is influenced by changes in DA reupt ake (see above; Appendices B-D; Olson and Justice, 1993, Smith and Justice, 1994; Shippenberg and Thompson, 1997). Taken together, development of the me solimbic system during adolescence and the dependency of DA recovery on DA degrada tion processes, underlie the importance of using quantitative microdialysis to measure extracellular DA levels in adolescent rats The aim of the present study was to measure basal and cocaine-induced extracellular DA levels in the NAcc of early adolescent (PND 35), late adolescent (PND 45) and young adult (PND 60) rats. Our results suggest th at there are ontogenetic differences in basal/cocaine-induced DA and Ed in the NAcc. Method and Materials Subjects. Ninety-three male Sprague-Dawley ra ts (Harlan Laboratories, IN) bred in our vivarium were used in the presen t study. Breeding and housing procedures are identical to that stated above in Experime nt 1. Rats used were divided into early adolescent (PND 35), late adolescent (PND 45) or young adults (PND 60). At the time of surgery, rats weighed between 200-340 g (PND 35, M = 110 g; PND 45, M = 225 g; PND 60, M = 299 g). Pre-exposure and surgical procedures. Experiments began at PND 29, PND 39 or PND 54. The experiment consisted of 4 phases: handling (days 1-2), pretreatment (days 3-6), surgery (afternoon of day 6), and dial ysis (day 7). All rats were handled daily in 3 minute sessions in order to decrease the stress of being handled by the experimenter during injections (Maldonado & Kirstein, 2005). For the next 4 consecutive days, rats received daily intraperitoneal injections of 5mg/kg cocaine or saline (0.9% NaCl). The 5mg/kg cocaine dose was chosen according to th e results of experiment 1 that showed

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25 ontogenetic differences in CPP. At least four hours following the last injection, rats were anesthetized using a ketami ne/ xylazine cocktail (1.0 and 0.15 mg/kg/ip). An incision was made over the skull and the rat was mounted on a stereotaxic inst rument for surgery. Two holes for skull screws and one for the gu ide cannula were drilled in the skull. The guide cannula was lowered into the brain usi ng appropriate age-defined coordinates to a site just above the NAcc (Phil pot et al, 2001). The guide can nula was affixed to the skull with cranioplast and the probe (2 mm membrane, 320 ODS, 30kDa MW cutoff, Bioanalytical Systems Inc., IN) was immediat ely lowered into the NAcc. Rats were singly housed in the dialysis testing environm ent overnight and allowed at least 18 h for recovery. Quantitative microdialysis procedures & neurochemical analyses. On the evening prior to dialysis, probe s were perfused continuously with artificial cerebrospinal fluid (145 mM NaCl, 2.4 mM KC l, 1.0 mM MgCl, 0.2 mM ascorbate, pH = 7.2) for at least twelve h prior to the start of sampling at a flow rate of 0.5 L/min. The following morning, DA solutions were prepared fresh from a 1 M stock solution in artificial cerebrospinal fluid to 1, 10 or 40 nM concen trations. Brain micr odialysis probes were connected through the use of a liquid switch to a 500 L Hamilton gastight syringe and a Bioanalytical syringe pump. The flow rate was increased to 2 L/min and the perfusion medium was then changed to 0, 1, 10 or 40 nM DA. After an equilibration period of 1.5 h, dialysates were collected by an automated fraction collector at 10-min intervals into refrigerated (4o C) microcentrifuge tubes containing 2.0 l of hydrochloric acid to prevent enzymatic breakdown. Three baseline dialysat e samples were taken from the NAcc after which animals received an injec tion of 5 mg/kg/ip cocaine or saline. Sampling continued

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26 for an additional 2 h. Dialysate samples (12.0 l) were either run immediately or quickly stored at -80o C until analyzed. Brains were removed, frozen and cut for histological verification of probe placemen t in the NAcc. Analyses of dialysate samples were performed by high performance liquid chroma tography with electrochemical detection (HPLC-EC) set to oxidize DA at 700 mV (Bioanalytical Systems, IN). A digital detector (Epsilon, Bioanalytical Systems, IN) was used with a radial flow carbon working electrode, referenced to an Ag/AgCl electrode. DA was el uted with a mobile phase consisting of 75 mM sodium phosphate, 1.4 mM octane sulfonic acid, 1 mM EDTA and 10% v/v acetonitrile with a pH = 2.9 and set at a flow rate of 60 l/min. Dialysate samples (6 l) were injected onto a C-18 microbore column, 100 x 1 m, 3 mm ODS for peak separation (Bioanalytical Systems, IN). The HPLC was calibrated with a standard curve consisting of 100 to 0.1 nM DA standard s. The range of detection was 1-10 nA and the average retention time for DA was 6 mi nutes. Peaks were verified by spiking one sample per rat with a DA standard. Data were recorded and quantified by Chromgraph on a Dell Dimension 2100. Design and analyses. Samples were analyzed by the equation DAin DAout = net DA, where DAin was the amount of DA pe rfused through the brain and DAout was the amount of DA obtained in the dialysate (Olson & Justice, 1993). The mean net DA was analyzed by linear regression and solved for basal extracellular DA. The slope of each regression line is equal to the recovery of DA and yields an indirect measure of DA reuptake, Ed. A three-way between subjects design was used to analyze age, drug and time course effects {[Age (3): PND 35, PND 45, PND 60] x [Treat ment (2): saline, cocaine] x [Time (2): Basal DA, Stimulated DA]. Paired t-tests were used to isolate age

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27 effects. A wilcoxon test was used to compare percent cocaine-induced DA increases to baseline (median percent DA increase versus 100%). All statistical analyses were determined significant at the 0.05 alpha level. Results Overall, there are significant age and treatment differences for basal and cocaine-induced DA. A significant Age x Dose x Time intera ction [F(2, 60) = 6.91, p < 0.05] was found. Basal dopamine Appendix E shows age-related di fferences in basal DA for both nave/cocaine treated rats {Age x Dose interaction [F(2, 12) = 14.01, p < 0.05]}. For nave rats, PND 35 rats had the lowest (0.4 nM), PND 45 rats had the greatest, and PND 60 rats had intermediate (1.3 nM) basal DA. In cocaine treated rats, basal DA levels decreased by 58% for PND 45 while there we re no changes in basal DA for PND 35 and PND 60 rats. These data suggest that basal DA varies as a function of both age in both nave and cocaine pretreated rats. Cocaine-induced dopamine. The time course effects (nM) of cocaine-induced DA for PND 35, PND 45, and PND 60 rats can be seen in Appendices F-H. However, because there were basal DA differences acro ss age, basal DA was normalized to 100% and changes in DA concentrations were expr essed as percent change from baseline. Time course effects for percent change in DA for PND 35, PND 45, and PND 60 can be seen in Appendices I-K. All three age groups demonstrated similar increases in percent DA. A wilcoxon test indicates that all three age groups show a significant increase in cocaine-induced DA over baselin e (100%). Cocaine increa sed DA to 318 %, 247 %, and 314 % over basal levels for P ND 35, PND 45, and PND 60 resp ectively. Interestingly, DA peaks and returns to baseline at a differe nt rate for each age [Age: F(2, 48) = 4.43, p

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28 < 0.05] with PND 35’s peaking quickly at 10 min post injection and PND 45 and PND 60 rats peaking at 20 min post injection (Appendi x L). The rate at which cocaine-induced DA returns to baseline differs across age w ith PND 35 quickly returning to baseline, PND 45 gradually returning to ba seline, and PND 60 stabilizing at a higher value than at baseline. These data suggest that extracellu lar DA remains elevated for variable amounts of time with younger rats exhibiting a rapi d decay and adults exhibiting prolonged elevations in cocaine -induced DA. Extraction fraction. Ed, an indirect measure of DA reuptake, varied by age, dose and time [Age x Time, F(2, 60) = 3.44, p< 0.05; Dose x Time, F(1, 60) = 7.84, p 0.05]. PND 45 (t(20.47), p < 0.05] and PND 60 [t(12.41) p < 0.05] cocaine treated rats show decreased basal Ed after cocaine pretreatme nt (Appendix M). Decreased basal Ed in response to administration of cocaine has b een previously shown in adults (Olson and Justice, 1993; Smith and Justice, 1994; Chef er et al., 2002). Ther e were no changes in basal Ed for cocaine treated PND 35 rats. These data suggest that the magnitude of cocaine blockade of the DAT incr eases as the rat matures. Basal Ed values were normalized to 100% and changes in Ed post challenge injection were compared across age (Appendix N). Cocaine-induced Ed varied with age and dose [Age x Dose: F(2, 48) = 4.79, p < 0.05] In cocaine treated rats, both the PND 35 [t(3.22), p < 0.05] and PND 60 [t(4.58), p < 0.05] rats show a decrease in Ed after a cocaine challenge, as expected because cocaine blocks the DAT Ed does not decrease in cocaine challenged PND 45’s, however, this ma y be influenced by a floor effect because basal DA and basal Ed levels have already decreased 58% and 25%, respectively. Adults show the greatest decrease in Ed in co mparison to younger rats suggesting a greater

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29 magnitude of cocaine-induced DAT blockade fo r adult rats. The time course effects of percent DA and percent Ed were compared at each age and are illustrated in Appendices O-Q. Ed follows the same temporal pattern as DA with Ed decreasing when DA peaks. This inverse relationship is supported by the f act that cocaine blocks the DAT, increases extracellular DA and decreases recovery of DA via a more gradual concentration gradient (Smith and Justice, 1994). Comparison of conventional and quantitative microdialysis. Rats only perfused with aCSF provided a conven tional microdialysis group so that conventional and quantitative microdialysis could be compared. DAout from the aCSF group was compared to extracellular va lues obtained via quantitativ e microdialysis (Appendices RT). Extracellular DA varied as a functi on of microdialysis method [Age x Time x Method: F(2, 124) = 8.32, p < 0.05]. Stimulated DA for PND 60 was underestimated by conventional microdialysis [Appendix T; Age x Time: F(1, 44) = 4.77, p < 0.05]. Underestimation of extracellular DA in the NA cc has been previously shown in the adult quantitative microdialysis liter ature (Olson and Justice, 1993). Interestingly, the opposite was found for PND 45 rats in that extracellular DA was overestimated by conventional microdialysis [Appendix S; Dose: F(1, 44) = 3.94, p < 0.05]. There were no differences in DA between the 2 microdialysis methods for PND 35 rats (Appendix R). Therefore, DA concentrations appear to vary depe nding on the type of microdialysis method employed. Further, as conventional microdial ysis neglects to control for DAT-probe interactions, these results provide additional evidence suggesting ontogenetic differences in the functioning of the DAT.

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30 Discussion Age-related differences in basal dopamine. The present study is the first to quantify basal extracellular DA levels in the NAcc of adolescent rats. There are differences in basal DA not only between a dolescents and adults but also within adolescence (Appendix E). Additionally, PND 45 rats show the greatest drug-induced plasticity in that they are the only age to show a decrease in basal DA after repeated cocaine pretreatment. A decrease in basa l DA after administration of a DA agonist is suggestive of change in one or more dopa minergic degradation processes such as upregulation of the DAT via increased tran sporter density and in creased rate of DA reuptake. Other DA degradation processe s, such as increased DA metabolism or facilitated negative feedback via supersensitive DA autoreceptors, coul d also be involved. Cocaine-induced changes in DA degradation pr ocesses are likely contributors to the decreased basal DA for PND 45 as ontogeneti c differences in DA metabolism (Philpot and Kirstein, 2004) and DA autor eceptors (Andersen et al., 1997) have been shown in the NAcc. Although cocaine blockade of th e DAT primarily affects DA degradation processes (i.e. reuptake, metabolism), change s in DA supply can alter basal DA as well by decreased DA synthesis or decreased tonic release of DA. All of the aforementioned synaptic changes would mediate decreased basal DA after repeated administration of cocaine in late adolescent rats. Differences in basal extracellular DA in a dolescent rats suggest fluctuations and instability of DA neuronal ac tivity in the mesolimbic system throughout development. Human adolescent brains undergo a physiological shift in primary brain activity from the limbic system during adolescen ce to more involvement of cortical areas in adulthood

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31 (Lewis et al, 1997). Together, the limbic me diated adolescent br ain and fluctuating DA levels in the NAcc would suppor t the hypothesis that adolescen ce is a time of transitional neuronal activity in the mesolimbic system. These data support studies comparing DA activity in the NAcc and PFC of adolescent rats. Spear ( 2000) eloquently discusses the inverse relationship between DA activity in th e NAcc and PFC with early adolescent rats (PND 30) showing greater PFC and less NAcc DA activity than older adolescent rats (PND 40). Similarly, rats that express high DA levels also have heightened basal firing patterns in the mesolimbic system (Gr ace et al., 1995). These heightened basal concentrations, or tonic DA release, are re gulated by glutamatergic afferents from the PFC, hippocampus and amygdala (Grace, 2000; O’Donnell et al, 1999). Fluctuating concentrations of extracellular DA in th e NAcc throughout adoles cence as shown by the findings of the present study may be mediated by differences in glutamatergic cortical regulation of the mesolimbic system throughout development. Ontogenetic differences in cocaine-induced dopamine. Cocaine-induced DA peaks faster for early adolescent rats than for the other 2 ages. DA peaks at 10 min for PND 35 while PND 45 and PND 60 show a delayed DA peak at 20 min post cocaine injection (Appendix L). Intere stingly, we have previously shown that early adolescents demonstrate a CPP for 5 mg/kg cocaine, while PND 45 and PND 60’s do not (Appendix A). A quick onset of cocaine-induced DA and prompt return to baseline values for early adolescent rats results in elevations in DA that are closely associated with the presentation of drug-related cues. Rather, for late adolescent and adult rats, DA is cleared less efficiently and el evations in DA are not closely time locked to drug cue presentation. Thus, associations between dr ugs and drug-related cues would be stronger

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32 in rats exhibiting quick on set of cocaine-induced DA. The present data supports expectancy theory (Montague et al., 1996; Schultz et al., 1997) which suggests changes in the concentration or onset of mesolim bic DA provides information on the magnitude of reward and the consistency of reward presentation. Others have shown VTA DA neurons fire in response to reward related cu es presented immediatel y prior to obtaining a reward (Carrelli and Ijames, 2000) and can be related to the quicker onset of cocaineinduced DA for PND 35 rats in the present study. For example, CPP conditioning trials are commonly 15 min. During place conditi oning, DA levels have already peaked and are starting to return to baseline for early adolescent rats while DA levels have not yet peaked in the late adolescent & adult rats. As a result, rats that have peak DA levels while still inside the CPP chamber will asso ciate drug effects with the chamber cues while other rats may not make a strong c onnection between the drug and chamber cues because DA is quite possibly peaking after co mpletion of the conditioning trial. The quick onset of DA observed for PND 35 ra ts in the present study may be the neurochemical mechanism mediating low dose co caine CPP in early adolescent rats. As all three age groups in the pr esent study had similar percen t increases in cocaine-induced DA, it may be the temporal changes in DA, and not absolute DA levels per se, that mediates adolescent vulnerability to drug addiction. Extraction fraction and dopamine reuptake. The present data demonstrate ontogenetic differences in basal and cocai ne inhibition of DA reuptake (Appendices MN). The findings in adults are similar to previous reports that both show decreases in basal Ed and a lack of change in DA levels af ter repeated cocaine pr etreatment (Chefer et al, 2002). The lack of change in basal Ed a nd small but significant decrease in cocaine-

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33 induced Ed for cocaine pretreated PND 35’s demonstrates less DAT inhibition. PND 45 rats illustrate a transitional stage betw een minimal cocaine induced effects on DA reuptake in early adolescent rats and a substa ntial inhibition of DA reuptake for adults. The Ed data provides additional evidence for the suggested differences in DA clearance rate as seen in the cocaine-induced DA fi ndings above. PND 35 rats clear DA quickly via facilitated DA reuptake while the oldest rats demonstrat e the greatest cocaine-induced DA effect via prolonged elevations of extr acellular DA and substantial inhibition of DA reuptake. Together, these da ta suggest age-related differences in DAT functioning with more efficient DA degradation in early adolescen t rats relative to older ages. Conventional vs. quantit ative microdialysis Adult data in the present study is similar to previous work that compared dial ysate and extracellular le vels in adult male Sprague-Dawley rats (Appendices R-T). Ol son and Justice (1993) found that animals perfused with aCSF had lower DA levels than measured through quantitative microdialysis, suggesting that basal extr acellular DA was underestimated using conventional microdialysis. Interestingly, the age differenc es found in the present study suggest that basal extracellu lar DA concentrations are dependent on the type of microdialysis technique employed. Alt hough both microdialysis techniques show significant age-related differences, dialysate le vels in late adolescents are overestimated (Appendix S) while adult levels are unde restimated (Appendix T). Therefore, conventional microdialysis may suggest much larger differences between age, thus creating false positives. Inte rpretation of conventional mi crodialysis studies should be carefully examined when applied to devel opmental work. These theoretical concerns directly relate to degradati on processes that are still developing throughout adolescence.

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34 Age-related differences in estimated dialys ate levels as measured via conventional microdialysis may likely be dependent on ont ogenetic differences in DA concentration gradients in the NAcc. In conclusion, there are ont ogenetic differences in ba sal/cocaine-induced DA and DA reuptake in the NAcc. Early adolescents have less and late adolescents have more basal DA than adults in the NAcc. All age gr oups demonstrated similar percent increases in cocaine-induced DA. DA reuptake, as m easured by Ed, varied as a function of age with early adolescent rats showing facilitated DA reuptak e relative to older rats. Fluctuations and changes in th e activity of DA in the NAcc may play a role in adolescent vulnerability to drug addicti on. It has been previously shown by our lab that early adolescent rats who demonstrate the quick est onset and decay of cocaine-induced DA will express a CPP for a low dose of cocaine (5 mg/kg), that which adults do not find rewarding. Together these findi ngs suggest that early adoles cent rats are vulnerable to developing a drug addiction after the repeated administration of co caine. Implications could be used to treat adolescent drug addict ion as well as provide insight into how the adolescent brain responds to dopaminergic drugs.

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35 Chapter Four Concluding Remarks and Implications In conclusion, the present st udies illustrate that there are ontogenetic differences in the rewarding properties of cocaine, basal DA and DA reuptake. Ea rly adolescent rats demonstrate a CPP for a low dose of cocaine suggesting an enhanced sensitivity to the rewarding properties of repeated cocaine. We also show a quick onset of cocaineinduced DA and facilitated DA reuptake for early adolescent rats which may allow cocaine-induced DA to be closely associated with the presentation of drug-related cues. For late adolescent and adult rats, DA may be cleared less efficiently causing cocaineinduced increases in DA to not be closely ti me locked to drug cue presentation. Thus, associations between drugs and drug-related cues would be stronger in rats exhibiting quick onset of cocaine-induced DA and fac ilitated clearing of excess DA from the synapse. The present data supports expectancy theory (Montague et al., 1996; Schultz et al., 1997) which suggests changes in the c oncentration or onset of mesolimbic DA provide information on the magnitude of reward and the consistency of reward presentation. Therefore, the overall activity of the mesolimbic system, and not absolute DA levels per se, may be the factor mediati ng adolescent vulnerabil ity to drug addiction as all three age groups in th e present study had similar pe rcent increases in cocaineinduced DA. The present findings suggest that human adolescents who repeatedly take cocaine may be particularly vulnerable to cue elicited cocaine craving if in fact they exhibit the same quick onset of cocaine-induced DA as de monstrated in the present study. It would be interesting to find if there were agedifferences in brain activity during drug cue

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36 presentation for adolescent and adult humans. The presen t findings may also provide insight for the treatment of adolescent a nd adult cocaine addict s. Clinical and experimental research should continue to focus on the development of the adolescent brain so to better understand how to treat adolescent substance abuse.

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37 References Adams JU, Careri JM, Efferen TR, Rotrosen J (2001) Differential effects of dopamine antagonists on locomotor activity, cond itioned activity and conditioned place preference induced by cocaine in rats. Behav Pharmacol 12: 603-611. Adriani W, Laviola G (2003) Elevated le vels of impulsivity and reduced place conditioning with d-amphetamine: two beha vioral features of adolescence in mice. Behav Neurosci 117: 695-703. Anderson SL, Dumont NL, Teicher MH (1997) Developmental differences in dopamine synthesis inhibition by (+/-)-7-OHDPAT. Naunyn Schmiedebergs Arch Pharmacol 356: 173-181. Anthony JC, Petronis KR (1995) Early-onset dr ug use and risk of later drug problems. Drug Alcohol Depend 40: 9-15. Bardo MT, Neisewander JL, Miller JS (1986) Repeated testing a ttenuates conditioned place preference with cocaine. Psychopharmacology (Berl) 89: 239-243. Blackburn JR, Pfaus JG, Phillips AG (1992) Dopamine functions in appetitive and defensive behaviours. Prog Neurobiol 39: 247-279. Cadoni C, Di Chiara G (1999) Reciprocal changes in dopamine responsiveness in the nucleus accumbens shell and core and in the dorsal caudate-putamen in rats sensitized to morphine. Neuroscience 90: 447-455. Calcagnetti DJ, Schechter MD (1993) Extinc tion of cocaine-induced place approach in rats: a validation of the "biased" cond itioning procedure. Brain Res Bull 30: 695700. Camp DM, Browman KE, Robinson TE (1994) The effects of methamphetamine and cocaine on motor behavior a nd extracellular dopamine in the ventral striatum of Lewis versus Fischer 344 rats. Brain Res 668: 180-193. Campbell JO, Wood RD, Spear LP (2000) Cocaine and morphine-induced place conditioning in adolescent and a dult rats. Physiol Behav 68: 487-493. Carelli RM, Ijames SG (2000) Nucleus accu mbens cell firing during maintenance, extinction, and reinstatement of cocaine self -administration behavi or in rats. Brain Res 866: 44-54. Chefer VI, Shippenberg TS (2002) Changes in basal and cocaine-e voked extracellular dopamine uptake and release in the rat nuc leus accumbens during early abstinence from cocaine: quantitative determination under transient conditions. Neuroscience 112: 907-919.

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44 Tanda G, Di Chiara G (1998) A dopamine-mu1 opioid link in the rat ventral tegmentum shared by palatable food (Fonzies) and non-psychostimulant drugs of abuse. Eur J Neurosci 10: 1179-1187. Tarazi FI, Tomasini EC, Baldessarini RJ (1998) Postnatal development of dopamine and serotonin transporters in rat caudateputamen and nucleus accumbens septi. Neurosci Lett 254: 21-24. Tarazi FI, Tomasini EC, Baldessarini RJ (1998) Postnatal development of dopamine D4like receptors in rat forebrain regions: comparison with D2-like receptors. Brain Res Dev Brain Res 110: 227-233. Tarazi FI, Tomasini EC, Baldessarini RJ (1999) Postnatal development of dopamine D1like receptors in rat cortical and striat olimbic brain regions: An autoradiographic study. Dev Neurosci 21: 43-49. Teicher MH, Andersen SL, Ho stetter JC, Jr. (1995) Evidence for dopamine receptor pruning between adolescence and adult hood in striatum but not nucleus accumbens. Brain Res Dev Brain Res 89: 167-172. Thompson AC, Zapata A, Justice JB, Jr., Vaughan RA, Sharpe LG, Shippenberg TS (2000) Kappa-opioid receptor activation modifies dopamine uptake in the nucleus accumbens and opposes the effects of cocaine. J Neurosci 20: 9333-9340. Tirelli E, Laviola G, Adriani W (2003) Ont ogenesis of behavioral sensitization and conditioned place preference induced by psyc hostimulants in laboratory rodents. Neurosci Biobehav Rev 27: 163-178. Tomkins DM, Joharchi N, Tampakeras M, Martin JR, Wichmann J, Higgins GA (2002) An investigation of the role of 5-HT(2 C) receptors in modifying ethanol selfadministration behaviour. Pharmacol Biochem Behav 71: 735-744. Tzschentke TM (1998) Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues. Prog Neurobiol 56: 613-672. Weiss F, Paulus MP, Lorang MT, Koob GF ( 1992) Increases in extracellular dopamine in the nucleus accumbens by cocaine are inversel y related to basal levels: effects of acute and repeated administ ration. J Neurosci 12: 4372-4380. White NM, Packard MG, Hiroi N (1991) Place conditioning with dopamine D1 and D2 agonists injected periphera lly or into nucleus accumbens. Psychopharmacology (Berl) 103: 271-276. Zahm DS, Heimer L (1990) Two transpallidal pathways originating in the rat nucleus accumbens. J Comp Neurol 302: 437-446.

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45 Appendices

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46 Appendix A: Cocaine Place Conditioning PND 35 PND 45 PND 60 0 100 200 3000 5 20 *=differs from saline #=differs from PND 45 & 60* * *#Ageseconds in l east preferred chamber post-pre All ages had a CPP for the high dose, 20 mg/kg cocaine. PND 35’s were the only age to have a CPP for 5 mg/kg cocaine. Each bar represents mean and SEM.

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47 Appendix B: Normal Dopamine Concentration Gradient Undisturbed DA

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48 Appendix C: Gradual Dopamine Concentration Gradient Undisturbed DA

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49 Appendix D: Steep Dopamine Concentration Gradient Undisturbed DA

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50 Appendix E: Basal Dopamine Levels salinecocaine 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0PND 35 PND 45 *PND 60 # # # # #DoseExtracellular DA (nM) For nave rats, PND 35 rats had the lowest (0.4 nM), PND 45 rats had the greatest (1.8 nM), and PND 60 rats had intermediate (1.3 nM ) basal DA. In cocaine treated rats, basal DA levels decreased by 58% for PND 45 while there were no changes in basal DA for PND 35 and PND 60 rats. Each bar represents mean and SEM.

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51 Appendix F: Dopamine and Repeated Cocaine Pretreament for PND 35 -30 -20 -10 0 10 20 30 40 50 60 70 80 90 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0saline cocaine TimeDA (nM) The time course effects (nM) of cocaine-i nduced DA for PND 35. The perpendicular line denotes the cocaine injection. Each data poi nt represents the x-intercept of a linear regression.

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52 Appendix G: Dopamine and Repeated Cocaine Pretreament for PND 45 -30 -20 -10 0 10 20 30 40 50 60 70 80 90 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0saline cocaine TimeDA (nM) The time course effects (nM) of cocaine-i nduced DA for PND 45. The perpendicular line denotes the cocaine injection. Each data poi nt represents the x-intercept of a linear regression.

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53 Appendix H: Dopamine and Repeated Cocaine Pretreament for PND 60 -30 -20 -10 0 10 20 30 40 50 60 70 80 90 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0saline cocaine TimeDA (nM) The time course effects (nM) of cocaine-i nduced DA for PND 60. The perpendicular line denotes the cocaine injection. Each data poi nt represents the x-intercept of a linear regression.

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54 Appendix I: Percent Change in Dopamine for PND 35 -30 -20 -10 0 10 20 30 40 50 6 0 70 8 0 90 0 50 100 150 200 250 300 350 400cocaine saline Time (min)Percent of Baseline (DA) The time course effects for percent change in baseline for PND 35. The perpendicular line denotes the cocaine injection.

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55 Appendix J: Percent Change in Dopamine for PND 45 -3 0 2 0 -10 0 10 2 0 30 40 50 60 70 80 90 0 50 100 150 200 250 300 350 400cocaine saline Time (min)Percent of Baseline (DA) The time course effects for percent change in baseline for PND 45. The perpendicular line denotes the cocaine injection.

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56 Appendix K: Percent Change in Dopamine for PND 60 -30 -20 -10 0 10 20 30 40 50 6 0 70 8 0 90 0 50 100 150 200 250 300 350 400cocaine saline Time (min)Percent of Baseline (DA) The time course effects for percent change in baseline for PND 60. The perpendicular line denotes the cocaine injection.

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57 Appendix L: Age-Related Differences in Cocaine-Induced Dopamine -3 0 -2 0 -10 0 1 0 2 0 30 4 0 5 0 60 70 8 0 90 0 50 100 150 200 250 300 350 400 450 500PND 35 PND 45 PND 60 Time (min)Percent of Baseline (DA) A comparison of the time course effects fo r percent change in baseline across age. Cocaine increased DA to 318 %, 247 %, and 314 % over basal levels for PND 35, PND 45, and PND 60 respectively. DA peaks and return s to baseline at a different rate for each age with PND 35’s peaking at 10 min and PND 45 and PND 60 rats peaking at 20 min post injection. The rate at which cocai ne-induced DA returns to baseline differs across age with PND 35 quickly returning to baseline, PND 45 gradually returning to baseline, and PND 60 stabilizi ng at a higher value than at baseline. The perpendicular line denotes the cocaine injection.

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58 Appendix M: Basal Extraction Fraction PND 35 PND 45 PND 60 50 75 100saline cocaine ## #* ** = differs from saline # = differs from PND 60 ## = differs from PND 45/PND60AgeEd (%) PND 45 and PND 60 cocaine treated rats s how decreased basal Ed after cocaine pretreatment. There were no changes in basa l Ed for cocaine treated PND 35 rats. Each bar represents mean and SEM.

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59 Appendix N: CocaineInduced Extraction Fraction PND 35 PND 45 PND 60 50 75 100 125saline cocaine *# #AgePercent of Baseline (Ed) In cocaine treated rats, both the PND 35 and PND 60 rats show a decrease in Ed after a cocaine challenge. Ed does not decrease in cocaine challenged PND 45’s. Each bar represents mean and SEM.

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60Appendix O: Extraction Fraction and Dopamine for PND 35 Figure 9a: PND 35 -30 -20 1 0 0 10 20 30 40 50 60 70 80 9 0 0 50 100 150 200 250 300 350 400 450 500Ed saline Ed cocaine DA saline DA cocaine 0 25 50 75 100 125Time (min)Percent of Baseline (DA)Percent of Baseline (Ed) The time course effects of percent DA and pe rcent Ed were compared for PND 35 rats. The perpendicular line denotes the cocaine injection.

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61 Appendix P: Extraction Fraction and Dopamine for PND 45 Figure 9b: PND 45 30 20 -10 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 0 50 100 150 200 250 300 350 400 450 500 0 25 50 75 100 125Ed saline Ed cocaine DA saline DA cocaine Time (min)Percent of Baseline (DA)Percent of Baseline (Ed) The time course effects of percent DA and pe rcent Ed were compared for PND 45 rats. The perpendicular line denotes the cocaine injection.

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62 Appendix Q: Extraction Fraction and Dopamine for PND 60 -30 -2 0 1 0 0 10 20 30 4 0 5 0 6 0 70 80 90 0 50 100 150 200 250 300 350 400 450 500 0 25 50 75 100 125Ed saline Ed cocaine DA saline DA cocaine Time (min)Percent of Baseline (DA)Percent of Baseline (Ed) The time course effects of percent DA and pe rcent Ed were compared for PND 60 rats. The perpendicular line denotes the cocaine injection.

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63 Appendix R: Conventional and Quantitative Microdialysis for PND 35 basalstimulated 0 1 2 3 conventional quantitative TimeDA (nM) There were no differences in DA between the 2 microdialysis methods for PND 35 rats. Each bar represents mean and SEM.

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64 Appendix S: Conventional and Quantitative Microdialysis for PND 45 basalstimulated 0 1 2 3* conventional quantitative = differs from conventionalTimeDA (nM) Extracellular DA was overestimated by convent ional microdialysis for PND 45 rats. Each bar represents mean and SEM.

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65 Appendix T: Conventional and Quantitative Microdialysis for PND 60 basal stimulated 0 1 2 3** conventional quantitative ** = differs from basal & conventionalTimeDA (nM) Cocaine-induced DA for PND 60 was underes timated by conventional microdialysis. Each bar represents mean and SEM.


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Badanich, Kimberly A.
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Age-related differences in cocaine place conditioning and cocaine-induced dopamine
h [electronic resource] /
by Kimberly A. Badanich.
260
[Tampa, Fla] :
b University of South Florida,
2005.
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ABSTRACT: In humans, adolescent exposure to illicit drugs predicts the onset of adult drug abuse and suggests that early drug use potentiates adolescent vulnerability to drug addiction. In experiment 1, it was hypothesized that adolescent rats would show a CPP for a low cocaine dose if in fact adolescents are more vulnerable to cocaine's rewarding effects. Place preferences were measured in early adolescent [postnatal day (PND) 35], late adolescent (PND 45) and young adult (PND 60) rats by injecting either 0, 5 or 20 mg/kg cocaine and conditioning them to environmental cues in a 2-chamber place conditioning apparatus. Significant cocaine preferences were found for all ages at the high dose. Interestingly, PND 35's were the only age group to have a CPP at the low dose suggesting that PND 35 rats are more sensitive than late adolescent and young adult rats to cocaine's rewarding effects.In Experiment 2, it was hypothesized that age-related differences in cocaine CPP may be mediated by differences in the mesolimbic dopaminergic (DA) system throughout development. Extracellular DA levels in the nucleus accumbens septi (NAcc) of early adolescent, late adolescent and adult rats were measured via quantitative microdialysis. PND 35, PND 45 and PND 60 rats were injected daily with either 5 mg/kg/ip or saline for 4 days, surgically implanted with a microdialysis probe aimed at the NAcc. Rats were perfused with either 0, 1, 10 or 40 nM DA and the extracellular DA concentration was measured. Our results show that adolescents differ from adults in basal DA with PND 35 rats having low basal DA (0.4 nM), PND 45 rats having high basal DA (1.8 nM) and PND 60 rats having intermediate basal DA (1.3 nM). PND 45 cocaine treated rats showed a 58% decrease in basal DA. All cocaine treated rats, regardless of age, showed a significant increase in DA over baseline in response to a cocaine challenge.Additionally, there were age-related differences in the extraction fraction (Ed), an indirect measure of DA reuptake, with PND 45 and PND 60's showing a decrease in basal Ed, an effect absent in PND 35's. Together these findings suggest that there are substantial ontogenetic differences in extracellular DA and DA reuptake and that these differences may provide an explanation for adolescent vulnerability to addiction. Future research should investigate DA supply and degradation processes in nave and cocaine treated adolescent rats and vulnerability to addiction.
502
Thesis (M.A.)--University of South Florida, 2005.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
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System requirements: World Wide Web browser and PDF reader.
Mode of access: World Wide Web.
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Title from PDF of title page.
Document formatted into pages; contains 65 pages.
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Adviser: Cheryl L. Kirstien, Ph.D.
653
Ontogeny.
Adolescent rat.
Nucleus accumbens.
Addiction.
Quantitiative in vivo microdialysis.
690
Dissertations, Academic
z USF
x Psychology
Masters.
773
t USF Electronic Theses and Dissertations.
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u http://digital.lib.usf.edu/?e14.1372