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The developing nucleus accumbens septi

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Title:
The developing nucleus accumbens septi susceptibility to alcohols' effects
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Language:
English
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Philpot, Rex Montgomery
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University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
nucleus accumbens
dopamine
alcohol
adolescence
addiction
Dissertations, Academic -- Psychology -- Doctoral -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: The mesolimbic dopamine (DA) system has been implicated in providing the basis of pleasure, guiding the general mechanism of reinforcement as well as motivation. Support for these roles have grown from neurochemical research in the field of addiction. It is now well known that DA activity increases in the nucleus accumbens septi (NAcc) with exposure to addictive substances. Moreover, pharmacological manipulation of this system produces predictable changes in the administration of drugs of abuse, as well as natural reinforcers. This system is responsive to natural reinforcers and addiction may be the transference of routine mesolimbic function to environmental stimuli predictive of drug administration. The role of the NAcc in addiction specifically appears to be the facilitation of attention to drug-paired stimuli and addiction may be the behavioral manifestation of conditioned NAcc DA reactivity to the presence of drug-related stimuli. Although these findings have been reported in adults, few studies have focused on adolescence, the time when drug use/abuse begins. Adolescents may be particularly susceptible to addiction when considered in the light of this hypothesis. Recent research has revealed that the mesolimbic system of periadolescent animals is undergoing dramatic transition in functional tone. DA receptor and transporter levels are up regulated, synthesis rates are altered, and innervation from prefrontal cortex (PFC), involved in regulating tonic and phasic DA activity, is increasing. Consequently, during adolescence there is a dramatic change in tonic DA levels, variations in phasic responses to acute drug administration and alterations in how the system adapts to repeated drug exposure. The present study utilizes the procedures of conditioned place preference, Novelty preference and in vivo microdialysis to determine how this conditioning process changes during the period of adolescence. The results indicate that adolescents are different from adults not only on behavioral measures associated with drug abuse, but in their neurochemical responsiveness to alcohol, and that these differences are related to a general developmental aspect of adolescence that renders them susceptible to addiction.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
Statement of Responsibility:
by Rex Montgomery Philpot.
General Note:
Includes vita.
General Note:
Title from PDF of title page.
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Document formatted into pages; contains 175 pages.

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University of South Florida Library
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University of South Florida
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aleph - 001478768
oclc - 56546836
notis - AJS2458
usfldc doi - E14-SFE0000410
usfldc handle - e14.410
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The Developing Nucleus Accumbens Septi: Susceptibility to Alcohols Effects by Rex Montgomery Philpot A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Psychology C ollege of Arts and Sciences University of South Florida Major Professor: Cheryl L. Kirstein Ph.D. Mark Goldman, Ph.D. Toru Shimizu, Ph.D. James Willott, Ph.D. Lynn Wecker, Ph.D. Date of Approval: May 20, 2004 Keywords: A ddiction, Adolescence, Alcohol, Dopamine, Nucleus Accumbens Copyright 2004, Rex Philpot

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Your spirit was my inspiration. More than friend and wife. Through the years I labored, lost in truth, you were my life.

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I would like to thank all of the facu lty and staff involved in my education at the University of South Florida. Y our knowledge and support have made this rocky road far, far smoother. In particular I would like to thank James Jenkins, for his endless nuggets of wisdom, many of which I took to heart much more tha n my behavior would suggest; Douglas Nelson, whos example as a professor I aspire to emulate ; and Lynn Wecker, who gave me a good shove when I really needed it most I would also like to thank Florencia Stanley, who has been here si nce I started and has always had the answers to my problem du jour. Thank you all. I would like to thank all the members of the Kirstien lab, past and present, who have shared the load through the years. There are too many to name here. I would not ha ve completed this project were i t not for all of your efforts. I would like to thank Cheryl Kirstien fo r her guidance You have been many things to me and you have helped me grow in more ways than as an academic. Many years ago I knocked on your door, a lost undergraduate looking for a n opportunity to prove my worth You gave me so much more than just an opportunity that day, you gave me a future I would like to thank my Mother and Father, for bringing me into this world and providing me with the means, and the tools, to achieve this goal. Knowing you would always be there if I needed you has given me more strength than you will ever know. And to my Brother and Sister, you have both been an inspiration to me, although as the youngest I try hard to hide my admiration, both of you cleared the path that I have been traveling. To my children, Rebecca and Grace, I am so grateful that I was given this time with you. Your joy for life has brought a smile to my face and warmed my heart at the end of ma ny a n otherwise horrid day. I love you both very much. To Elizabeth, thank you for being my friend and companion through it all. There are no words for what you mean to me, or what you meant when I needed you most. You will always be near to my heart

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i TABLE OF CONTENTS LIST OF FIGURES iv LIST OF TABLES v LIST OF DIAGRAMS vi ABSTRACT vii INTRODUCTION 1 The Problem of Adolescent Substance Use 1 Sensation Seeking and Substance Use 3 Conditioned Place Preference and Drug Induced Motivation 4 Neurochemistry, Reward and Drugs of Abuse 5 Adolescence, NP, CPP and Neurochemistry 7 Summary 9 ADOLESCENT TRANSITIONS IN NOVELTY PREFERENCE 11 Abstract 11 Introduction 13 Experiment One 18 Methods 18 Subjects 18 Apparatus 18 Training 19 Behavioral Testing 19 Analysis 20 Results 20 Baseline Analysis 20 Novelty Preference 23 Discussion 23 Experiment Two 24 Methods 24 Subjects 24 Apparatus 24 Training 25

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ii Behavioral Testing 25 Analysis 25 Results 26 Baseline Analysis 26 Novelty Preference 27 Discussion 27 General Discussion 28 References 32 PLACE CONDITIONING: AGE RELATED CHANGES IN THE REWARDING AND AVERSIVE EFFECTS OF ALCOHOL 35 Abstract 35 Introduction 37 Methods 43 Subjects 43 Apparatus 43 Ages 44 Training 44 Testing 44 Design and Analyses 45 Results 46 Discussion 48 References 53 EFFECTS OF REPEATED ETHANOL ON BASAL DOPAMINE LEVELS 59 Abstract 59 Introduction 61 Methods 62 Results and Discussion 64 References 66 ETHANOL MEDI ATED DOPAMINE RELEASE IN THE NUCLEUS ACCUMBENS SEPTI OF ADOLESCENT ANIMALS 67 Abstract 67 Introduction 68 Methods 72 Animals 72 Surgery 72 Dialysate Analysis 73 Ethanol Induced NAcc DA Release Across Age 73 Results 74 Basal Levels 74

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iii DOPAC 74 DA 76 DOPAC/DA 76 Treatment Effects 80 DOPAC 80 DA 81 DOPAC/DA 81 Temporal Effects 81 DA 81 Saline 81 Acute (Figures Seven A D) 83 0.2 g/kg EtOH 83 0.5 g/kg EtOH 83 1.0 g/kg EtOH 84 2.0 g/kg EtOH 86 Repeated (Figures Eight A D) 86 0.2 g/kg EtOH 86 0.5 g/kg EtOH 87 1.0 g/kg EtOH 87 2.0 g/kg EtOH 88 Expectancy (Figures Nine A D) 90 0.2 g/kg EtOH 90 0.5 g /kg EtOH 90 1.0 g/kg EtOH 91 2.0 g/kg EtOH 91 Discussion 93 References 99 GENERAL DISCUSSION 104 Conclusions 107 LITERATURE CITED 110 APPENDICE S 126 Appendix A: Stereotaxic Localization of the Developing Nucleus Accumbens Septi 127 Appendix B: The Attentional Model of the Nucleus Accumbens Septi and Addiction 143 ABOUT THE AUTHOR End Page

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iv LIST OF FIGURES ADOLESCENT TRANSITIONS IN NOVELTY PREFERENCE Figure One: Novelty Preference Across Age 4 Familiarization Trials 22 Figure Two: Novelty Preference Across Age 8 Familiarization Trials 26 PLACE CONDITIONING: AGE RELATED CH ANGES IN THE REWARDING AND AVERSIVE EFFECTS OF ALCOHOL Figure One: Activity Levels Across Age 46 Figure Two: Alcohol Place Conditioning 48 EFFECTS OF REPEATED ETHANOL ON BASAL DOPAMINE LEVELS Figure One: Basal DA in Dialysate (nM) Across Age 64 Figure Two: Basal DOPAC/DA Ratio Across Age 65 ETHANOL MEDIATED DOPAMINE RELEASE IN THE NUCLEUS ACCUMBENS SEPTI OF ADOLESCENT ANIMALS Figure One: Basal DOPAC Levels in the NAcc 75 Figure Two: Basal DA Levels in the NAcc 77 Figure Three: Basal DOPAC/DA Levels in the NAcc 78 Figure Four: DOPAC Peak Area Under the Curve Following Ethanol Administration 79 Figure Five: DOPAC Area Under the Curve Following Ethanol Age X Treatment 80 Figure Six: DA Area Under the Curve Following Ethanol Treatment 82 Figure Seven: Effects of Acute EtOH 85 Figure Eight: Effects of Repeated EtOH 89 Figure Nine: Effects of Expected EtOH 92 APPENDICES: Appendix A Figure One: Interaction of Gender and Age on Weight 137 Figure Two: Anterior Coordinates by Weight 138 Figure Three: Lateral Coordinates by Weight 139 Figure Four: Ventral Coordinates by We ight 140

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v LIST OF TABLES ADOLESCENT TRANSITIONS IN NOVELTY PREFERENCE Table One: Figurines 19 APPENDICES: Appendix A Table One: Optimal Coordinates be Mean Weight 137

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vi LIST OF DIAGRAMS APPENDICES: Appendix B Diagram One 153 Diagram Two 154 Diagram Three 155 Diagram Four 157 Diagram Five 158

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vii The Developing Nucleus Accumbens Septi: Suceptibility to Alcohols Effects Rex Montgomery Philp ot ABSTRACT The mesolimbic dopamine (DA) system has been implicated in providing the basis of pleasure, guiding the general mechanism of reinforcement as well as motivation. Support for these roles have grown from neurochemical research in the field of add iction. It is now well known that DA activity increases in the nucleus accumbens septi (NAcc) with exposure to addictive substances. Moreover, pharmacological manipulation of this system produces predictable changes in the administration of drugs of abuse, as well as natural reinforcers. This system is responsive to natural reinforcers and addiction may be the transference of routine mesolimbic function to environmental stimuli predictive of drug administration. The role of the NAcc in addiction specificall y appears to be the facilitation of attention to drug paired stimuli and addiction may be the behavioral manifestation of conditioned NAcc DA reactivity to the presence of drug related stimuli. Although these findings have been reported in adults, few stud ies have focused on adolescence, the time when drug use/abuse begins. Adolescents may be particularly susceptible to addiction when considered in the light of this hypothesis. Recent research has revealed that the mesolimbic system of periadolescent anima ls is undergoing dramatic transition in functional tone. DA receptor and transporter levels are up regulated, synthesis rates are altered, and innervation from prefrontal cortex (PFC),

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viii involved in regulating tonic and phasic DA activity, is increasing. Con sequently, during adolescence there is a dramatic change in tonic DA levels, variations in phasic responses to acute drug administration and alterations in how the system adapts to repeated drug exposure. The present study utilizes the procedures of condit ioned place preference, Novelty preference and in vivo microdialysis to determine how this conditioning process changes during the period of adolescence. The results indicate that adolescents are different from adults not only on behavioral measures assoc iated with drug abuse, but in their neurochemical responsiveness to alcohol, and that these differences are related to a general developmental aspect of adolescence that renders them susceptible to addiction.

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1 INTRODUCTION The Problem of Adolescent Substance Use The initiation of drug use increases dramatically during adolescence. By twelfth grade, approximately 80.3% of U.S. adolescents have used alcohol at some time, an increase from 51.7% for 8 th graders (SAMHSA 2003a) Adult lifetime prevalence data indicate 81.3% have had experience with alcohol, a rate only slightly higher than in adolescence, suggesting that during adolescence most ind ividuals have their first experience with the drug. Importantly, the rate of initiation of alcohol use among those 18 and younger nearly doubled from 1990 to 2000 (SAMHSA 2003b) revealing a decade long rise in the initiation of use in the adolescent population Rece nt statistics reveal that 17.3% of 12 17 year olds are currently using alcohol, an increase from 16.4% in 2000, and the rate of current use increases tremendously during this time (SAMHSA 2003b) A reported 2.6% of 12 year olds have used alcohol within the past mont h. However, by 17 years of age this rate has increased to nearly 35.0% (SAMHSA 2003a) Additionally, 10.6% and 2.5% of those in this age range are binge users or heavy drinkers respectively, and the rate of substance abuse or dependence is 8.9%, and an estimated 1.5 million at this age in need of treatment for an alcohol problem (SAMHSA 2003b) These data indicate both a significant initiation of use in early adolescence, a rapid increase in use and a significant risk of addiction during the adolescent period.

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2 Many reports have indicated a disturbing relationship between age of drug use initiation and subsequent rates of substance related problems (Kandel 1982; Kandel and Logan 1984; Robins and Przybeck 1985; Kandel and Davies 1992; Breslau et al. 1993; Anthony and Petronis 1995; DeWit et al. 2000) The lifetime prevalence of drug dependence problems is significantly higher in those initiating drug use prior to the age of 15, than in those initiating use after this time (Robins and Przybeck 1985) In 2002 17.9% of adults whose first experience with alcohol was prior to 15 years of age were classified wi th abuse of, or dependence on, alcohol as adults; This is in contrast to 3.7% of adults whose first alcohol experience was after the age of 18 (SAMHSA 2003b) These results do not appear related to differences in the total duration of use. Anthony and Petronis (Anthony and Petronis 1995) report that the highest probability of developing drug related problems within one year of initiation is among 15 year olds (19.01%) and is lowest for those 18 or older (9.36%). Additionally, the probability of d eveloping problems within seven years of initial use decreases as a function of age, 68.07% for those 12 years or younger compared to 26.71% for those 18 or older. These statistics can be interpreted as those predisposed to addiction tend to initiate drug use earlier in life, however an alternate interpretation is available. There is some evidence to suggest that personality characteristics associated with substance abuse and addiction (risk taking, impulsivity, novelty seeking) exhibit a developmental traj ectory that peaks in adolescence and that this association may be propelled by the physiological changes of puberty (Martin et al. 2002) This suggests that a biological transition produces behavioral p atterns that increase the probability of initial substance use and that this use

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3 may interfere with the normal developmental trajectory, increasing the probability of substance abuse or dependence. Sensation Seeking and Substance Use The personality trait of sensation seeking has a well established association with drug use initiation, regular drug use and addiction vulnerability (Kilpatrick et al. 1976; Zuckerman 1979; Zuckerman and Neeb 1979; Zuckerman 1983; Pedersen 1991; Bates et al. 1994; Zuckerman 1994; Franques et al. 2000) This trai t is characterized by the seeking of varied, novel, complex, and intense sensations and experiences, and the willingness to take physical, social, legal, financial risks for the sake of such experiences (Zuckerman 1994) Recent reports suggest that sensation seeking can be shaped by experiences (Bardo et al. 1996) and more importantly, that it exhibits a developmental trajectory, with demonstrable elevations in adolescence (Zuckerman 1994) As previously mentioned, several reports indicate an increased risk of substance abuse or addiction cont ingent upon the age of use initiation (Kandel and Logan 1984; Anthony and Petronis 1995; DeWit et al. 2000) Recent studies have demonstrated a relationship between developmental increases in sensation seeking and current and long term drug use in the adolescent population (Martin et al. 2002; Crawford et al. 2003) Research in rodents has attempted to establish the relationship between sensation seeking, drug addiction and developmental neurobiology. Using novelty preference as a measure, researchers have det ermined that novel stimuli activate the same brain regions [the mesolimbic dopamine (DA) system] that mediate the rewarding effects of addictive compounds (Bardo et al. 1996) Attraction to novelty is a quantifiable component of sensation seeking that is useful in drawing parallels between the human condition and

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4 animal models of substance abuse. The preference for novelty has been successfully utilized as an indicator of sensation seeking in humans (Gunnarsdottir et al. 2000) while research in rodents reveals a strong relationship between novelty preference and sens itivity to psychomotor stimulants. Rats with higher novelty preferences: 1) demonstrate greater sensitivity to the locomotor effects of amphetamine; 2) are more easily conditioned with amphetamine in a conditioned place preference (CPP) paradigm (discussed below) and 3) are more sensitive to and more accurately discriminate amphetamine doses (Bevins et al. 1997; Kleba ur and Bardo 1999) supporting the contention that the novelty preference paradigm, like sensation seeking in humans, is a reliable indicator of addiction vulnerability. Conditioned Place Preference and Drug Induced Motivation The CPP procedure allows for the measurement of the motivational capacity of an associated stimulus. In this paradigm subjects are not reinforced for a behavior, but rather receive multiple pairings between a neutral context and an unconditioned stimulus. The tendency to approach or a void the stimulus paired context when give free access is presumed to represent the appetative or aversive capacity of the unconditioned stimulus. Therefore, in the case of drug induced conditioning, the demonstration of a CPP or conditioned place aversion (CPA) represents the motivational capacity of the examined drug (Bardo and Bevins 2000) In adults, associative conditioning procedures induce de monstrable place preferences using natural rewards such as food (Papp 1988; Guyon et al. 1993; Perks and Clifton 1997) or sexu al stimuli (Miller and Baum 1987; Hughes et al. 1990; Mehrara and Baum 1990) Further, a CPP is routinely reported using psychomotor stimulants (cocaine

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5 or amphetamine) as the Unconditioned Stimulus (US) (for review see (Tzschentke 1998) However, in rats there have only been limited reports of ethanol producing a CPP and the effectiveness has been dependent upon pre exposure (Reid et al. 1985; Gauvin and Holloway 1992; Holloway et al. 1992; Bienkowski et al. 1995) extensive pairings (Bozarth 1990) or the use of selectively bred ethanol preferring lines (Colombo et al. 1990) Induction of an ethanol CPP has been more successful in mice {for discussion see (Cunningham et al. 1993) }. However, regardless of species, without additional ma nipulation ethanol typically induces a CPA (Stewart and Grupp 1986, 1989; Gauvin and Hollo way 1992; Holloway et al. 1992; Schechter and Krimmer 1992) or has no effect (Asin et al. 1985) on place preference. Neurochemistry, Reward and Drugs of Abuse A common feature of all drugs that induce a CPP is the ability to alter neurochemical activity in the midbrain and limbic system (White 1996; Koob and Nestler 1997; Leshner and Koob 1999) In the mid 1950's researchers James Olds and Peter Milner discovered that electrical stimulation of the medial forebrain bundle (MFB) produced a CPP, suggesting that ac tivation of these projection fibers was rewarding (Olds and Milner 1954) This discovery implicated the neurot ransmitter systems that comprise the MFB (dopamine, norepinephrine, serotonin) as central to the reward process (Fouriezos et al. 1978; Speciale et al. 1978; Wise 1978; Olds and Fobes 1981) The use of histofluorescence techniq ues has revealed a strong correspondence between brain stimulation reward sites and dopamine (DA) systems that pass through the MFB (Dahlstrom and Fuxe 1964; Fuxe 1965; Arbuthnott et al. 1970; Wise 1981) suggesting that DA systems are the critical components for the reward mediating capacity of the

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6 MFB. Substantial evidence supports the notion that DAergic systems mediate the motivational component of hedonic behaviors such as drinking, eating and sexual activity (Heffner et al. 1977; Wise et al. 1978; Zigmond et al. 1980; Xenakis and Sclafani 1981; Geary and Smith 1985; Hoebel 1985; Schneider et al. 1986; Blackburn et al. 1987; Smith and Schneider 1988; Weatherford et al. 1990; Tyrka and Smith 1993; Hsiao and Chen 1995; Hsiao and Smith 1995) Additionally, systemic or central administration of DA agonists can produce a CPP and DA agonists and antagonists can alter operant behaviors in a fashion indicative of altered reinfo rcement (Yokel and Wise 1975; Phillips and Fibiger 1978; Gallistel and Karras 1984; Koob and Hubner 1988) These data suggest a central role of DA in the administration of abusable drugs, specifically that animals can monitor drug induced states and alterations in drug efficacy related to DA systems, modifying behavioral output to compensate for neurochemical changes. One DA system implicated in these motivated behaviors is the mesocorticolimbic pathway, ori ginating in the ventral tegmental area (VTA) and projecting to the limbic system (e.g. nucleus accumbens septi (NAcc), amygdala, hippocampus, septum, olfactory bulb, bed nucleus of the stria terminalis) and prefrontal cortex (Le Moal and Simon 1991) T he mesolimbic structure most frequently implicated in mediating these DAergic processes is the NAcc, which receives DAergic input from the VTA and constitutes one of the projection areas of the MFB. Manipulations that directly stimulate the DA receptors in the NAcc reinforce many behaviors (Olds and Fobes 1981) Electrical stimulation of the NAcc itself, or any of the pathways which result in increased DA efflux within the NAcc, produces behavioral reinforcement and animals will lever press for this stimulation (Arbuthnott et al. 1970; Crow 1971; Anlezark et al. 1972; Crow

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7 1972a, 1972b; Anlezark et al. 1973; Crow 1973; Anlezark et al. 1974; Anlezark et al. 1975; Ranaldi and Beninger 1994) Additionally, injections of DA agonists into the NAcc have rewarding effects producing a CPP (Hoebel et al. 1983) This region also appears to be directly involved in the reinforcing effects of cocaine (Moghaddam and Bunney 1989) and the administration of numerous drugs, including alcohol, all elicit a significant increase in DA levels in the NAcc (Phillips et al. 1983; Koob 1992a, 1992b; Koob et al. 1994; Koob 1996; Phillips and Shen 1996; Koob 1999, 2000) These data implicate dopaminergic (DAergic) activity, specifically in the NAcc, as critical in the process of drug induced reinforcement and possible a ddiction and implicates DA and the NAcc as key areas for investigation regarding the unique profiles observed in adolescent drug use. Adolescence, NP, CPP and Neurochemistry Adolescence in the rodent has been defined using various factors indicative of de velopmental transition in human adolescents. These factors include changes in behavioral patterns, in hormonal patterns, and/or in primary sexual characteristics. In a comprehensive review (Spear 2000) argues that adole scence cannot be defined based solely on the characteristics we associate with puberty and sexual maturation, but that adolescence is a period of soft events that should be viewed as a period of transitions that cannot be clearly delineated. Using the ap pearance of growth spurt, pruning of excitiatory synapses as well as unique behavioral transitions in the rat (increased peer interaction and play; exploratory behavior in the wild) Spear defines adolescence broadly from postnatal day (PND) 28 to PND 42, w ith the acknowledgement that in males some traits may appear as late as PND 55 (Spear 2000) Other reviewers report similar broad classifications defined by the appearance of mature hormonal cycling (PND 28 30) and

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8 the subsequent appearance of reproductive maturity (as late as PND 60 in males) (Smith 2003) Within this broad range some researchers have identified t he categories of early (PND 21 34), mid (PND 34 46) and late (PND 46 59) adolescence centered around the appearance of puberty (between PND 33 44) and bracketed by weaning (PND 21) and reproductive maturity (PND 59) (Tirelli et al. 2003) Using this temporal frame to study adolescence, there appears to be parallel developmental trajectories in the NP of rodents and sensation seeking in human adolescents. Adolescent mice demonstrate greater novelty preference (Adriani et al. 1998) and human adolescents exhibit elevated sensation seeking scores (Zuckerman 1994) than adult counterparts. The presence of these developmental patterns in laboratory animals suggest a biologically driven developmental trajectory underlying sensation seeking that is in dicative of increased risk of drug use and subsequent addiction in the adolescent population. A recent report by Martin et al (Martin et al. 2002) supports the adolescent transition and biological basis o f sensation seeking in humans. Programmed changes in the central nervous system structures involved in reward and reinforcement may underlie this developmental pattern in NP, however the use of CPP to measure transitions in reward in the adolescent has bee n a neglected area. Amphetamine and cocaine have been shown to induce a CPP in animals as young as 3 weeks of age and into adolescence (Laviola et al. 1992; Cirulli and Laviola 2000; T irelli et al. 2003; Schramm Sapyta et al. 2004) Additionally, nicotine has been demonstrated to induce a CPP in adolescent animals (Vastola et al. 2002) or alleviate an aversion These studies substantiate the effectiveness of the paradigm i n evaluating drug conditioning in the

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9 developing animal. However, prior to this project no published studies have demonstrated ethanol mediated place conditioning in the adolescent animal. Research has examined the developmental patterns of DA (Anderson et al. 1997) DA receptors (Hedner and Lundborg 1985; Andersen and Gazzara 1994; Andersen and Teicher 2000; Andersen et al. 2002) and transporters ( Coulter et al. 1996, 1997) through adolescence. Neurochemically, basal DA synthesis in the NAcc are lower in PND 30 than PND 40 rats and turnover rates for PND30 animals is less than reported in adults (Anderson et al. 1997) Research on DA receptor populations indicates a pattern of overproduction and pruning that occurs across adolescence in a sex specific manner (Teicher et al. 1995; Andersen et al. 1997) This pattern is true in humans as well (Seeman et al. 1987) The density of D1, D2, and D4 receptors in the NAcc increases to a peak at PND 28, then declin es significantly to adult levels at PND 60 (Tarazi and Baldessarini 2000) Furthermore, D3 receptor numbers appear to increase monotonically, with some reports finding adult levels at weaning (Demotes Mainard et al. 1996) but others finding D3 levels in weanlings far lower than adults (Stanwood et al. 1997) In conjunction with receptor density changes, D1 stimulatory and D2 inhibitory effects on adenylyl cyclase production are less apparent in adolescence than adults (Andersen and Teicher 2000) Parallel to these changes, DA transporter levels are undergoing developmental changes, increasing in concentration in the NAcc to adult levels through adolescence (Coulter et al. 1996, 1997) Summary The data are clear that the adolescent is unique with respect to drug use tendencies and vulnerability to addiction. The characteristic of sensation seeking has long been

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10 reported as an indicator of increased risk for addiction and there is a developmental transition in this trait, peaking in adolescence. This suggests the possibility of a developmental transition in the rewarding or reinforcing efficacy of drugs of abuse that peaks in adolescence. The use of CPP procedures in adolescent animals has provided some evidence, albeit limited, that adolescents find abused substances more rewarding. This may be due to the adolescent development of the mesolimbic DA syste m, the NAcc and brain regions that modulate its activity. Significant evidence indicates that the NAcc is a central player in the reward response to drugs of abuse and a growing body of literature indicates that these regions are neurochemically distinct i n the adolescent. Spear (2000) has suggested that this neurochemical transition represents a developmental trajectory within which the adolescent animal begins to explore its potential and develop independence. It is suggested here that because drugs of ab use act through this developing system, that exposure to alcohol during this time can alter the programmed pattern of development, rendering the individual at increased risk to develop subsequent alcohol related problems. The present study utilized the met hods of NP, CPP and microdialysis to evaluate adolescent transitions in sensation seeking, ethanol reward and ethanol induced effects, acute, repeated and expected, on reward related behavior and neurochemistry.

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ADOLESCENT TRANSITIO NS IN NOVELTY PREFER ENCE Abstract Recent research has revealed a strong relationship between a rodent's preference for novelty and sensitivity to psychomotor stimulants. Animals with exhibiting a high response to novelty are more easily conditioned with amphetamine in a co nditioned place preference (CPP) paradigm, and are more sensitive to and more accurately discriminate amphetamine doses. In humans, novelty preference (NP) is used as an indicator of sensation seeking which is strongly correlated with addiction vulnerabil ity. Evidence suggests that preference for novelty and drug taking behaviors are mediated by the mesolimbic dopamine (DA) system, specifically the nucleus accumbens septi (NAcc). During adolescence there are substantial developmental changes in the mesoli mbic system, with significant over production and pruning of DA receptors, changes in DA synthesis, increases in DA transporter levels, and differential activation of DA regulated second messenger systems. The behavioral measure of NP appears to be an ind icator of drug sensitivity. Thus, the present study used a playground maze procedure to measure changes in NP across age. The present findings demonstrate a significant preference for novel stimuli in developing animals. Preadolescent, postnatal day 24 (P ND 24) animals exhibited a significant preference for novelty that was not present in early adolescent (PND 34), or early adult animals (PND 59). Late adolescent (PND 44) animals exhibited a significant aversion to novel stimuli. An increase in habituation trials resulted in a

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12 similar pattern of reduced NP into adolescence, however the increased exposures attenuated the late adolescent aversion and resulted in a preference for novelty in adult animals. The data indicate strong behavioral differences in NP b etween early adolescence and adulthood that may be related to a developmental increase in contextual regulation of behavior.

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13 Introduction Drug use begins early in development. For example, approximately 80.3% of U.S. adolescents have used alcohol by 12 t h grade, an increase from 51.7% for 8 th graders (SAMHSA 2003a) Adult lifetime prevalence data indicate 81.3% have had experience with alcohol, a rate only slightly higher than in adolescence, suggesting that during adolescence most individuals have their first experience with the drug. Importantly, the rate of initiation of alcohol use among the 12 17 age group increased from 111.0/1,000 potential new users to 158.8/1,000 from 1991 to 1996 (Johnston et al. 2002) revealing a recent rise in the initiation of use in the adolescent population The numbers for use and initiati on are similar for illicit substances. For example, 8.6% of 12 th graders have used cocaine, an increase from 4.5% for 8 th graders (Johnston et al. 2002) These data in adolescents compare to a lifetime prevalence of 10.6% in adults (SAMHSA 2003b) suggesting that most users initially experience cocaine well before adulthood. T he age specific rate of new use of cocaine for ages 12 17 has climbed steadily from 1.2 in 1992 to 5.6 in 1997 (SAMHSA 2003b) again indicating a trend toward adolescent initiation in drug use The National Household Survey (SAMHSA 2003b) for the age group 18 25 indicates that 48.1% have experienced some illicit drug in their lifetime. Further, the Monitoring the Future Study (Johnston et al. 2002) a detailed study of youth trends, indicates that by 12 th grade 54.1% have used some form of illicit drug. Additionally, among youths ages 12 13, 2.9% were current illicit drug users with the highest rates found among young people ages 16 17 (16.4%), and ages 18 20 (19.9%) (Johnston et al. 2002) These data indicate not only a significant i nitiation of use in early adolescence but also a rapid increase in use during the adolescent period. Together, these data

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14 demonstrate the need for critical study into the dynamics of drug exposure and abuse potential among the adolescent population. Arnet t (Arnett 1999) has shown a relative tendency towards sensation seeking in adolescence, a factor that Zuckerman associates with increased likelihood of risk taking behaviors (Zuckerman 1986) including drug use or initiation. Measures of sensation seeking are highly correlated with approach to novelty or NP in humans (McCourt et al. 1993) Given these findings, preference for novelty appears to be a valid measure of risk taking behavior probability, specifically, drug use initiation. This assumption has been born out in the animal literature. Numerous studies have demonstrated a strong correlation between behavioral reactivity to novel stimuli and both the reinforcing efficacy of psychomotor stimulants and self administration rates in animals. Specifically, (Klebaur and Bardo 1999) have shown that novelty seeking behavior in rats is r elated to the reinforcing efficacy of psychomotor stimulants, with high responders for novelty displaying higher amphetamine induced conditioned place preference (CPP). Additionally, self administration probabilities for amphetamine are directly related t o behavioral reactivity to a novel stimulus. In this study, animals that exhibited greater motor activity in the presence of novelty established self administration patterns more readily (Piazza et al. 1990) These data suggest a strong relationship between sensation seeking, novelty seeking, drug self administration and drug related reinforcement, relationships which may be mediated by the functions of the mesolimbic dopamine (DA) system. There is considerable evidence that the mesolimbic DA system is involved in the establishment and maintenance of a range of behaviors. Initial studies implicating the

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15 nucleus accumbens septi (NAcc) date back to (Olds and Milner 1954) who disc overed that electrical stimulation of the medial forebrain bundle could produce a CPP. Further research specifically implicated DA efflux in the NAcc as crucial to this process. Intracranial self stimulation of the ventral tegmental area (VTA) supports l ever pressing behavior in rats, a behavior mediated by dopaminergic output in the NAcc (Mogenson et al. 1980) Specifically, injections of DA antagonists in the NAcc attenuates or blocks intracranial self stimulation behavior These data indicate that accumbal DA is critical in the maintenance of reinforced behavior and may be the underlying source of behavioral activation in novel situations. Studies with drugs of abuse parallel these findings. Drugs of abuse have a common ability to induce dopaminergic activity in the mesocorticolimbic system (Koob 1992) a quality that, given the role of the NAcc in reinforcement, suggests a strong possibility for a role in addictive behavior. Specifically, rats will readily self administer drugs of abuse directly into the NAcc and self admi nistration behavior can be altered by co injecting a dopaminergic antagonist into the NAcc (Caine et al. 1995) Additionally, infusing amphetamine into the NAcc produces a CPP suggesting elevations of accumbal DA are reinforcing (McBride et al. 1999) It is important t o note that novel stimuli have been shown to elevate DA in the NAcc. Rebec et.al. (Rebec et al. 1997) have reported enhanced DA efflux in the NAcc during exposure to a novel e nvironment. Specifically, in a familiar environment, animals exhibited lower levels of NAcc DA efflux in comparison to dopaminergic activity in a novel environment. Additionally, lesioning the NAcc attenuated locomotor activity in response to a novel sti mulus, indicating a relationship between the NAcc and behavioral

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16 reactivity to novelty (Bardo et al. 1996) Injections of 6 OHDA into the NAcc reduced both mesolimbic DA and subsequent NP. This suggests that dopaminergic activity of the mesolimbic system mediates the behavioral aspects of NP in rats. Further, microdialysis studies have shown a direct correlation between reaction to novelty and amount of "drug stimulated DA release in the NAcc" (Bradberry et al. 1991) Moreover, high locomotor responsivity to a novel environment has been correlated with enhanced amphetamine induced dopaminergic activity in the NAcc (Hooks and Kalivas 1995) Therefore, it appears there is a strong relationship between the behavioral reac tivity to novelty and the neurochemical effects of drugs and it appears that these processes share a common neural substrate. It is clear that adolescence is a period of tremendous experimentation and risk taking (Spe ar 2000) a pattern that, in the arena of drug abuse, manifests itself as first time drug use and potentially drug abuse (Zuckerman 1974) It is likely that neurophysiological changes in the mesolimbic system during development may mediate the initiation of drug use and potentiate the likelihood of abuse during adolescence. Specifically, it is clear that in bot h human and rodent populations the mesolimbic DA systems are undergoing tremendous transition. For example, basal DA synthesis in the NAcc is lower in postnatal day 30 (PND 30) than PND 40 rats and turnover rates for PND 30 animals are less than those rep orted in adults (Andersen et al. 1997) Receptor populations also are in flux during development, with a pattern of overproduction and pruning that occurs across adol escence in a sex specific manner (Teicher et al. 1995; Andersen et al. 1997; Andersen and Teicher 2000) Males exhibit greater levels across age and greater over production of D1 and D2 receptor types than

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17 females. This pattern is similar in humans as well (Seeman et al. 1987) In rats, the d ensity of D1, D2, and D4 receptors in the NAcc increase and reach peak levels at PND 28, and then decline significantly to adult levels at PND 60 (Tarazi and Baldessarini 2000) Additionally, D3 receptor numbers appear to increase monotonically, with some reports finding adult levels a t weaning (i.e., PND 21) (Demotes Mainard et al. 1996) but others finding D3 levels in weanlings far lower than those observed in adults (Stanwood et al. 1997) In conjunction with receptor de nsity changes, D1 stimulatory and D2 inhibitory effects on adenylyl cyclase production are less apparent in adolescence than in adults (Andersen and Teicher 1999) DA transporter levels are also undergoing substantial change, increasing in concentration in the NAcc to adult levels through adolesce nce (Coulter et al. 1996, 1997) This dynamic transition during adolescence suggests that processes that are mediated by the mesolimbic DA system are unlikely to manifest themselves similarly in adults and adolescents. Moreover, across adolescence there may be tremendous transitions in reactivity to stimuli (e.g., novel stimuli or drugs) that act on these systems. As previously mentioned, novelty and novel stimuli produce profiles in the NAcc that are simil ar to those caused by drugs of abuse. Additionally, behavioral measures of novelty responsiveness has a strong relationship with behavioral measures of drug conditionability and underlying neurochemical responsivity to drugs of abuse. Therefore, a prefere nce for novelty across adolescence can be viewed as an indicator of the potential for abuse and addiction liability if use is initiated during this time. In the present studies, preferences for novelty were measured across the periadolescent period using the

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18 playground maze paradigm developed by Nicholls et al. (Kleb aur and Bardo 1999) to determine potential differences in developing adolescent animals. Experiment One Methods Subjects Fourty Three Sprague Dawley (Zivic Miller Laboratories) rat pups weighing 60 300g at the time of testing were used as subjects in the se experiments. No more than one male and one female per litter were used in a given condition (pups were derived from 10 separate liters). Pups were sexed and culled to 10 pups per litter on postnatal day 1 (PND 1). Pups remained housed with their resp ective dams in a temperature and humidity controlled vivarium on a 12:12h light: dark cycle (07:00 h/19:00 h) until PND 21, pups were weaned and individually housed. Apparatus The NP apparatus and procedure were adapted from Nicholls et al. (Nicholls et al. 1992) Animals were tested on a white plastic circular platform (216 cm in diameter) standing 70 cm from the ground. Eight black circles (28 cm in diameter) were evenly spaced outlining the perimeter of the tabletop. Each black circle was situated 30 cm away from the edge of the tabletop and 55 cm from the center. Eight different plastic figurines were adhered to the middle of each black circle with Velcro (see Table 1 for a list of the 10 figurines). The figurine's average size ranged from about 5x2x2 cm to 2x2x7 cm. A video camera was hung directly over the table to record the animal's behavior for later scoring (see analysis).

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19 Table One: Figurines Figurine Number Figurine Identity 1 Baseball Player 2 Race Car 3 Coral 4 Whistle 5 Yellow Bird 6 Bottle 7 Chair 8 Salt Shaker 9 Dolphin 10 Scuba Diver Table One : Listing of object number and corresponding characteristics Training Rats were handled on either PND 21, 31, 41 or 56 for one three minute session to minimize stress levels due to handling. For the next three consecutive days (PND 22 24, 32 34, 42 4 4 and 57 59) each rat was placed on the playground maze facing away from the experimenter and allowed to freely explore the novel environment for three minutes. The experimenter left the room during the three minute session. The table and figurines were w iped down with alcohol between each session to control for olfactory cues. Each day, the eight figurines were randomly distributed among the black circles. Behavioral Testing On the fourth consecutive day, rats were exposed to a familiarization trial. A nimals were be placed in the familiar apparatus for 3 min, removed for 1 min while a novel object was placed instead of a random familiar object. Rats were again placed on

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20 the table facing away from both the experimenter and their novel object for three m inutes to freely explore the familiar environment. The novel object was randomized for each litter; however, one male and one female per litter received the same object as novel. Analysis A video recorder hanging above the table taped behavior during tr aining and testing sessions. The length of time each rat spent investigating each figurine was recorded (i.e., when the animal's head was within the black circle). Number of entries and duration of entries were recorded. Four separate 2 (Sex) x 4 (Age) x 4 (Familiarization day) x 8 (Zone or Object) analyses were performed to determine any baseline differences in time or entries in a given zone and time or entries for a given object. Analysis of NP used a 2 (Sex) x 4 (Age) x 8 (Zone) ANOVA with preferenc e score (adjusted percent time in novel zone minus mean adjusted percent time in familiar zones) as the dependent variable. Fisher Protected LSD tests were used to isolate significant effects. Results Baseline Analysis Analyzing for baseline pr eference revealed significant main effects of Age, F (3, 126) = 16.921, p < 0.05, and Zone (time per zone) F (7, 882) = 5.901, p< 0.05. Subsequent post hoc analyses of Age using Fisher's PLSD revealed significant differences in mean zone time between ages PND 25 (x = 9.835), 35 (x = 6.539), 45 (x = 6.468) and 60 (x = 3.529), between PND 35 and 60, and between PND 45 and 60. Subsequent planned comparisons of Zone indicated there were significant differences in baseline preference between locations, with zo nes 1, 2 and 8 being preferred over other zones. Although not statistically significant, there was a trend for an Age X Zone

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21 interaction, F (21, 882) = 1.555, p = 0.0532, with young animals exhibiting larger zone preferences than adults. Analyzing for th e number of entries into each zone revealed significant main effects for Age, F (3, 126) = 16.722, p < 0.05 and Zone, F (7, 882) = 11.367, p< 0.05. Subsequent post hoc analyses of Age using Fisher's Protected LSD indicated significant differences in zone entries between ages PND 25 (x = 3.800) and 60 (x = 2.381), between PND 35 (x = 4.119) and 45 (x = 3.289) or 60, and between PND 45 and PND 60. Subsequent planned comparisons of Zone indicated there were significant differences in the number of entries pe r zone, with zones 1, 2 and 8 being entered more frequently. There was also a significant Zone X Age interaction, F (21, 882) = 1.635, p< 0.05, with adult animals generally exhibiting fewer entries than younger animals with little preference for any speci fic zone. Analysis of time spent at each object revealed significant main effects of Age, F (3, 126) = 15.783, p < 0.05 and Object, F (9, 1134) = 7.781, p< 0.05. Subsequent post hoc analyses of Age using Fisher's PLSD indicated significant differences in mean time per object between ages PND 25 (x = 9.278) 35 (x = 6.382), 45 (x = 6.521) or 60 (x = 4.340), between PND 35 and 60, and between PND 45 and 60. Subsequent planned comparisons of Object indicated there were significant differences between objects with objects 5, 6 and 7 being preferred over other objects. Analysis of the number of entries for each object revealed significant main effects of Age, F (3, 126) = 16.189, p < 0.05 and Object, F (9, 1134) = 8.40, p< 0.05. Subsequent post hoc analyses of Age indicated significant differences in object entries between ages PND 25 (x = 3.769) and 60 (x = 2.663), between PND 35 (x = 4.020) and

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22 45 (x = 3.403) or 60, and between PND 45 and 60. Subsequent planned comparisons of Object indicated there were si gnificant differences between objects, with objects 5, 6 and 7 having more entries than other objects. There was also a significant Object X Age interaction, F (27, 1134) = 1.963, p < 0.05, with animals at every age showing different object preferences. There was also a significant Object X Age X Familiarization Day interaction, F (81, 1134) = 1.613, p < 0.05, with animals at every age showing different object preferences on the four different familiarization days. Figure One : Developmental differences in novelty preference as a function of age with 4 habituation trials. Adjusted preference score represents the total time spent with the novel object/zone minus the mean time spent with the familiar objects/zones with a correction for age related basal dif ferences in zone preference. Preadolescent animals (PND 24) exhibit a significant preference for novelty, while late adolescent (PND 44) animals exhibit a significant aversion. Additionally, late adolescent and adult animals (PND 44, 59) are significantly different from preadolescent animals.

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23 Novelty Preference Baseline object preference across familiarization days was mediated by zone preferences across age. Therefore, only 'novel object zone preference' was used for analysis of NP to allow for baseline correction. To eliminate any zone bias due to baseline preferences, scores for each zone were adjusted by the baseline zone preference for that age. This adjusted score was used to compute NP. The mean adjusted time spent in the familiar object zones wa s subtracted from the adjusted time spent in the novel object zone to generate the preference score. Using this formula there was a significant main effect for Age, F (3, 39) = 6.165, p < 0.05. Subsequent post hoc analyses of age using Fisher's PLSD reve aled significant differences between ages PND 24 (x = 26.74) and 44 (x = 7.123) or PND 59 (x = 0.3624). Analysis for preference or aversion revealed a trend toward NP (p = 0.0674) in PND 24 animals and a significant novelty aversion [t(13) = 4.050, p < 0 .05] in PND 44 animals (See Figure One). Discussion Postnatal day 24 animals demonstrated greater exploration of objects than any other age, while adolescent animals (i.e., PND 34,44) explored the objects more than the adults did. All animals entered mor e often and spent more time in zones 1, 2 and 8. These zones were near objects in the room that were in close visual proximity to the playground maze. NP scores were calculated by correcting for this baseline preference. In the original study by Nicholl s et al. (Nicholls et al. 1992) no zone preferences were observed, howev er that study used adult animals only. It is interesting to note that this effect was less pronounced in adult animals in the present study. Both preadolescent (i.e., PND 25) and adolescent (i.e., PND 35, 45) animals entered zones more frequently than

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24 adu lt animals on training days. Examining the responses to objects on training days showed that 25 day olds explored more objects than all other ages while adolescent animals explored more than adult animals did. To verify and extend the results of this stud y a visual barrier was employed to eliminate the influence of extra apparatus visual stimuli on behavior. Additionally, consideration was given to the influence of rate of habituation on the outcome of Experiment One. Given the short temporal parameters of this study it is possible that the results were affected by an age related deficit in acquisition rates. In short, animals that were not familiarized with 4 exposures would not react to a new stimulus in the same fashion as animals that have completely ha bituated. This result, however, would not constitute a lack of NP. Therefore, to reduce stress during the first exposure to the playground maze, the number of handling sessions prior to familiarization was increased to 6 sessions over 3 days. Additionally, the number of daily exposures to the apparatus was doubled, increasing the total number of familiarization trials to 8 prior to testing. The reduction of stress on trial one due to increased handling and the increased number of trials should allow suffici ent time to fully habituate to the objects. Experiment Two Methods Subjects Eighty two Sprague Dawley (Zivic Miller Laboratories) rat pups weighing 60 300g at the time of testing were used as subjects in these experiments. Animal care and environmental co nditions were identical to those in Experiment One. Apparatus The NP apparatus was modified from Experiment One to include a white vinyl curtain that encircled the NP platform to prevent any potential influence of external

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25 visual stimuli on the distributi on of behavior within the open field. All other conditions were identical to Experiment One. Training To test the hypothesis that reduced preference for novelty in adolescence was due to a lack of habituation the number of object familiarization trials was doubled. Rats were handled beginning on either PND 18, 28, 38 or 53 for three minute sessions, twice a day (9:00 hr and 15:00 hr) for three consecutive days, to minimize stress levels due to handling. Over the next four days (PND 21 24, 31 34, 41 44 and 56 59) each rat was placed in the playground maze, twice per day (9:00 hr and 15:00 hr), facing away from the experimenter and allowed to freely explore the novel environment for three minutes. The table and figurines were wiped down with alcohol between each session to control for olfactory cues. Each day, the eight figurines were randomly distributed among the black circles. The experimenter left the room during the three minute session. Behavioral Testing On the afternoon of the fourth day, rats wer e exposed to a familiarization trial. Animals were placed in the familiar apparatus for 3 min, removed for 1 min while a novel object replaced a random familiar object. Rats were again placed on the table facing away from both the experimenter and their novel object for three minutes to freely explore the familiar environment. The novel object was randomized for each litter; however, one male and one female per litter received the same object as novel. Analysis All data were quantified using a behavior al tracking system (Noldus Ethovision) coupled to a digital video camera suspended above the playground maze. The length of time each rat spent investigating each figurine was recorded (i.e., when the animal's head

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26 was within the black circle). Four separ ate 2 (Sex) x 4 (Age) x (Familiarization trial) x 8 (Zone or Object) analyses were performed to determine any baseline differences in time in a given zone and with a given object. Analysis of NP used a 4 (Age) x 8 (Zone) ANOVA of preference score (adjus ted percent time in novel zone minus mean adjusted percent time in familiar zones). Fisher Protected LSD tests were used to isolate significant effects. Results Baseline Analysis No differences in basal zone or object preference were observed using the enclosed playground maze. Figure Two : Developmental differences in novelty preference as a function of age with 8 habituation trials. Adjusted preference score represents the total time spent with the novel object/zone minus the mean time spent with the f amiliar objects/zones. Preadolescent (PND 24) and early adult (PND 59) animals exhibit a significant preference for novelty, while early and late adolescent and animals (PND 34, 44) demonstrate no response to novelty.

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27 Novelty Preference There was a signi ficant main effect for Age, F (3, 71) = 3.976, p < 0.05. Subsequent post hoc analyses of age using Fisher's PLSD revealed significant differences between ages PND 24 (x = 15.33) and PND 35 (x = 0.639) or PND 45 (x = 0.887). Analysis for NP revealed a sign ificant preference in PND 24 [t(21) = 2.233, p < 0.05] and 59 animals [t(21) = 2.344, p < 0.05] (See Figure Two). Discussion The addition of a visual barrier successfully eliminated basal zone preferences within the playground maze. The findings are consi stent with the results from Experiment One, indicating a reduction in NP in adolescence compared to preadolescent counterparts. It was hypothesized that the absence of significant NP in adolescents and adults in Experiment One resulted from limited habitua tion trials and that with more thorough familiarization a clear response to novelty would be manifest. However, the overall pattern varied with increased habituation. Preadolescent (PND 24) and early adolescent (PND 34) animals demonstrated a reduction, wh ile late adolescent (PND 44) and early adult (PND 59) animals demonstrated an increase, in novelty preference scores with increased habituation. Although Experiment Two was designed to examine the influence of habituation on the demonstration of NP, the re sults are suggestive of an elevation in experimental anxiety through adolescence and into adulthood. The presence of additional handling sessions as well as increased experience with the experimental context affected preadolescent animals minimally. Althou gh PND 24 animals did not demonstrate a statistically significant NP with 4 habituation trials, the absolute preference score was larger than 8 trial counterparts and did not differ statistically. NP in PND 34 animals also

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28 did not differ with increased hab ituation trials, however, increasing the number of trials did result in a difference between PND 24 and PND 34 animals with early adolescent demonstrating significantly lower scores. Interestingly, the addition of habituation trials served to shift scores toward a preference in late adolescent and early adult animals, alleviating an aversion in the former and inducing a preference in the latter. NP has traditionally been considered as a behavioral correlate to sensation seeking in the human population and a fundamental component of this trait is risk taking or reduced perceived risk. These results suggest a developmental increase in perceived risk that hinders the demonstration of NP with age and this can be attenuated with experience. General Discussion The present results demonstrate an age specific transition in the effectiveness of novel stimuli to attract and sustain exploratory behaviors. Early after weaning, preadolescent pups have a significant preference for new stimuli, and this response to novelty changes in early adolescence. By late adolescence and on to early adulthood there is a dramatic drop in NP with relatively few familiarization trials. In fact, late adolescent animals exhibit a novelty induced aversion (i.e., neophobia) relative to famili ar stimuli. These patterns suggest that there is a greater likelihood of exploration and experimentation with unfamiliar objects, environments and conditions in early adolescence as compared to late adolescence or adulthood. From a developmental perspect ive, a shift from NP to aversion has been explained as the typical functional adolescent transition during development. Facilitation of active exploring of the environment in the physically capable being increases environmental skills and abilities that a re necessary for and/or increase the probability of

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29 competitive success and survival (Spear 2000) However, once sufficient time has passed, and presumably sufficient learning has occurred, it is detrimental to enter n ew situations rather than remain in more comfortable domains, given that in the experienced animal such situations are more likely to present higher survival risk than contribute to competitive benefit. Such behavioral changes are likely mediated by devel opmental transitions in a number of brain structures known to mediate motivational and behavioral processes. Of specific interest to this research are the NAcc, and the prefrontal cortex (PFC). These structures have been repeatedly demonstrated to mediat e the initiation and maintenance of a range of behaviors. Further, substantial evidence is emerging that these structures are anatomically and functionally dissimilar in adolescents and adults, and that the transition process of these structures appears to occur during the adolescent period. For example, the PFC declines in volume during adolescence (Jernigan et al. 1991) exhibits reduced glutamatergic input (Virgili et al. 1990) and increased DA input (Rosenberg and Lewis 1994, 1995; Lewis et al. 1998) Further, as mentioned previously there are substantial changes occurring in these dopaminergic systems as well (Andersen and Gazzara 1996; Coulter et al. 1996; Andersen et al. 1997; Stanwood et al. 1997) The observed shift in NP for during adolescence may be directly related to the developmental transitions r eported in these structures. There appears to be a transition in the regulatory role of the PFC through adolescence, with early periadolescent behavior being predominantly impulsive, or affectively regulated, while later adolescent behavior appears to be more contextually regulated (Spear 2000) This behavioral transition may result from a shift in the ability of the PFC or amygdala to modulate activity in the NAcc.

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30 Spear has suggested that elevated DA in the PFC i n the preadolescent may result in disinhibition of amygdalar inputs to the NAcc while simultaneously inhibiting medial PFC input to the NAcc. The net result of such a shift in functional regulation of NAcc activity is reduced contextual and increased emot ional regulation of NAcc activity given that the amygdala appears to be involved in the processing of emotionally salient events. However, as the neurochemical projections to the PFC and PFC interconnections with the NAcc develop during adolescence there is increased regulation of attention and behavior to contextually important, rather than affectively salient stimuli [for discussion see Spear (2000)]. The relationship between behaviors like novelty seeking and drug use (Klebaur and Bardo 1999) may be mediated by these developing structures and developmental changes in novelty seeking may not only indicate the likelihood of drug use but reflect ongoing changes in the functional interconnections of the mesocorticolimbic system. Sustained drug use during this transitional period may result in a greater probability of addiction later in life by effectively altering the course of development of these circuits, sustaining a more affectively regulated motivational system. Therefore, these data suggest a transitional period in neural development in which the initiation of drug use is both more likely and potentially more costly. Such outcomes are born out in the human literature given that addiction is twice as likely if use starts before the age of 15, than if initiation occurs after 18 years of age. Further, these data indicate that the likelihood of addiction is not mediated by the length of use, but rather by when use was initiated (Anthony and Petronis 1995) Interestingly, the estimated probability of future addiction exhibits it steepest asce nt across 15 18 years of age, regardless of initial age of initiation,

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31 suggesting some critical component to the combination of drug use and age during this developmental period. The present data provide a measure of evidence for a distinct biological tran sition in NP during adolescence. Prior evidence has shown that sensation seeking and novelty seeking are predictors of substance abuse liability in humans (Zuckerman 1986; McCourt et al. 1993) This predictive relationship has also been demonstrated in adult rodents (Klebaur and Bardo 1999) Moreover, novelty seeking and substance abuse share some underlying neural substrates (Bradberry et al. 1991; Bardo et al. 1996) Given these findings, it seems likely that changes in novelty preference resulting from ongoing developmental processes can provide a simple behavioral measure for increased risk of addicti on as a function of age. Using procedures such as this to study development and the processes involved in adolescence and drug abuse is a critical area of research.

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32 References Andersen, S. L. and R. A. Gazzara (1996). Effects of ( ) s ulpiride on dopamine release in striatum of developing rats: Degree of depolarization influences responsiveness. J Neurochem 67 (5): 1931 7. Andersen, S. L., M. Rutstein, J. M. Benzo, J. C. Hostetter and M. H. Teicher (1997). Sex differences in dopamine rec eptor overproduction and elimination. Neuroreport 8 (6): 1495 8. Andersen, S. L. and M. H. Teicher (1999). Cyclic adenosine monophosphate (camp) changes dramatically across periadolescence and region. Society for Neuroscience Abstracts 25 : 1471. Andersen, S L. and M. H. Teicher (2000). Sex differences in dopamine receptors and their relevance to adhd. Neurosci Biobehav Rev 24 (1): 137 41. Anthony, J. C. and K. R. Petronis (1995). Early onset drug use and risk of later drug problems. Drug Alcohol Depend 40 (1) : 9 15. Arnett, J. J. (1999). Adolescent storm and stress, reconsidered. Am Psychol 54 (5): 317 26. Bardo, M. T., R. L. Donohew and N. G. Harrington (1996). Psychobiology of novelty seeking and drug seeking behavior. Behav Brain Res 77 (1 2): 23 43. Bradberr y, C. W., R. J. Gruen, C. W. Berridge and R. H. Roth (1991). Individual differences in behavioral measures: Correlations with nucleus accumbens dopamine measured by microdialysis. Pharmacol Biochem Behav 39 (4): 877 82. Caine, S. B., S. C. Heinrichs, V. L. Coffin and G. F. Koob (1995). Effects of the dopamine d 1 antagonist sch 23390 microinjected into the accumbens, amygdala or striatum on cocaine self administration in the rat. Brain Res 692 (1 2): 47 56. Coulter, C. L., H. K. Happe and L. C. Murrin (1996). Postnatal development of the dopamine transporter: A quantitative autoradiographic study [published erratum appears in brain res dev brain res 1996 jan 2;98(1):150]. Brain Res Dev Brain Res 92 (2): 172 81. Coulter, C. L., H. K. Happe and L. C. Murrin (1997 ). Dopamine transporter development in postnatal rat striatum: An autoradiographic study with [3h]win 35,428. Brain Res Dev Brain Res 104 (1 2): 55 62. Demotes Mainard, J., C. Henry, Y. Jeantet, J. Arsaut and E. Arnauld (1996). Postnatal ontogeny of dopamin e d3 receptors in the mouse brain: Autoradiographic evidence for a transient cortical expression. Brain Res Dev Brain Res 94 (2): 166 74. Hooks, M. S. and P. W. Kalivas (1995). The role of mesoaccumbens -pallidal circuitry in novelty induced behavioral acti vation. Neuroscience 64 (3): 587 97. Jernigan, T. L., J. R. Hesselink, E. Sowell and P. A. Tallal (1991). Cerebral structure on magnetic resonance imaging in language and learning impaired children. Arch Neurol 48 (5): 539 45.

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33 Johnston, L. D., P. M. O'Malle y and J. G. Bachman (2002). Monitoring the future national results on adolescent drug use: Overview of key findings, 2001. Bethesda, MD, National Institute on Drug Abuse : NIH Publication No. 02 5105. Klebaur, J. E. and M. T. Bardo (1999). Individual diffe rences in novelty seeking on the playground maze predict amphetamine conditioned place preference. Pharmacol Biochem Behav 63 (1): 131 6. Koob, G. F. (1992). Drugs of abuse: Anatomy, pharmacology and function of reward pathways. Trends Pharmacol Sci 13 (5): 177 84. Lewis, D. A., S. R. Sesack, A. I. Levey and D. R. Rosenberg (1998). Dopamine axons in primate prefrontal cortex: Specificity of distribution, synaptic targets, and development. Adv Pharmacol 42 : 703 6. McBride, W. J., J. M. Murphy and S. Ikemoto (1 999). Localization of brain reinforcement mechanisms: Intracranial self administration and intracranial place conditioning studies. Behav Brain Res 101 (2): 129 52. McCourt, W. F., R. J. Gurrera and H. S. Cutter (1993). Sensation seeking and novelty seekin g. Are they the same? J Nerv Ment Dis 181 (5): 309 12. Mogenson, G. J., D. L. Jones and C. Y. Yim (1980). From motivation to action: Functional interface between the limbic system and the motor system. Prog Neurobiol 14 (2 3): 69 97. Nicholls, B., A. Springh am and J. Mellanby (1992). The playground maze: A new method for measuring directed exploration in the rat. J Neurosci Methods 43 (2 3): 171 80. Olds, J. and P. Milner (1954). Positive reinforcement produced by electrical stimulation of septal area and othe r regions of rat brain. J Comp Physiol Psychol 47 (6): 419 27. Piazza, P. V., J. M. Deminiere, M. le Moal and H. Simon (1990). Stress and pharmacologically induced behavioral sensitization increases vulnerability to acquisition of amphetamine self adminis tration. Brain Res 514 (1): 22 6. Rebec, G. V., J. R. Christensen, C. Guerra and M. T. Bardo (1997). Regional and temporal differences in real time dopamine efflux in the nucleus accumbens during free choice novelty. Brain Res 776 (1 2): 61 7. Rosenberg, D. R. and D. A. Lewis (1994). Changes in the dopaminergic innervation of monkey prefrontal cortex during late postnatal development: A tyrosine hydroxylase immunohistochemical study. Biol Psychiatry 36 (4): 272 7. Rosenberg, D. R. and D. A. Lewis (1995). Postn atal maturation of the dopaminergic innervation of monkey prefrontal and motor cortices: A tyrosine hydroxylase immunohistochemical analysis. J Comp Neurol 358 (3): 383 400. SAMHSA (2003a). Alcohol use by persons under the legal drinking age of 21, Substanc e Abuse and Mental Health Services Administration.

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34 SAMHSA (2003b). Results from the 2002 national survey on drug use and health: National findings. Rockville, Substance Abuse and Mental Health Services Administration. Seeman, P., N. H. Bzowej, H. C. Guan, C. Bergeron, L. E. Becker, G. P. Reynolds, E. D. Bird, P. Riederer, K. Jellinger, S. Watanabe and et al. (1987). Human brain dopamine receptors in children and aging adults. Synapse 1 (5): 399 404. Spear, L. P. (2000). The adolescent brain and age related b ehavioral manifestations. Neurosci Biobehav Rev 24 (4): 417 63. Stanwood, G. D., S. McElligot, L. Lu and P. McGonigle (1997). Ontogeny of dopamine d3 receptors in the nucleus accumbens of the rat. Neurosci Lett 223 (1): 13 6. Tarazi, F. I. and R. J. Baldessa rini (2000). Comparative postnatal development of dopamine d(1), d(2) and d(4) receptors in rat forebrain. Int J Dev Neurosci 18 (1): 29 37. Teicher, M. H., S. L. Andersen and J. C. Hostetter, Jr. (1995). Evidence for dopamine receptor pruning between adole scence and adulthood in striatum but not nucleus accumbens. Brain Res Dev Brain Res 89 (2): 167 72. Virgili, M., O. Barnabei and A. Contestabile (1990). Regional maturation of neurotransmitter related and glial markers during postnatal development in the ra t. Int J Dev Neurosci 8 (2): 159 66. Zuckerman, M. (1974). The sensation seeking motive. Prog Exp Pers Res 7 : 79 148. Zuckerman, M. (1986). Sensation seeking and the endogenous deficit theory of drug abuse. NIDA Res Monogr 74 : 59 70.

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35 PLACE CONDITIONING: AGE RELATED CHANGES IN T HE REWARDING AND AVERSIVE EFFECTS OF ALCOHOL Abstract Alcohol abuse levels are very high in adolescen ts creating a significant societal issue It has been shown that people who begin alcohol use as adolescents are more likely to become addicts than people who initiate alcohol use as adults. Importantly, the development of addiction in humans is more rapid with initiation in adolescen ce than in adult hood In order to determine changes in the reinforcing efficacy of alcohol as a fu nction of adolescent development we used a place conditioning paradigm. In this study we assessed the ability of ethanol to support a conditioned place preference (CPP) or aversion (CPA). Animals (postnatal days; PND 25, 35, 45 and 60) were tested for alco hol induced conditioning in response to a range of ethanol doses (0.2, 0.5, 1.0, 2.0 g/kg/i.p. or saline ). In general, there was a trend for alcohol to produce an aversion to the ethanol paired compartment at higher doses. These patterns differed significa ntly as a function of age. Younger animals, PND 25 exhibited a CPP to a low dose and an aversion at high doses. Late adolescent (PND 45) animals exhibited a CPP at two moderate doses, but a CPA at the highest dose. PND 35 and 60 animals did not exhibit a C PP at any examined dose and PND 60 exhibited a progressive aversion with increasing dose. The data show that the developmental processes of adolescence influence general responsiveness to alcohol. Specifically, late adolescent animals (PND 45) appear to pr efer doses of alcohol that are either not reinforcing (0.5) or aversive (1.0) at other ages.

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36 These processes need to be examined thoroughly in order to understand the development of addiction in adolescence. This is especially important given that alcohol abuse in adolescence may interfere with the usual pattern of brain development as it relates to alcohol reinforcement.

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37 Introduction Initiation of alcohol use in adolescence has unique consequences on the development of addiction in adulthood. For exampl e, incidences of alcohol dependency are higher among those who initiate heavy drinking (5 drinks per occasion) before the age of 18 (Johnston et al., 2002) Individuals who begin drinking before the age of 15 are four times as likely to b e alcohol dependent adults than those who begin at 21. Importantly, total number of years of alcohol abuse does not impact the development of addiction as strongly as early alcohol use initiation (De Wit et al., 1999) indicating the impor tant relationship between age of initiation and addiction. Generally, a lcohol dependent adults initiate drinking at an earlier age and drink more frequently throughout adolescence than non dependent counterparts (Guo et al., 2000) Young er age of initiation of alcohol use is highly associated with addiction severity as measured by the Addiction Severity Index (Tam et al., 2000) and age of alcohol initiation is inversely correlated with the magnitude of alcohol abuse in la te adolescence (Hawkins et al., 1992, Grant and Dawson, 1998, DeWit et al., 2000) Importantly, adolescent initiation of alcohol use amplifies the effects social risk factors have on the development of addiction (e.g., parental drinking, proactive parenting, peer alcohol use, ethnicity) (Hawkins et al., 1997) Together these data suggest that alcohol use in adolescence has unique implications on the development of alcohol dependency. A notable consideration often over looked when examining the correlational links between adolescent use and addiction is the direction of effect. It is reasonable to assume that individuals prone to alcoholism are also those most likely to initiate drinking early in life, therefore produci ng a strong relationship between the two factors. However,

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38 behavioral characteristics common to the addictive personality and the adolescent period (e.g., see below) give rise to the plausible hypothesis that adolescent alcohol use subsequently increases the risk of alcohol abuse and addiction. Most likely, there is a dynamic interaction between these and other factors that ultimately determine the risk of addiction. However, of importance here is the consideration of biological changes in the central ne rvous system (CNS) of the adolescent that may serve to motivate initial use and as a consequence, alter CNS development in a way the promotes prolonged use. Clearly, psychological, biological and environmental factors all influence the development of addic tion. Psychological factors, including specific personalities traits (impulsiveness (Myers et al., 1995) sensation seeking, rebelliousness [ (Zuckerman et al., 1984) impaired emotional well being, low self esteem (Kandel, 1980) and non conformity to traditional morals and values (Kandel, 1980, Zuckerman et al., 1984) ], Importantly, some of these traits are characteristic of adolescents in that they engage in risky behaviors more often t han adults (Teichman et al., 1989) It may be that adolescents and addicts share specific biological commonalities that underlie the presence of similar personality and behavioral characteristics. Special importance has been placed on sensation seeking behavior, a personality trait common to both alcohol dependents and adolescents (Zuckerman et al., 1984) (Teichman et al., 1989) Researchers have hypothesized that high sensation seekers enjoy the mind altering experiences a drug offers whereas low sensation seekers are stressed by these experiences and therefore avoid them (Zuckerman et al., 1968) To examine this hypothesis, Klebaur and Bardo (1999) divided animals into high and low n ovelty preference groups and patterns of amphetamine conditioned place preference

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39 (CPP) and self administration were investigated (Klebaur et al., 2001) The results indicated that preference for novelty was directly related to the degree of conditionability with amphetamine (Klebaur and Bardo, 1999) Further, high novelty preference was directly related to amphetamine intake in a self administration paradigm (Klebaur et al., 2001) Studies in adolescent mice have shown increased novelty/sensation seeking in adolescent animals in comparison to younger and older counterparts (Adriani et al., 1998) However, these studies have not been extended to make comparisons between these behaviors an d drug effects. Adolescence is a period of increased social activity in general (Primus and Kellogg, 1989) and hyperactivity when exposed to a novel environment (Bronstein, 1972, Spear and Brake, 1983) Using the novelt y seeking paradigm, relationships between ages, novelty preference and alcohol responses are currently being investigated in our laboratory. The biological factor linking sensation/novelty seeking, adolescent development and alcohol addiction may be the d eveloping mesolimbic DA system. The mesolimbic pathway, particularly the dopaminergic (DAergic) projection from the ventral tegemental area (VTA) to the nucleus accumbens septi (NAcc), has been implicated in behavioral reinforcement and is activated by al cohol administration (Johanson and Schuster, 1975, Stewart, 1984, Hernandez and Hoebel, 1988, Di Chiara and Imperato, 1988, Bergman et al., 1989, Hubner and Koob, 1990) Microdialysis studies have shown alcohol induced increases in accumb al DA levels in alcohol preferring rats (Katner and Weiss, 2001, Engleman et al., 2000) There is substantial evidence that this system mediates motivation in general, although the specific mechanism (e.g., attention, reward, motor, antic ipation/expectancy) has been debated. Rats will self administer DA re uptake

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40 inhibitors directly in the NAcc, suggesting the involvement of mesolimbic DA in the process of reinforcement (Kuhar et al., 1991) In addition, electrophysiolog ical studies show heightened activation of DA neurons in the VTA during drug self administration (Carelli et al., 2000) and activational patterns suggest an anticipatory or predictive role in that firing occurs before drug delivery once se lf administration is established. Further, lesioning any part of the mesolimbic pathway alters drug taking behavior (Hubner and Koob, 1990) in a fashion indicative of loss of reinforcement. However, drug addiction relapse occurs along wi th, and can be induced by, reactivation of DA neurons (Stewart, 1984) suggestive of a motivational or predictive role. The neural mechanisms underlying alcohol addiction are more complex than many abused substances given that alcohol affe cts multiple interactive neurotransmitter systems in addition to DA (e.g., serotonin, GABA, opioid)(See (Koob, 1992) However, activation of the mesolimbic DA system is a common substrate of all addictive substances and therefore a point of focus in neurobiological research on alcohol addiction. Although many studies have shown that changes in DA activity in the mesolimbic system affect the expression of substance use patterns in adult animals, relatively few have focused on adolescence, t he time when drug use/abuse it typically initiated. In rodents adolescence is broadly defined as postnatal days (PND) 28 to 55 based on the onset of hormonal changes that initiate puberty (Ojeda and Urbanski, 1994, Odell, 1990) Behavior al change s that resemble those seen in human adolescence, such as increases in peer interaction, increased exploration, risk taking and play behavior appear from PND 28 to 42 (Spear, 2000) These changes provide a more precise development al time frame that is congruent with both the physical and behavioral changes occurring in human

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41 adolescents. Studies that have focused on responsiveness to drugs in adolescents have shown a decreased responsiveness to dopaminergic agonists (Bauer and Evey, 1981, Bolanos et al., 1998, Lanier and Isaacson, 1977, Spear and Brick, 1979, Infurna and Spear, 1979) and increased responsiveness to a DA antagonist (Spear et al., 1980) Recent studies examining the long term effect s of alcohol during adolescence have found no baseline differences in their measures but significant changes in response to a subsequent ethanol challenge. Specifically, adult animals that were exposed to alcohol during adolescence have decreased EEG resp onsiveness with higher doses (Slawecki, 2002) decreased behavioral measures of intoxication during subsequent ethanol challenge (Slawecki, 2002, White et al., 2002) altered electrophysiology (Slawecki et al., 2001) and working memory impairments (White et al., 2000) Young adolescent animals also show a differential sensitivity to binge pattern alcohol induced brain damage (Crews et al., 2000) These studies clearly dem onstrate a unique susceptibility of the adolescent brain to alcohols effects. Further studies are needed to examine how the mesolimbic DA pathway may be susceptible to alcohol induced alterations during adolescence. Dynamic changes occur during this tim e period (Stanwood et al., 1997) including receptor overproduction and pruning (Andersen and Teicher, 2000; Tarazi and Baldessarini, 2000) specifically in the prefrontal cortex and limbic regions (for review see Spear, 2000) Overproduct ion and pruning occur in the dopaminergic, serotonergic, GABAergic and glutamatergic systems. Earlier studies reported D2 and D3 receptor densities to be similar in rats PND 21 and PND 60, but failed to include the adolescent period a time of great chang e in the DA system (Stanwood et al., 1997) There is evidence for receptor population overproduction and pruning during adolescence (Andersen and Teicher, 2000,

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42 Tarazi and Baldessarini, 2000) An important aspect for inve stigation in adolescence is the role drug related environmental cues play in addiction. Such cues have been shown to induce alcohol conditioned drug seeking behavior, craving and relapse (Weiss et al., 2001) Evidence suggests that repea ted pairings of alcohol and specific environmental cues produce conditioned associations and expectancies in the drinker (Goldman, 2002) as well as physiological responses (Glautier, 2000) Animal models have also demonst rated conditioning to environmental cues associated with alcohol. Through classical conditioning, an animal associates not only the administration of a drug with it's physiological effects, but also environmental cues that are repeatedly present during dr ug administration (York and Regan, 1982) and these cues may come to motivate future use. The conditioned place preference (CPP) paradigm measures reward value of a drug utilizing drug related environmental cues/context (C arr et al., 1989) The reinforcing effects of a drug are determined by the amount of time the animal spend s in the drug paired chamber in comparison to non drug paired chamber Mice spend more time in a chamber in which they received alcohol through jugu lar catherization, suggesting a preference for the alcohol paired chamber (Kelley et al., 1997) Moreover, animals selectively bred to exhibit stronger behavioral effects of alcohol show stronger conditioned place preferences (Cunningham et al., 1991, Risinger et al., 1994) In adult animals, CPP has been demonstrated with many drugs including cocaine (Spyraki et al., 1982a, Spyraki et al., 1982b) apomorphine (Wise et al., 1976) and morph ine (Sherman et al., 1980) However, results have been variable in alcohol place conditioning in that some studies find a CPP (Stewart and Grupp, 1986, Stewart and Grupp, 1989, Gauvin and Holloway, 1992, Schechter, 1992, M orse et al., 2000, Cunningham and Henderson,

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43 2000, Holloway et al., 1992, Ciccocioppo et al., 1999) and others find a conditioned place aversion (CPA) depending upon dose, (Cunningham and Henderson, 2000) strain (Schechter 1992) and drug chamber timing interval (Stewart and Grupp, 1989) (Holloway et al., 1992, Ciccocioppo et al., 1999) The only studies that have examined place conditioning in adolescent animals have shown comparable pla ce preferences between adolescents and adult animals with both cocaine and morphine (Campbell et al., 2000) Other studies investigating adolescents only demonstrate a CPP with cocaine but not morphine (Bolanos et al., 199 6) To date, no one has investigated differences in alcohol induced CPP across age. The present study investigate d the relationship of age and alcohol induced CPP in order to determine if adolescents are unique in their responsiveness to the rewarding e fficacy of alcohol. Methods Subjects Offspring (n=227 derived from 59 litters ) of Sprague Dawley breeding pairs (Zivic Miller Laboratories) weighing from 50 300 g (PND 25, 60 90g; PND 35, 120 160g; PND 45, 180 240; PND 60, 220 350g) at th e time of testing were used in this study. No more than one male and one female per litter were used in a given condition. Pups were sexed and culled to 10 pups per litter on postnatal day one (PND 1). Pups remained housed with their respective dams in a temperature and humidity controlled vivarium on a 12:12h light: dark cycle (07:00h/ 19:00h). On PND 21, pups were weaned and individually housed. Apparatus The apparatus consisted of three visually and tactilely distinctive chambers: A large (21W x 36L x 21H, in cm.) neutral chamber made of black Plexiglas and a floor

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44 of black Plexiglas bisecting two end chambers (21W x 15L x 21H, in cm.) with distinct tactile and visual cues. End chambers contained either a checkerboard pattern on the walls with a wir e mesh floor or a floral pattern on the walls with a rubberized floor The chambers were separated by removable Plexiglas doors. Ages To determine the reinforcing efficacy of alcohol across adolescence, animals were divided into 4 age categories (PND 21 2 5, 31 35, 41 45, 56 60) and trained and tested according to the procedures outlined below. Training Animals were trained over a period of 4 days and tested 1 day following the final day of training in an unbiased CPP paradigm Each morning animals were c onfined to either the Paired or the Unpaired compartment for 5 minutes following the administration of ethanol (0.2, 0.5, 1.0 or 2.0 g/kg i.p.) or saline. At each age half of the animals received saline and half received ethanol as their first exposure Animals were placed in the alternate compartment in the afternoon and received a corresponding injection (eg., animals that received ethanol in the morning received saline in the afternoon and vice versa) The environment designated as the drug paired environment was counterbalanced across all ages and conditions. An equal number of animals received ethanol in the checkers environment as did animals receiving ethanol in the floral environment. This procedure was repeated for a total of 4 training days (4 Paired, 4 Unpaired exposures). Testing On day 5 (i.e., PND 25, 35, 45 or 60) drug free animals were placed in the central portion of the neutral compartment and allowed free access to all three chambers for a

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45 total of 5 minutes. Time spent in each chamber was recorded and preference was determined by comparing time spent in the Paired compartment vs. Unpaired compartment. Both absolute and relative comparisons were made. Relative comparisons divided drug paired chamber time by combined time in t he drug paired and unpaired chambers. Design and Analyses Chamber entries were analyzed using a 3 factor mixed design ANOVA for Age (4; 25, 35,45 and 60) by Dose (5; Saline, 0.2, 0.5, 1.0 and 2.0 g/kg alcohol) with Chamber as a repeated measure Preferen ce scores were analyzed using a 2 factor ANOVA for Age (4; 25, 35,45 and 60) by Dose (5; Saline, 0.2, 0.5, 1.0 and 2.0 g/kg alcohol) with adjusted difference score as the dependent measure. Subsequent planned comparisons (Fischer PLSD; t tests) were used to isolate effects Preference scores were calculated as relative ratios to prevent any influence of the novel neutral chamber on the balanced conditioning across the paired and unpaired chambers. The relative ratio calculation serves to control for variat ions produced in paired and unpaired times as a result of time spent in the third, intermediate/novel chamber. The experimental question to be answered is whether animals prefer the drug paired chamber to the saline paired chamber. The animals response t o the introduction of a third chamber, the novel runway, should not influence these results. The relative ratio comparison allows for this direct comparison by removing neutral chamber scores and setting the total test time to the time in the two experime ntal chambers. However, because removing neutral scores will result in different total test times for each animal, the paired and unpaired scores must be converted to a percentage of total time to produce

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46 an honest comparison uninfluenced by time spent in the novel neutral zone. It is important to note that such a conversion cannot make an aversion a preference or vice versa but merely removes the influence a novel third zone has on difference scores. Figure One : Animals exhibited increased activity in t he testing apparatus as a function of age. PND 25 animals exhibited fewer grid crossings than all other ages while PND 60 exhibited the most. = Different from PND 35, 45 and 60 (p < 0.05). = Different from PND 25, 35 and 45 (p < 0.05). Results Activ ity, measured by the total number of chamber entries differed as a function of age ( F (3, 110) = 14.24, p < 0.05 ) There was no main effect of dose. Post hoc Fishers LSD collapsing across dose indicated that P ND 60 animals were more active than all other ages as demonstrated by total number of chamber entries (Figure 1) Pre adolescent Figure One: Activity Levels Across Age 15 20 25 30 35 40 25 35 45 60 AGE (Postnatal Day) Chamber Entries

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47 animals (PND 25) made significantly less chamber entries than all other ages and adolescent animals (PND 35, 45) were intermediate in comparison to younger and older animal s Postnatal day 25 animals were significantly less active than PND 35 or 45 animals. Postnatal day 60 animals exhibited increased chamber entries (more activity) in comparison to all other ages. There were no baseline preferences or aversions (ie., sid e bias) for either chamber in saline controls at any age. Preference scores for chamber were calculated as ratios of paired (P) and unpaired (UP) chamber times (% time in P UP/total time in P and UP) to control for any age or dose differences in activity i n the novel neutral chamber. As can be see n in Figure 2 there was a significant interaction of Age x Dose (F ( 12, 208) = 1.79, p=0.05 ) The overall patterns were similar to those observed with raw difference scores (P UP time), however, neutral chambe r activity did appear to influence the raw scores somewhat. Using ratio scores PND 25 animals exhibited a preference at 0.2 g/kg and an aversion at 1.0 g/kg, with other doses not significantly affecting preference. Adolescent animals exhibited aversions at 0.2 g/kg (PND 45) and 1.0 g/kg (PND 35). A preference was observed in PND 45 animals at 0.5 and 1.0 g/kg. Adult animals exhibited a progressive reduction in preference score with increasing dose, with all doses producing a CPA, 0.2 g/kg the mildest, 0 .5 and 1.0 intermediate and 2.0 producing the greatest aversion to the drug paired chamber. For clarity, only the relative to baseline comparisons are denoted in Figure 2, however, comparisons across age follow here. Again, adolescent animals were unique in their responsiveness to alcohol. PND 35 and PND 45 animals exhibited an aversion at 0.2 g/kg greater than PND 60 animals. However, PND 45 animals exhibited a preference at both the 0.5 and 1.0 g/kg doses, while the PND 35 animals showed only an

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48 avers ion at the 1.0 g/kg dose. Despite the strong preference observed at 1.0 g/kg for PND 45 animals, astrong aversion was seen for PND 45 animals at the 2.0 g/kg dose. Preference scores did not differ significantly among the saline injected control groups at any age [see Figure 2; Saline (0 g/kg)] Figure Two : Preference scores were calculated as ratios of total time spent in the two conditioning chambers: (P UP)/(P + UP). Preference scores different (p < 0.05) from zero were observed at 0.2 g/kg for PND 25 animals and 0.5 & 1.0 g/kg for PND 45 animals. Aversions relative to zero were observed at 1.0 g/kg for PND 25 and 35 animals and 2.0 for PND 45 animals. Discussion The use of chamber entries as a measure of activity in control animals revealed that a dult animals were more active during testing than adolescent animals and that adolescent animals were, in turn more active than preadolescent animals. While the Figure Two: Alcohol Place Conditioning -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0 0.2 0.5 1 2 Alcohol Dose (g/kg/ip) Preference Score (P vs. UP Time) PND 25 PND 35 PND 45 PND 60 * * = Different from Control *

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49 PND 60 animals exhibited more chamber entries, this effect is due to the fact that they spen t less time in the neutral zone and more time in the end chambers relative to other ages. Preference scores were determined to control for this finding given that they are unaffected by time in the neutral zone. We have recently examined activity in sali ne injected control animals and found that all ages exhibited equivalent velocity and total distance traveled scores. In this study, adult animals again exhibited more chamber entries and interestingly the adolescent animals spent more time in the novel neutral zone (manuscript in preparation). Together these findings suggest that using chamber entries as a measure of activity per se, may not be useful in ontogenetic studies. The results of the present study reveal that PND 45 rats are unique in their r esponsivity to alcohol, preferring doses that are non preferred or aversive in younger and older animals. Postnatal day 25 animals exhibited a place preference for the alcohol paired chamber when conditioned with the 0.2 g/kg dose of ethanol only, while 4 5 day olds exhibited preferences at both the 0.5 and 1.0 g/kg doses. No other age by dose relationship produced an alcohol related CPP. This demonstrates that these ages are unique in reactivity to alcohol, as the general finding in non selectively bred a nimals is an alcohol induced aversion (Morse et al., 2000, Cunningham and Henderson, 2000, Gauvin et al., 1994, Stewart and Grupp, 1986, Stewart and Grupp, 1989) as seen in our PND 60 animals. Low and moderate doses of alcohol vary in th eir ability to establish a preference as a function of age suggesting that the process of development itself can contribute powerfully to the reinforcing aspects of alcohol. Further, since alcohol was aversive at all doses in adult animals these data sugge st that adolescence, the age of initiation at greatest

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50 risk for sustained alcohol use, is a critical period. Delayed initiation of alcohol intake may be protective in the absence of other intervening psychosocial variables. The dose of 1.0 g/kg produced the most dramatic spread of behavior across ages, with all ages exhibiting a unique conditioned response. Postnatal day 45 animals exhibited a preference for the drug paired chamber while PND 60, PND 35 and PND 25 animals exhibited increasing degrees of a version, making this moderate dose optimal for further examination of age related alcohol effects. Interestingly, prior data from our lab has shown that PND 25 animals exhibit significant increases in NAcc DA when given 1.0 g/kg of ethanol i.p. (Philpot and Kirstein, 1998) Viewing dopaminergic activity in the NAcc as strictly mediating reward, these data would appear to be in conflict with the alcohol place aversion observed at this age. However if DAergic activity is viewed as mediatin g reinforcement by way of increasing environmental salience of both appetitive and aversive stimuli this conflict does not occur. For example, the strength of the association (as measured by the stimulis influence on behavior), and not the direction of t he association, would be intertwined with NAcc DA concentrations, while some other system(s) may mediate the emotional valence of the stimulus in question. If this is the case DAergic responses to 1.0 g/kg would be largest in the PND 25 animals (mean pref erence score of 0.363), followed by the PND 35 and PND 45 animals (preference of 0.197 and 0.215 respectively) and smallest in PND 60 animals ( 0.115). Preliminary data from our lab confirm this hypothesis. Acute administration of 1.0 g/kg ethanol in P ND 25 animals does indeed produce the largest relative elevations in accumbal DA, with PND 35 and 45 animals intermediate and PND 60 animals exhibiting the smallest relative increases among these ages. Importantly, PND 45 animals appear unique,

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51 demonstrat ing a lack of DAergic tolerance to repeated 1.0 g/kg ethanol administration (Kirstein and Philpot, 2002) The present data suggest increased risk to the reinforcing aspects of alcohol for adolescents when compared to young adults and thes e effects do not appear to be mediated by metabolic processes as evidenced in research by Silveri and Spear (Silveri and Spear, 1999, Silveri and Spear, 2000) Although other studies have been able to produce a CPP in adult animals, these studies usually involve extensive training or a preexposure period to reduce the aversive properties of ethanol (Gauvin and Holloway, 1992) By contrast, the present study demonstrates a rapid place conditioning in late adolescence. Spec ifically, a CPP occurred in a period of 4 days without the aid of a preexposure phase, indicating a unique susceptibility. Further, this study replicates results in adults consistent with reports of an ethanol induced aversion (Heinrichs e t al., 1995, Morse et al., 2000, Cunningham and Henderson, 2000, Cunningham et al., 1998, Gauvin et al., 1994, Holloway et al., 1992, Stewart and Grupp, 1986, Stewart and Grupp, 1989) further validating the unique nature of the PND 45 animals. The implica tion is clear, adolescence represents a unique risk period for alcohol addiction. Data from the Monitoring the Future Studies (Johnston et al., 2002) and The National Center on Addiction and Substance Abuse (Califano, 200 2) have suggested adolescence as a risk period based on population data. The present data confirm observations made in the human population and further, because this is an animal model of addiction, suggest the distinct possibility that basic biological factors may be fundamental in the manifestation of adolescent alcohol use and subsequent addiction. The results suggest the possibility that the biological transition that occurs during adolescence may manifest itself, in part, as increased preference for the appetitive aspects

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52 of alcohol. This could result in an increased probability of initiation/use. Further, given the substantial amount of data that suggests higher risk of alcoholism in those initiating alcohol use in adolescence (DeWi t et al., 2000, De Wit et al., 1999, Grant and Dawson, 1998, Hawkins et al., 1992, Hawkins et al., 1997, Guo et al., 2000) ; it is important to determine how alcohol exposure during this time alters the systems which mediate alcohol induced reinforcement. The present study demonstrates that reinforcement mechanisms do not function identically in late adolescent and young adult animals. Alcohol use during this critical period likely alters the natural transition of these reinforcement mechanisms as adultho od approaches. Studies examining the effects of repeated drug exposure on the subsequent responsiveness of the adolescent mesolimbic DA system are currently being examined in our laboratory.

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53 References Adriani, W., Chiarotti, F. and Lavio la, G. (1998) Elevated novelty seeking and peculiar d amphetamine sensitization in periadolescent mice compared with adult mice. Behav Neurosci 112, 1152 66. Andersen, S. L. and Teicher, M. H. (2000) Sex differences in dopamine receptors and their relevan ce to ADHD. Neurosci Biobehav Rev 24 137 41. Bauer, R. and Evey, L. (1981) Differential effects of l amphetamine on ontogeny of active avoidance, intertrial responses, and locomotor activity. Psychopharmacology 75 299 304. Bergman, J., Madras, B. K., J ohnson, S. E. and Spealman, R. D. (1989) Effects of cocaine and related drugs in nonhuman primates. III. Self administration by squirrel monkeys. J Pharmacol Exp Ther 251 150 5. Bolanos, C. A., Garmsen, G. M., Clair, M. A. and McDougall, S. A. (1996) Ef fects of the kappa opioid receptor agonist U 50,488 on morphine induced place preference conditioning in the developing rat. Eur J Pharmacol 317 1 8. Bolanos, C. A., Glatt, S. J. and Jackson, D. (1998) Subsensitivity to dopaminergic drugs in periadolesc ent rats: a behavioral and neurochemical analysis. Brain Res Dev Brain Res 111 25 33. Bronstein, P. M. (1972) Repeated trials with the albino rat in the open field as a function of age and deprivation. J Comp Physiol Psychol 81 84 93. Califano, J. A. ( 2002),The National Center on Addiction and Substance Abuse at Columbia University, New York. Campbell, J. O., Wood, R. D. and Spear, L. P. (2000) Cocaine and morphine induced place conditioning in adolescent and adult rats. Physiol Behav 68 487 93. Carel li, R. M., Ijames, S. G. and Crumling, A. J. (2000) Evidence that separate neural circuits in the nucleus accumbens encode cocaine versus "natural" (water and food) reward. J Neurosci 20 4255 66. Carr, G. D., Fibiger, H. C. and Phillips, A. G. (1989) In The Neuropharmacological Basis of Reward (Eds, Liebman, J. M. and Cooper, S. J.) Clarendon Press, Oxford, pp. 264 319. Ciccocioppo, R., Panocka, I., Froldi, R., Quitadamo, E. and Massi, M. (1999) Ethanol induces conditioned place preference in genetically s elected alcohol preferring rats. Psychopharmacology (Berl) 141 235 41. Crews, F. T., Braun, C. J., Hoplight, B., Switzer, R. C., 3rd and Knapp, D. J. (2000) Binge ethanol consumption causes differential brain damage in young adolescent rats compared with adult rats. Alcohol Clin Exp Res 24 1712 23. Cunningham, C. L., Hallett, C. L., Niehus, D. R., Hunter, J. S., Nouth, L. and Risinger, F. O. (1991) Assessment of ethanol's hedonic effects in mice selectively bred for sensitivity to ethanol induced hypoth ermia. Psychopharmacology 105 84 92.

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55 Heinrichs, S. C., Menzaghi, F., Schulteis, G., Koob, G. F. and Stinus, L (1995) Suppression of corticotropin releasing factor in the amygdala attenuates aversive consequences of morphine withdrawal. Behav Pharmacol 6 74 80. Hernandez, L. and Hoebel, B. G. (1988) Food reward and cocaine increase extracellular dopamine in the nucleus accumbens as measured by microdialysis. Life Sci 42 1705 12. Holloway, F. A., King, D. A., Bedingfield, J. B. and Gauvin, D. V. (1992) Role of context in ethanol tolerance and subsequent hedonic effects. Alcohol 9 109 16. Hubner, C. B. and Koo b, G. F. (1990) The ventral pallidum plays a role in mediating cocaine and heroin self administration in the rat. Brain Res 508 20 9. Infurna, R. N. and Spear, L. P. (1979) Developmental changes in amphetamine induced taste aversions. Pharmacol Biochem Behav 11 31 5. Johanson, C. E. and Schuster, C. R. (1975) A choice procedure for drug reinforcers: cocaine and methylphenidate in the rhesus monkey. J Pharmacol Exp Ther 193 676 88. Johnston, L. D., O'Malley, P. M. and Bachman, J. G. (2002),National In stitute on Drug Abuse, Bethesda, MD, pp. NIH Publication No. 02 5105. Kandel, D. B. (1980) Drug use by youth: an overview. NIDA Res Monogr 38 1 24. Katner, S. N. and Weiss, F. (2001) Neurochemical characteristics associated with ethanol preference in sel ected alcohol preferring and nonpreferring rats: a quantitative microdialysis study. Alcohol Clin Exp Res 25 198 205. Kelley, B. M., Bandy, A. L. and Middaugh, L. D. (1997) A study examining intravenous ethanol conditioned place preference in C57BL/6J m ice. Alcohol Clin Exp Res 21 1661 6. Kirstein, C. L. and Philpot, R. M. (2002) In Society For Neuroscience, 30th Annual Meeting Vol. 28 Orlando, pp. (Submitted). Klebaur, J. E. and Bardo, M. T. (1999) Individual differences in novelty seeking on the pla yground maze predict amphetamine conditioned place preference. Pharmacol Biochem Behav 63 131 6. Klebaur, J. E., Bevins, R. A., Segar, T. M. and Bardo, M. T. (2001) Individual differences in behavioral responses to novelty and amphetamine self administra tion in male and female rats. Behav Pharmacol 12 267 75. Koob, G. F. (1992) Neural mechanisms of drug reinforcement. Ann N Y Acad Sci 654 171 91. Kuhar, M. J., Ritz, M. C. and Boja, J. W. (1991) The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci 14 299 302. Lanier, L. P. and Isaacson, R. L. (1977) Early developmental changes in the locomotor response to amphetamine and their relation to hippocampal function. Brain Res 126 567 75.

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56 Morse, A. C., Schulteis, G., Holloway, F. A. and Koob, G. F. (2000) Conditioned place aversion to the "hangover" phase of acute ethanol administration in the rat. Alcohol 22 19 24. Myers, M. G., Brown, S. A. and Mott, M. A. (1995) Preadolescent conduct disorder behaviors predict relapse and p rogression of addiction for adolescent alcohol and drug abusers. Alcohol Clin Exp Res 19 1528 36. Odell, W. D. (1990) In Control of onset of puberty. (Eds, Grumbach, M. M., Sizonenko, P. C. and Aubert, M. L.) Williams and Wilkins, Baltimore, pp. 183 210. Ojeda, S. R. and Urbanski, H. F. (1994) In The Physiology of Reproduction. (Eds, Knobil, E. and Neill, J. D.) Raven Press, New York, pp. 363 409. Philpot, R. M. and Kirstein, C. L. (1998) The effects of repeated alcohol exposure on the neurochemistry of the periadolescent nucleus accumbens septi. Neuroreport 9 1359 63. Primus, R. J. and Kellogg, C. K. (1989) Pubertal related changes influence the development of environment related social interaction in the male rat. Dev Psychobiol 22 633 43. Risinger, F O., Malott, D. H., Prather, L. K., Niehus, D. R. and Cunningham, C. L. (1994) Motivational properties of ethanol in mice selectively bred for ethanol induced locomotor differences. Psychopharmacology (Berl) 116 207 16. Schechter, M. D. (1992) Locomoto r activity but not conditioned place preference is differentially affected by a moderate dose of ethanol administered to P and NP rats. Alcohol 9 185 8. Sherman, J. E., Pickman, C., Rice, A., Liebeskind, J. C. and Holman, E. W. (1980) Rewarding and avers ive effects of morphine: temporal and pharmacological properties. Pharmacol Biochem Behav 13 501 5. Silveri, M. M. and Spear, L. P. (1999) Ontogeny of rapid tolerance to the hypnotic effects of ethanol. Alcohol Clin Exp Res 23 1180 4. Silveri, M. M. an d Spear, L. P. (2000) Ontogeny of ethanol elimination and ethanol induced hypothermia. Alcohol 20 45 53. Slawecki, C. J. (2002) Altered EEG responses to ethanol in adult rats exposed to ethanol during adolescence. Alcohol Clin Exp Res 26 246 54. Slawec ki, C. J., Betancourt, M., Cole, M. and Ehlers, C. L. (2001) Periadolescent alcohol exposure has lasting effects on adult neurophysiological function in rats. Brain Res Dev Brain Res 128 63 72. Spear, L. P. (2000) The adolescent brain and age related beh avioral manifestations. Neurosci Biobehav Rev 24 417 63. Spear, L. P. and Brake, S. C. (1983) Periadolescence: age dependent behavior and psychopharmacological responsivity in rats. Dev Psychobiol 16 83 109.

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57 Spear, L. P. and Brick, J. (1979) Cocaine in duced behavior in the developing rat. Behav Neural Biol 26 401 15. Spear, L. P., Shalaby, I. A. and Brick, J. (1980) Chronic administration of haloperidol during development: behavioral and psychopharmacological effects. Psychopharmacology 70 47 58. Sp yraki, C., Fibiger, H. C. and Phillips, A. G. (1982a) Cocaine induced place preference conditioning: lack of effects of neuroleptics and 6 hydroxydopamine lesions. Brain Res 253 195 203. Spyraki, C., Fibiger, H. C. and Phillips, A. G. (1982b) Dopaminergi c substrates of amphetamine induced place preference conditioning. Brain Res 253 185 93. Stanwood, G. D., McElligot, S., Lu, L. and McGonigle, P. (1997) Ontogeny of dopamine D3 receptors in the nucleus accumbens of the rat. Neurosci Lett 223 13 6. Stew art, J. (1984) Reinstatement of heroin and cocaine self administration behavior in the rat by intracerebral application of morphine in the ventral tegmental area. Pharmacol Biochem Behav 20 917 23. Stewart, R. B. and Grupp, L. A. (1986) Conditioned place aversion mediated by orally self administered ethanol in the rat. Pharmacol Biochem Behav 24 1369 75. Stewart, R. B. and Grupp, L. A. (1989) Conditioned place aversion mediated by self administered ethanol in the rat: a consideration of blood ethanol le vels. Pharmacol Biochem Behav 32 431 7. Tam, T. W., Weisner, C. and Mertens, J. (2000) Demographic characteristics, life context, and patterns of substance use among alcohol dependent treatment clients in a health maintenance organization. Alcohol Clin E xp Res 24 1803 10. Tarazi, F. I. and Baldessarini, R. J. (2000) Comparative postnatal development of dopamine D(1), D(2) and D(4) receptors in rat forebrain. Int J Dev Neurosci 18 29 37. Teichman, M., Barnea, Z. and Rahav, G. (1989) Sensation seeking, state and trait anxiety, and depressive mood in adolescent substance users. Int J Addict 24 87 99. Weiss, F., Martin Fardon, R., Ciccocioppo, R., Kerr, T. M., Smith, D. L. and Ben Shahar, O. (2001) Enduring resistance to extinction of cocaine seeking beh avior induced by drug related cues. Neuropsychopharmacology 25 361 72. White, A. M., Bae, J. G., Truesdale, M. C., Ahmad, S., Wilson, W. A. and Swartzwelder, H. S. (2002) Chronic intermittent ethanol exposure during adolescence prevents normal developmen tal changes in sensitivity to ethanol induced motor impairments. Alcohol Clin Exp Res 26 960 8. White, A. M., Ghia, A. J., Levin, E. D. and Swartzwelder, H. S. (2000) Binge pattern ethanol exposure in adolescent and adult rats: differential impact on sub sequent responsiveness to ethanol. Alcohol Clin Exp Res 24 1251 6.

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58 Wise, R. A., Yokel, R. A. and DeWit, H. (1976) Both positive reinforcement and conditioned aversion from amphetamine and from apomorphine in rats. Science 191 1273 5. York, J. L. and Re gan, S. G. (1982) Conditioned and unconditioned influences on body temperature and ethanol hypothermia in laboratory rats. Pharmacol Biochem Behav 17 119 24. Zuckerman, M., Ballenger, J. C. and Post, R. M. (1984) The neurobiology of some dimensions of pe rsonality. Int Rev Neurobiol 25 391 436. Zuckerman, M., Persky, H., Link, K. E. and Basu, G. K. (1968) Responses to confinement: an investigation of sensory deprivation, social isolation, restriction of movement and set factors. Percept Mot Skills 27 3 19 34.

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59 EFFECTS OF REPEATED ETHANOL ON BASAL DOP AMINE LEVELS Abstract Recent research indicates that alcohol use/abuse is often initiated during the adolescent period and that brain reinforcement pathways [e.g., the mesolimbic dopamine (DA) pathway] are undergoing developmental transition. Our research focuses on the effects of ethanol administration on neural mechanisms associated with addiction in preadolescent (postnatal day; PND 25), adolescent (PND 35, PND 45) and young adult (PND 60) animals. U sing conditioned place preference (CPP) testing we have shown that adolescent animals are unique in their responses to ethanol. CPP has been associated with contextually conditioned incentive motivation, our results suggest that younger animals may be more vulnerable to addiction. The present data reveal that adolescent animals are neurochemically distinct in response to ethanols effects. Using in vivo microdialysis within the nucleus accumbens septi (NAcc) we have determined the dopaminergic (DAergic) resp onse across development. Results reveal that the basal levels of DA transition during the adolescent period and differ from preadolescent or adult animals. Specifically, PND 45 animals exhibited significantly higher, and PND 25 significantly lower, basal D A levels than all other ages examined. Further, repeated exposure to ethanol elevated basal DA levels significantly regardless of age or dose. Basal DOPAC/DA ratio also differed as a function of age, with PND 35 and PND 60 animals

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60 demonstrating the highes t ratios, and PND 45 animals producing the lowest baseline levels. Repeated ethanol exposure produced significant changes in basal ratios as a function of age. Interestingly, PND 45 animals exhibited no change in ratios with repeated exposure, while all ot her ages demonstrated a dose dependent rise in DOPAC/DA ratios. These data indicate an age dependant difference in the homeostatic alterations of mesolimbic systems in response to repeated ethanol treatment, an effect that may manifest itself as difference s in behavioral responsivity and conditionability to the alcohol and its effects.

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61 Introduction Adolescence is a complex developmental period involving increased socialization, risk taking, cognitive and sexual development and rapid growth. This period i s characterized by exploring potentials, challenging protective barriers to transition from a dependent to an independent organism. A natural consequence of adolescence is tremendous experimentation and risk taking (for review see Spear, 2 000) a pattern that, in the arena of drug abuse often manifests itself as first time drug use and the potential for drug abuse (Zuckerman 1974) Drug use typically begins early in the maturational process, increasing dramatically in adol escence. By twelfth grade, approximately 80.3% of U.S. adolescents have used alcohol at some time, an increase from 51.7% for 8 th graders (Johnston 2000) Adult lifetime prevalence data indicate 81.3% have had some experience with alcoho l, a rate only slightly higher than that reported for 12 th graders, suggesting that during adolescence most individuals have their first experience with the drug. Of particular importance, the rate of initiation of alcohol use among the 12 17 year old age group has increased in recent years (SAMHSA 1999) Developmental changes in the mesolimbic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens septi (NAcc), may mediate the behavioral changes associated w ith adolescence. Consequently increasing the probability of drug use initiation as well as potentiate the likelihood of abuse. It is clear that in both human and rodent populations, mesolimbic dopamine (DA) systems are undergoing tremendous transition. Basal DA synthesis in the NAcc is lower in postnatal day 30 (PND 30) than PND 40 rats and turnover rates for PND 30 animals are less than reported in adults (Andersen, Rutstein et al. 1997) DA receptor populations also change

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62 exhibiting a pattern of overproduction and pruning across adolescence (Teicher, Andersen et al. 1995; Andersen, Rutstein et al. 1997; Andersen and Teicher 2000) This pattern is similar in humans as well (Seeman, Bzowej et al. 1987) In rats, the density of D1, D2, and D4 receptors in the NAcc increases to a peak at PND 28, and then declines significantly to adult levels at PND 60 (Tarazi and Baldessarini 2000) Parallel with these changes, DA transporter levels ar e undergoing substantial change, increasing in concentration in the NAcc to adult levels through adolescence (Coulter, Happe et al. 1996; Coulter, Happe et al. 1997) The dynamic changes in the mesolimbic DA system during adolescence sugge sts that processes mediated by this system are unlikely to manifest themselves similarly in adults. Across adolescence there may be tremendous transitions in reactivity to stimuli (eg., drugs) that act on these systems. Particularly, developmental transi tions in this pathway may mediate the increased likelihood of engaging in drug use initiation (risk taking) during adolescence. Thus, drugs of abuse may exhibit unique profiles of action in these systems during adolescence and resultantly increase the pro bability for the development of addiction among adolescents. Methods To examine the influence of ethanol exposure on NAcc DA, animals were placed into one of two treatment conditions: acute or repeated ethanol, and administered one of five doses: saline, 0.2, 0.5, 1.0 or 2.0 g/kg ethanol (17% v/v). Neurochemical responses to treatment were measured using in vivo microdialysis following 4 days of treatment. Coordinates for probe placement in the NAcc of PND 25, 35, 45 and 60 animals were determined using weight based regression lines established in a prior study (Philpot,

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63 McQuown et al. 2001) All animals received injections (b.i.d.) at one of 4 age ranges: 21 24 (preadolescent), 31 34, 41 44 (periadolescent) or 56 59 (young adult) to exa mine developmental differences in response profile. Four to eight animals were used per group, for a total of 315 animals. The probe was perfused with artificial cerebrospinal fluid (145 mM NaCl, 2.4 mM KCl, 1.0 mM MgCl 2 1.2 mM CaCl 2 and 0.2 mM ascorb ate, pH = 7.4) at a flow rate of one l/min and inserted (under anesthesia). Animals were placed within a BAS Raturn Apparatus overnight to allow for elimination of anesthetic. Sampling began 24 hr following placement of the probe. Samples were collecte d (1l/min) every 10 min using a refrigerated fraction collector (BAS) and acidified with 0.25 N HClO 4 A total of six baseline samples were collected the final three of which were used for the calculation of baseline levels. Drop sites were verified his tologically to ensure placement in the NAcc shell region. Extracellular levels of DA, and 3,4 dihydroxyphenylacetic acid (DOPAC) were determined using HPLC EC. Direct injections of dialysis samples were separated by a Microbore HPLC system (BAS) with a mobile phase consisting of monochloroacetic acid (0.15 M), sodium octyl sulfate (0.50 mM), EDTA (1.0 mM) and acetonitrile (4.0%) (pH = 2.9). Peaks were detected using a BAS LC 4C electrochemical detector (Bioanalytical Systems, West Lafayette, IN) couple d to a radial flow electrode referenced at 0.800 V. Data were analyzed using a 4 (Age) x 2 (Treatment) x 4 (Dose) ANOVA with subsequent simple effects analyses and Fisher LSDs to isolate effects.

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64 Basal DA in Dialysate (nM) Across Age PND 25 PND 35 PND 45 PND 60 0 1 2 3 PND 25 PND 35 PND 45 PND 60 u u P45/P60 P25/P35/P60 Figure One AGE Figure One : Analysis of basal DA revealed a significant main effect of Age 3,279 F = 17.171, P < 0.05. Post Hoc analysis revealed that P25 animals exhibited significantly less basal DA in dialysate than PND 45 or PND 60, while PND 45 animals demonstrated higher basal levels th an all other ages examined. Resu lts and Discussion The present findings indicate late adolescent animals (ie., PND 45) produce substantially greater quantities of DA in extracellular fluid than younger and older animals (Figure One). Given evidence suggesting that NAcc DA is related to both responsiveness to novel stimuli and to drug abuse liability, and that these two factors are correlated, it is reasonable to assume that differences in basal DA activity in the NAcc may manifest itself as transitional differences in behavior. The dev elopmental elevation in DA may serve to produce the increased exploratory and risk taking typically observed in adolescence. The present study shows a lack of alteration in DOPAC/DA turnover at this age (PND 45; Figure Two), suggesting a failure to adapt to repeated ethanol, or at a minimum a unique fashion of adaptation at this age that may result in increased reactivity

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65 to ethanol following repeated use relative to other ages. Further, the unique status of the DA system during this time, coupled to its unique response to repeated ethanol, may result in a system that develops differentially as a result of alcohol exposure during adolescence. This may, in turn, increase the likelihood of long term ethanol use, abuse and dependence. Basal DOPAC/DA Ratio Across Age PND 25 PND 35 PND 45 PND 60 0 100 200 300 Naive Repeated Naive * Figure Two AGE Figure Two : Analysis of basal DOPAC/DA ratio revealed a significant Age X Pretreatment interaction 3, 279 F = 7.637, p < 0.05. Analysis of simple effects in Nave animals revealed that PND 60 animals produce higher DOPAC/DA ratios than all other ages, and that PND 45. With Rep eated exposure, PND 25 animals exhibited lower turnover values than PND 35 or PND 60, and PND 45 animals exhibited lower turnover than all ages. Importantly, PND 45 animals failed to demonstrate an elevation in DOPAC/DA ratio following repeated ethanol.

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66 R eferences Andersen, S. L., M. Rutstein, et al. (1997). Sex differences in dopamine receptor overproduction and elimination. Neuroreport 8 (6): 1495 8. Andersen, S. L. and M. H. Teicher (2000). Sex differences in dopamine receptors and the ir relevance to ADHD. Neurosci Biobehav Rev 24 (1): 137 41. Coulter, C. L., H. K. Happe, et al. (1996). Postnatal development of the dopamine transporter: a quantitative autoradiographic study [published erratum appears in Brain Res Dev Brain Res 1996 Jan 2;98(1):150]. Brain Res Dev Brain Res 92 (2): 172 81. Coulter, C. L., H. K. Happe, et al. (1997). Dopamine transporter development in postnatal rat striatum: an autoradiographic study with [3H]WIN 35,428. Brain Res Dev Brain Res 104 (1 2): 55 62. Johnsto n, L. D. (2000). Monitoring the future : national survey results on drug use, 1975 1999. Bethesda, Md., National Institute on Drug Abuse, U.S. Dept. of Health and Human Services, National Institutes of Health. Philpot, R. M., S. McQuown, et al. (2001). St ereotaxic localization of the developing nucleus accumbens septi. Brain Res Dev Brain Res 130 (1): 149 53. SAMHSA (1999). Summary of findings from the 1998 national household survey on drug abuse. Rockville, Md, U.S. Dept. of Health and Human Services : 128 Seeman, P., N. H. Bzowej, et al. (1987). Human brain dopamine receptors in children and aging adults. Synapse 1 (5): 399 404. Spear, L. P. (2000). The adolescent brain and age related behavioral manifestations [In Process Citation]. Neurosci Biobehav Rev 24 (4): 417 63. Tarazi, F. I. and R. J. Baldessarini (2000). Comparative postnatal development of dopamine D(1), D(2) and D(4) receptors in rat forebrain. Int J Dev Neurosci 18 (1): 29 37. Teicher, M. H., S. L. Andersen, et al. (1995). Evidence for do pamine receptor pruning between adolescence and adulthood in striatum but not nucleus accumbens. Brain Res Dev Brain Res 89 (2): 167 72. Zuckerman, M. (1974). The sensation seeking motive. Prog Exp Pers Res 7 : 79 148.

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67 ETHANOL MEDIATED DOP AMINE RELEA SE IN THE NUCLEUS ACCUMBENS SEPTI OF A DOLESCENT ANIMALS Abstract The mesolimbic dopamine (DA) system has been implicated as central to motivated behaviors. This system has been demonstrated to mediate the motivational aspects of natural reinforcers. It is now well known that DA activity increases in the nucleus accumbens septi (NAcc) with exposure to addictive substances and manipulation of this response alters the motivational capacity of drugs of abuse. Recent research has revealed that the mesolimbic system of periadolescent animals is undergoing dramatic transition in functional tone. DA receptor and transporter levels are up regulated, synthesis rates are altered, and innervation from prefrontal cortex (PFC), involved in regulating tonic and phasic D A activity, is increasing. Consequently, during adolescence there is a dramatic change in tonic DA levels, variations in phasic responses to acute drug administration and alterations in how the system adapts to repeated drug exposure. These data suggests t hat adolescents may be particularly susceptible to addiction. The present study examined the responsiveness of the NAcc to the administration of ethanol in adolescent and adult animals. The results indicate that adolescents are different from adults in th eir neurochemical responsiveness to alcohol and suggest that late adolescent animals are particularly vulnerable to the rewarding effects of repeated ethanol administration.

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68 Introduction Drug use typically begins early in the maturational process and incr eases dramatically in adolescence. Adult lifetime prevalence data indicate 81.3% have had some experience with alcohol, a rate only slightly higher than that reported for high school students in 12 th grade, suggesting that during adolescence most individua ls have their first experience with the drug. Approximately 10.7 million people aged 12 to 20 were current alcohol users in 2002. From 12 years to 17 years of age current use rates for alcohol increased from 2.6% to 36.2% (SAMHSA 2003b) Within the ages 12 to 15 rates of recent alcohol use increased to 21.5%, increasing to more than 8 times in a four year span (SAMHSA 2003a) Of particular importance, the numbers of initial alcohol users among the 12 17 year old age group increased from 2 .2 to 3.1 million between 1995 and 2000 revealing a recent rise in the initiation of use in the adolescent population (SAMHSA 2003c) These data indicate both a significant initiation of use in early adolescence as well as a rapid increase in using during the adoles cent period. Adolescence is a complex developmental period involving increased socialization, risk taking, cognitive development, sexual development and rapid growth. Its temporal boundaries are difficult to define as adolescence involves the occurrence of a range of events over a broad period. In humans, adolescence is a period in which the developing organism begins to explore potentials, to challenge protective barriers and structure in order to transition from a dependent child to an independent adu lt (Spear 2000a) A natural consequence of this developmental strategy is tremendous exp erimentation and risk taking a pattern that can manifests itself as first time drug use and subsequent drug abuse (Zuckerman 1974)

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69 Interestingly, many of the biological and behavioral characteristics of human adolescence are paralleled in the rodent population. O'dell (1990) has defined the period of adolescence in the rat as between PND 20 and 55 based on hormonal changes associated with sexual development. During this broad timeframe, behavioral characteristics emerge that are similar to tho se observed in the human adolescent. Rodents increase play behavior with conspecifics during this time and show increased activity and exploratory behavior. Continuing development of the central nervous system (CNS) may underlie observed behavioral chang es in both humans and rodents (Spear 2000b) The CNS structure most frequently implicated in mediating addictive processes is the nucleus accumbens septi (NAcc), which receives dopaminergic (DAergic) input from the ve ntral tegmental area (VTA). Manipulations that directly stimulate dopamine (DA) receptors in the NAcc reinforce many behaviors (Olds and Fobes 1981) including place preference and lever pressing. Electrical stimulation of the NAcc itself, or any of the pathways which result in increased DA efflux within the NAcc, produce behavioral r einforcement and animals will lever press for this stimulation (Crow 1971; Anlezark et al. 1972; Crow 1972, 1973; Anlezark et a l. 1974) Additionally, injections of DA agonists into the NAcc have rewarding effects, producing a conditioned place preference (CPP) in treated animals (Hoebel et al. 1983) Animals will also lever press for microinjections of DA or amphetamine directly int o the NAcc (Hoebel et al. 1983) indicating that drug stimulation of this region is sufficient to establish and maintain stimulant use. This region also appears to be directly involved in the reinforcing effects of cocaine (Moghaddam and Bunney 1989) and the administration of numerous drugs,

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70 including alcohol, all elicit a significant increase in DA levels in the NAcc (Phillips et al. 1983; Koob 1992a, 1992b, 1992c, 1996b, 1996a; Phillips and Shen 1996) Decreasing DA release in the NAcc results in higher stimulation thresholds for elec trical brain self stimulation, mimicking the effect of systemic administration of DA antagonists. Direct injections of DA receptor blockers into the NAcc necessitate a large increase in the amount of electrical stimulation necessary to maintain self stimu lation behavior (Stellar et al. 1983) Additionally, experiments using animals which are trained to self administer cocaine or amphetamine have shown that microinjections of DA antagoni sts (directly into the NAcc) decrease self administration (Koob 1992a) when administered in sufficient quantities. Moreover, following 6 hydroxydopamine (6 OHDA) lesions, animals will no longer lever press to administer the DAergic agonists cocaine or amphetamine (Zito et al. 1985; Roberts 1989) However, they will vigorously respond if apomorphine (DA receptor agonist) is infused into the NAcc during the self administration procedure, producing post synaptic activity despite the lesion. Taken together, these studies show the central role that DA activation of the postsynaptic receptors of the NAcc plays in a broad range of reinforcing behaviors. It is likely that neurophysiological changes in the mesolimbic system during development modulate the initiation of drug use and potentiate the likelihood of abuse during adolescence. Specifically, it is clear in both human and rodent populations that the mesolimbic DA systems are undergoing tremendous transition during adolescence. Neurochemically, basal DA synthesis in the NAcc is lower in postnatal day 30 (PND 30) than PND 40 rats and turnover rates for PND 30 animals are less than those reported in adults (Andersen et al. 1997) Research on DA receptor populations indicates a pat tern of

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71 overproduction and pruning that occurs across adolescence in a sex specific manner (Teicher et al. 1995; Andersen et al. 1997; Andersen and Teicher 2000) with males exhibiting greater levels across age and greater over production of D1 and D2 receptor types. This pattern is similar in humans as well (Seeman et al. 1987) In rats, the density of D1, D2, and D4 receptors in the NAcc increases to peak at PND 28, and then declines significantly to adult levels at PND 60 (Tarazi and Baldessarini 2000) Furthermore, D3 receptor numbers appear to increase monotonically, w ith some reports finding adult levels at weaning (i.e., PND 21) (Demotes Mainard et al. 1996) but others find D3 levels in weanlings far lower than those observed in adults (Stanwood et al. 1997 ) In conjunction with receptor density changes, D1 stimulatory and D2 inhibitory effects on adenylyl cyclase production are less apparent in adolescence than in adults (Andersen and Teicher 1999) Simultaneously, DA transporter levels are undergoing substantial change, increasing in concentrati on in the NAcc to adult levels through adolescence (Coulter et al. 1996, 1997) This dynamic transition during adolescence suggests that processes that are mediated by the mesolimbic DA system are unlikely to manifest themselves similarly in adults and adolescents and that across adolescence there may be tremendous transitions in reactivity to stimuli that act on these systems. Of particular importance, developmental transitions in this system may mediate the increased likelihood of engaging in drug use (risk taking) during adolescence and drugs of abuse may exhibit unique profiles of action in these systems during adolescence that underlie the increased probability for the development of addiction among ad olescents. The present study utilized in vivo microdialysis to examine ethanols effects on DA levels in the NAcc of adolescent and adult animals.

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72 Methods Animals One hundred ninety two rats (Zivic Miller Laboratories) weighing from 50 300 g (PND 25, 60 90g; PND 35, 120 160g; PND 45, 180 240; PND 60, 220 350g) at the time of dialysis were used in this study. No more than one male and one female per litter were used in a given condition. Pups were sexed and culled to 10 pups per litter on postnatal day one (PND 1). Pups remained housed with their respective dams in a temperature and humidity controlled vivarium on a 12:12h light: dark cycle (07:00h/ 19:00h). On PND 21, pups were weaned and pair housed. Surgery Our laboratory has recently determined th e effective coordinates for microdialysis probe placement in PND 25, 35, 45 and 60 animals (Philpot et al. 2001) for use in these procedures. P readolescent (i.e., PND 24), periadolescent (i.e., PND 34, 44) and adult (PND 59) animals were anesthetized using a xylazine/ketamine cocktail (0.15.and 1.0 mg/kg respectively). An incision was made over the skull, the guide cannula af fixed with cyanoacrylate and cranioplast and the dialysis probe lowered to the NAcc shell. Anchor screws were used to insure sufficient support and topical lidocaine was applied to the wound edge to reduce potential discomfort. The probe was perfused wit h artificial cerebrospinal fluid (145 mM NaCl, 2.4 mM KCl, 1.0 mM MgCl 2 1.2 mM CaCl 2 and 0.2 mM ascorbate, pH = 7.4) at a flow rate of one l/min and inserted (under anesthesia) following solidification of the cranioplast. Animals were placed within a B AS Raturn Apparatus overnight to allow for elimination of anesthetic. Sampling began 24 hr following placement of the probe. Six baseline samples were collected prior to drug manipulation, the final three of which were used for the

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73 calculation of basel ine levels. After the collection of baseline samples animals received an injection of saline or drug (as outlined in the specific experiments below). Samples were collected (1l/min) every 10 min for a total duration of 120 min after injection (60 minute s prior to injection) using a refrigerated fraction collector (BAS) and acidified with 0.25 N HClO 4 Animals were overdosed with Nembutal (80 mg/kg), decapitated, the probe removed and the brain frozen for sectioning to verify probe placement in the NAcc shell region. Dialysate Analysis Extracellular levels of DA and the metabolite 3,4 dihydroxyphenylacetic acid (DOPAC) were determined using High Performance Liquid Chromatography with Electrochemical Detection (HPLC EC). After acidification, samples we re run or stored at 80 O c. Direct injections of dialysis samples were separated by a Microbore HPLC system (BAS) with a mobile phase consisting of monochloroacetic acid (0.15 M), sodium octyl sulfate (0.50 mM), EDTA (1.0 mM) and acetonitrile (4.0%) (pH = 2.9). Peaks were detected using a BAS LC 4C electrochemical detector (Bioanalytical Systems, West Lafayette, IN) coupled to a radial flow electrode referenced at 0.800 V. Peak detection limits for catechols using this system is 150 fg injected (Pers. Comm., BAS) at 1 nA sensitivity with peak signal to noise ratio of at least 2:1. The recoveries of DA and DOPAC through the dialysis membrane are 10 20% as measured in vitro at 23 O C. Ethanol Induced NAcc DA Release Across Age To examine the responsivity tolerance and conditionability of the developing NAcc to alcohol, animals were placed into one of three treatment conditions: acute, repeated or expectancy, at one of four doses: saline, 0.5, 1.0 or 2.0 g/kg alcohol, and neurochemical responses were meas ured. All animals received injections (b.i.d.) in one

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74 of 4 ages: 21 25 (preadolescent), 31 35, 41 45 (periadolescent) or 56 60 (young adult) to examine developmental differences in response profile. Four animals were used per group, for a total of 192 an imals. Animals in the acute condition received injections (b.i.d.) of saline followed by a drug challenge at the respective dose on the day of dialysis. Animals in the repeated condition received repeated drug injections and received the respective dose on the day of testing. Animals in the expectancy condition received repeated drug injections and were challenged with saline in the environment previously paired with ethanol on the day of testing Basal differences in nM concentrations were analyzed with a 4 (Age) x 4 (Dose) ANOVA to determine age related and drug exposure induced changes in DA and DOPAC. Drug effects on DA and DOPAC were analyzed using 4 (Age) x 4 (Dose) Area Under the Curve (AUC) analysis of both absolute (nM) and percent relative to b aseline response profiles. Temporal drug effects on DA and DOPAC were analyzed using a 4 (Age) x 3 (Treatment) x 4 (Dose) x 13 (Time) factorial ANOVA with subsequent Newman Keuls planned comparisons to isolate effects over time. Results Basal Levels DOP AC Analysis of basal DOPAC revealed significant main effects of Age 3,279 F = 6.887, P < 0.05; Pretreatment 1,279 F = 49.251, p < 0.05; and Dose 4, 279 F = 2.719, p < 0.05. A significant Dose X Pretreatment interaction 3, 279 F = 3.690, p < 0.05 was also observed.

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75 Basal DOPAC (nM) in Dialysate Across Age PND 25 PND 35 PND 45 PND 60 0 50 100 150 200 250 300 350 400 450 PND35/PND60 A. AGE Basal DOPAC (nM) Following Repeated Treatment Saline 0.2 0.5 1.0 2.0 0 100 200 300 400 500 600 n Saline or 0.2 g/kg o All other doses o n n B. DOSE Figure One: Basal DOPAC Levels in the NAcc Figure One : PND 25 and PND 45 animals possessed significantly less DOPAC in dialysate than their PND 35 and PND 60 counterparts (A). Following Repeated treatment with 0.5, 1.0 or 2.0 g/kg EtOH DOPAC levels were elevated over Saline or 0.2 g/kg E tOH treated animals. 2.0 g/kg treatment produced elevations over 0.5 and 1.0 g/kg EtOH (B).

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76 Post Hoc analysis demonstrated that PND 25 and PND 45 animals possessed significantly less DOPAC in dialysate than their PND 35 and PND 60 counterparts (Figure One: A). Analysis of simple effects revealed that following Repeated treatment with 0.5, 1.0 or 2.0 g/kg EtOH DOPAC levels were elevated over Saline or 0.2 g/kg EtOH treated animals. Further, 2.0 g/kg treatment produced elevations over 0.5 and 1.0 g/kg EtOH ( Figure One: B). DA Analysis of basal DA revealed significant main effects of Age 3,279 F = 17.171, P < 0.05; and Pretreatment 1, 279 F = 28.071, p < 0.05. Post Hoc analysis revealed that PND 25 animals exhibited significantly less basal DA in dialysate tha n PND 45 or PND 60, while PND 45 animals demonstrated higher basal levels than all other ages examined (Figure Two: A). Additionally, animals pretreated with alcohol (Repeated and Expectancy groups) demonstrated higher basal levels than those receiving on ly saline during the pretreatment phase (Figure Two: B). DOPAC/DA Analysis of basal DOPAC/DA ratio revealed significant main effects of Age 3,279 F = 30.728, P < 0.05; Pretreatment 1, 279 F = 48.495, p < 0.05 and Dose 4, 279 F = 4.248, p < 0.05 ;a signific ant Age X Pretreatment 3, 279 F = 7.637, p < 0.05 and Dose X Pretreatment interaction 3,279 F = 3.589, p < 0.05. Post Hoc analysis revealed that PND 45 animals had significantly lower DOPAC/DA ratios than all other ages, and that PND 25 animals demonstrate d lower ratios than PND 35 or PND 60 (Figure Three: A).

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77 Basal DA in Dialysate (nM) Across Age PND 25 PND 35 PND 45 PND 60 0 1 2 3 u u P45/P60 P25/P35/P60 A. AGE Basal DA in Dialysate (nM) Following Repeated Ethanol Naive Repeated 0 1 2 3 l l Naive B. PreTreatment Figure Two: Basal DA Levels in the NAcc Figure Two : PND 25 animals exhibited significantly less basal DA in dialysate than PND 45 or PND 60. PND 45 animals demonstrated higher basal levels than all other ages examined (A). Animals pretr eated with alcohol demonstrated higher basal levels than saline treated animals (B).

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78 Basal DOPAC/DA in Dialysate (nM) PND 25 PND 35 PND 45 PND 60 0 100 200 u PND 35 or PND 60 All other ages u A. AGE Basal DOPAC/DA Ratio Across Age PND 25 PND 35 PND 45 PND 60 0 100 200 300 Naive Repeated Naive * B. AGE Basal DOPAC/DA Ratio Across Age by Dose Saline 0.2 0.5 1.0 2.0 0 50 100 150 200 250 Naive Repeated Naive Saline Saline, 0.2 or 0.5 g/kg * C. DOSE Figure Three: Basal DOPAC/DA Levels in the NAcc Figure Three : PND 45 animals had significantly lower DOPAC/DA ratios than all other ages, and PND 25 animals demonstrated lower ratios than PND 35 or PND 60 (A). In nav e animals, PND 60 animals demonstrated higher DOPAC/DA ratios than all other ages, and that PND 35 animals exhibited greater ratios than PND 45. With repeated exposure, PND 25 animals exhibit lower turnover values than PND 35 or PND 60, and PND 45 animals exhibited lower turnover than all ages. PND 45 animals did not show the elevation in turnover following repeated EtOH observed in all other ages (B). Repeated ethanol exposure shifted turnover rates. 0.5, 1.0 and 2.0 g/kg produced significant increases in DOPAC/ DA ratio over Saline. 1.0 and 2.0 g/kg elevated turnover over 0.2 and 0.5 g/kg (C).

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79 Analysis of Simple effects in Nave animals revealed that PND 60 animals demonstrated higher DOPAC/DA ratios than all other ages, and that PND 35 animals exhibited greater ratios than PND 45. However, with Repeated exposure, PND 25 animals exhibit lower turnover values than PND 35 or PND 60, and PND 45 animals exhibited lower turnover than all ages. PND 45 animals did not show an elevation in turnover following repea ted EtOH which was observed in all other ages (Figure Three: B). Additionally, following Repeated ethanol exposure there was a shift in turnover rates with 0.5, 1.0 and 2.0 g/kg producing significant increases in DOPAC/ DA ratio over Saline, and 1.0 and 2. 0 g/kg elevating turnover over 0.2 and 0.5 g/kg as well (Figure Three: C). DOPAC Peak Area Under the Curve Following Ethanol Administration Saline 0.2 0.5 1.0 2.0 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Dose Figure Four 0.5, 1.0, 2.0 g/kg 1.0, 2.0 g/kg Figure Four : Saline was significantly different from 0.5, 1.0 and 2.0 g/kg i.p., while 0.2 and 0.5 g/kg were significantly different from 1.0 and 2.0 g/kg i.p.

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80 Treatment Effects DOPAC Peak DOPAC (%) AUC varied significantly as a function of Treatment, F (2, 265) = 14.989, p < 0.05 and Dose, F (3, 265) = 8.770, p < 0.05 and exhibited an Age X Treatment, F (6, 265) = 2.947, p < 0.05 interaction. Subsequent Fishers PLSD revealed th at Saline was significantly different from 0.5, 1.0 and 2.0 g/kg i.p., while 0.2 and 0.5 g/kg were significantly different from 1.0 and 2.0 g/kg i.p. (Figure Four) Analysis of simple effects revealed that for PND 25 animals, Saline, Repeated and Expectancy were significantly different from Acute while for PND 35 animals Saline and Expectancy significantly differed from Acute (Figure Five). DOPAC Area Under The Curve Following Ethanol Age X Treatment Saline Acute Repeated Expectancy 0 5000 10000 15000 PND 25 PND 35 PND 45 PND 60 Figure Five Saline, Repeated, Expectancy Saline, Expectancy Treatment Figure Five : For PND 25 animals, Saline, Repeated and Expectancy differed from Acute. For PND 35 animals Saline and E xpectancy differed from Acute.

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81 DA Peak DA (%) AUC varied significantly as a function of Treatment, F (2, 265) = 14.316, p < 0.05 and Dose, F (3, 265) = 5.963, p < 0.05 and exhibited an Treatment X Dose, F (6, 265) = 2.820, p < 0.05 interaction. Analysis of simple effects revealed that for Acute treatment Saline and 0.2 g/kg were significantly different from 1.0 and 2.0 g/kg i.p. and 0.5 g/kg was significantly different from 2.0 g/kg i.p. For Repeated treatment 0.2 g/kg significantly differed from 1.0 and 2.0 g/kg i.p. Additionally, for 0.5 g/kg i.p. Saline differed significantly from Acute and Repeated, and Expectancy differed significantly from Acute. For 1.0 and 2.0 g/kg i.p. Saline differed significantly from Acute, Repeated and Expectancy, Expectanc y differed significantly from Acute and Repeated and Repeated differed significantly from Acute (Figure Six). DOPAC/DA Peak DOPAC/DA (%) AUC exhibited a significant Age X Treatment F (6, 265) = 2.769, p < 0.05 interaction. Analysis of simple effects revea led that for Acute treatment PND 25 animals were significantly different from PND 35, 45 and 60. Additionally, for PND 25 animals Saline and Acute differed significantly from Repeated and Expectancy treatments. Temporal Effects DA SALINE For PND 25 animal s Area Under the Curve analysis revealed two peaks following Saline challenge, 10 (AUC = 304.1) and 90 (AUC = 276.7) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 35 an imals Area Under the Curve analysis revealed no peaks

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82 DA Area Under the Curve Following Ethanol Treatment Acute Repeated Expectancy 0 5000 10000 15000 Saline 0.2 0.5 1.0 2.0 Figure Six 1.0, 2.0 g/kg 2.0 g/kg u Saline n Acute Repeated u u u u u u u u n n n n Treatment n Figure Six : For Acute treatment, Saline and 0.2 g/kg were significantly different from 1.0 and 2.0 g/kg i.p. and 0.5 g/kg was significantly different from 2.0 g/kg i.p. For Repeated treatment 0.2 g/ kg significantly differed from 1.0 and 2.0 g/kg i.p. For 0.5 g/kg i.p. Saline differed significantly from Acute and Repeated, and Expectancy differed significantly from Acute. For 1.0 and 2.0 g/kg i.p. Saline differed significantly from Acute, Repeated an d Expectancy. Expectancy differed significantly from Acute and Repeated and Repeated differed significantly from Acute. following Saline challenge. For PND 45 animals Area Under the Curve analysis revealed one peak following Saline challenge, 80 (AUC = 428.7) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 60 animals Area Under the Curve analysis revealed two peaks following Saline challenge, 70 (AUC = 508.7) and 110 (A UC = 418.5) minutes post

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83 injection. However, there were no significant elevations over baseline values for a given ten minute interval. No peaks differed significantly across age. ACUTE (Figure Seven A D) 0.2 G / KG E T OH For PND 25 animals Area Under the C urve analysis revealed one peak following 0.2 g/kg EtOH challenge, 40 (AUC = 1490) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 35 animals Area Under the Curve analysis revealed one peak following 0.2 g/kg EtOH challenge, 70 (AUC = 3157) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 45 animals Area Under the Curve analysis revealed on e peak following 0.2 g/kg EtOH challenge, 80 (AUC = 4597) minutes post injection. This value was significantly elevated over Baseline and over PND 25 animals at 80 minutes post injection. For PND 60 animals Area Under the Curve analysis revealed one peak following 0.2 g/kg EtOH challenge, 80 (AUC = 4008) minutes post injection. This value was significantly elevated over PND 25 animals at 80 minutes post injection but was not significantly different from baseline. 0.5 G / KG E T OH For PND 25 animals Area Und er the Curve analysis revealed one peak following 0.5 g/kg EtOH challenge, 40 (AUC = 7837) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 and 60 minutes post injection. For PND 35 animal s Area Under the Curve analysis revealed one peak following 0.5 g/kg EtOH challenge, 20 (AUC = 10384) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 45 animals Area Unde r the Curve analysis revealed one peak

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84 following 0.5 g/kg EtOH challenge, 50 (AUC = 4290) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 60 animals Area Under the Curve analysis revealed one peak following 0.5 g/kg EtOH challenge, 60 (AUC = 4503) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 50 and 60 minutes post injection. 1.0 G / KG E T OH For PND 25 anim als Area Under the Curve analysis revealed one peak following 1.0 g/kg EtOH challenge, 50 (AUC = 10572) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 60 minutes post injection. For PN D 35 animals Area Under the Curve analysis revealed one peak following 1.0 g/kg EtOH challenge, 60 (AUC = 15288) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 50 70 minutes post injection For PND 45 animals Area Under the Curve analysis revealed one peak following 1.0 g/kg EtOH challenge, 40 (AUC = 11734) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 60 animals Area Under the Curve analysis revealed one peak following 1.0 g/kg EtOH challenge, 60 (AUC = 5539) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 60 minutes post injection.

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85 0.2 g/kg EtOH -100 100 200 300 400 500 PND 25 PND 35 PND 45 PND 60 Time (Min) Acute 0.2 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE 0.5 g/kg EtOH -100 100 200 300 400 500 PND 25 PND 35 PND 45 PND 60 Time (Min) Effects of Acute EtOH Acute 0.5 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE Figure Seven A. C. 1.0 g/kg EtOH -100 100 200 300 400 500 PND 25 PND 35 PND 45 PND 60 Time (Min) Acute 1.0 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE 2.0 g/kg EtOH -100 100 200 300 400 500 PND 25 PND 35 PND 45 PND 60 Time (Min) Acute 2.0 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE D. C. B. Fig ure Seven : Acute EtOH administration elevated NAcc DA levels in all ages in a dose dependent fashion. PND 45 animals were unique in demonstration peak elevations at 1.0 g/kg. All other ages demonstrated either peak elevations at 2.0 g/kg or, in the case of PND 35 animals, a plateau at 1.0 and 2.0 g/kg. Temporally, for all ages, DA elevations occurred between 30 and 70 minutes post injection.

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86 2.0 G / KG E T OH For PND 25 animals Area Under the Curve analysis revealed one peak following 2.0 g/kg EtOH challenge, 6 0 (AUC = 14969) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 50 and 60 minutes post injection. For PND 35 animals Area Under the Curve analysis revealed one peak following 2.0 g/kg EtOH c hallenge, 50 (AUC = 15097) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 30 70 minutes post injection. For PND 45 animals Area Under the Curve analysis revealed one peak following 2.0 g/ kg EtOH challenge, 40 (AUC = 8060) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 50 minutes post injection. For PND 60 animals Area Under the Curve analysis revealed one peak following 2.0 g/kg EtOH challenge, 40 (AUC = 11122) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 50 minutes post injection. REPEATED (Figure Eight A D) 0.2 G / KG E T OH For PND 25 animals Area Under the Curve analysis revealed one peak following 0.2 g/kg EtOH challenge, 20 (AUC = 4132) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 35 animals Area Under the Cu rve analysis revealed one peak following 0.2 g/kg EtOH challenge, 20 (AUC = 4133) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 45 animals Area Under the Curve analysis revealed one peak following 0.2 g/kg EtOH challenge, 20

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87 (AUC = 4199) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 60 animals Area Under the Curve analysis revealed on e peak following 0.2 g/kg EtOH challenge, 40 (AUC = 2730) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. 0.5 G KG E T OH For PND 25 animals Area Under the Curve analysis revealed one peak following 0.5 g/kg EtOH challenge, 30 (AUC = 5553) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 20 40 minutes post injection. For PND 35 animals Area Under the Curve analysis revealed one peak following 0.5 g/kg EtOH challenge, 50 (AUC = 8274) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 45 animals Area Under the Curve analysis revealed one peak following 0.5 g/kg EtOH challenge, 40 (AUC = 4474) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 50 minutes post injection. For PND 60 animals Area Under the Curve analysis reve aled one peak following 0.5 g/kg EtOH challenge, 40 (AUC = 3742) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. 1.0 G / KG E T OH For PND 25 animals Area Under the Curve analysis r evealed one peak following 1.0 g/kg EtOH challenge, 40 (AUC = 7648) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 20 50 minutes post injection. For PND 35 animals Area Under the Curve an alysis revealed two peaks following 1.0 g/kg EtOH challenge, 40 (AUC = 7724) and 100 (AUC = 322.5) minutes

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88 post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 30 40 minutes post injection. For PND 45 animals Area Under the Curve analysis revealed one peak following 1.0 g/kg EtOH challenge, 40 (AUC = 8135) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 minutes post injection. For PND 60 animals Area Under the Curve analysis revealed one peak following 1.0 g/kg EtOH challenge, 80 (AUC = 5057) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. 2.0 G / KG E T OH For PND 25 animals Area Under the Curve analysis revealed one peak following 2.0 g/kg EtOH challenge, 30 (AUC = 6764) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 30 40 minutes post injection. For PND 35 animals Area Under the Curve analysis revealed one peak following 2.0 g/kg EtOH challenge, 30 (AUC = 8488) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 30 40 minutes post inj ection. For PND 45 animals Area Under the Curve analysis revealed one peak following 2.0 g/kg EtOH challenge, 60 (AUC = 6921) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 60 animals Area Under the Curve analysis revealed one peak following 2.0 g/kg EtOH challenge, 60 (AUC = 7345) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval.

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89 0.2 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Repeated 0.2 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE 0.5 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Effects of Repeated EtOH Repeated 0.5 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE Figure Eight 1.0 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Repeated 1.0 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE 2.0 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Repeated 2.0 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE D. C. B. A. Figure Eight : Repeated EtOH administration elevated NAcc DA levels in all ages. For PND 25 and 35 animals, effects asymptote 0.5 g/kg. PND 45 animals reached plateau at 1.0 g/kg while PND 60 animals demonstrated a dose dependent increase. Temporally, effects occurred b etween 20 and 80 minutes post injection.

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90 EXPECTANCY (Figure Nine A D) 0.2 G / KG E T OH For PND 25 animals Area Under the Curve analysis revealed one peak following Saline challenge, 10 (AUC = 4849) minutes post injection. However, there were no significant e levations over baseline values for a given ten minute interval. For PND 35 animals Area Under the Curve analysis revealed three peaks following Saline challenge, 10 (AUC = 1269), 50 (AUC = 124.6) and 110 (AUC = 213.8) minutes post injection. However, the re were no significant elevations over baseline values for a given ten minute interval. For PND 45 animals Area Under the Curve analysis revealed one peak following Saline challenge, 60 (AUC = 9317) minutes post injection. However, there were no signific ant elevations over baseline values for a given ten minute interval. For PND 60 animals Area Under the Curve analysis revealed one peak following Saline challenge, 80 (AUC = 2185) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. 0.5 G / KG E T OH Area Under the Curve analysis revealed one peak following Saline challenge, 40 (AUC = 1198) minutes post injection. However, there were no significant elevations over baseline values for a giv en ten minute interval. For PND 35 animals Area Under the Curve analysis revealed three peaks following Saline challenge, 30 (AUC = 2413), 80 (AUC = 57.14) and 110 (AUC = 390.5) minutes post injection. Analysis for significance versus baseline (Dunnetts ,) revealed a significant elevation 30 minutes post injection. For PND 45 animals Area Under the Curve analysis revealed one peak following Saline challenge, 30 (AUC = 5043) minutes post injection. However, there were no significant elevations over basel ine values for a given ten minute interval. For

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91 PND 60 animals Area Under the Curve analysis revealed one peak following Saline challenge, 20 (AUC = 766.8) minutes post injection. However, there were no significant elevations over baseline values for a g iven ten minute interval. 1.0 G / KG E T OH For PND 25 animals Area Under the Curve analysis revealed one peak following Saline challenge, 30 (AUC = 3019) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant el evation 30 minutes post injection. For PND 35 animals Area Under the Curve analysis revealed two peaks following Saline challenge, 50 (AUC = 5431) and 110 (AUC = 967.7) minutes post injection. Analysis for significance versus baseline (Dunnetts,) reveal ed a significant elevation 50 minutes post injection. For PND 45 animals Area Under the Curve analysis revealed one peak following Saline challenge, 40 (AUC = 2409) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 50 minutes post injection. For PND 60 animals Area Under the Curve analysis revealed two peaks following Saline challenge, 40 (AUC = 2312) and 80 (AUC = 179.8) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 minutes post injection. 2.0 G / KG E T OH For PND 25 animals Area Under the Curve analysis revealed one peak following Saline challenge, 30 (AUC = 3039) minutes post injection. Analysis for significance versu s baseline (Dunnetts,) revealed a significant elevation 30 minutes post injection. For PND 35 animals Area Under the Curve analysis revealed one peak following Saline challenge, 40 (AUC = 2265) minutes post injection. However, there were no significant

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92 0.2 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Expectancy 0.2 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE 0.5 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Effects of Expected EtOH Expectancy 0.5 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE Figure Nine A. 1.0 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Expectancy 1.0 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE 2.0 g/kg EtOH -100 100 200 300 PND 25 PND 35 PND 45 PND 60 Time (Min) Expectancy 2.0 EtOH 0 10000 20000 PND 25 PND 35 PND 45 PND 60 AGE D. C. B. Figure Nine : All animals exhibited a Saline induced elevation in NAcc DA following EtOH pretreatment.

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93 elevations over baseline values for a given ten minute interval. For PND 45 animals Area Under the Curve analysis revealed one peak following Sali ne challenge, 60 (AUC = 3920) minutes post injection. However, there were no significant elevations over baseline values for a given ten minute interval. For PND 60 animals Area Under the Curve analysis revealed one peak following Saline challenge, 40 (A UC = 4210) minutes post injection. Analysis for significance versus baseline (Dunnetts,) revealed a significant elevation 40 and 60 minutes post injection. Discussion The initiation of drug use and subsequent abuse liability is an issue of tremendous soc ial significance in the adolescent population (SAMHSA 2003c, 2003a) Rates of drug use initiation are highest during the adolescent period and the risk of subsequent addiction is directly relat ed to the age of use onset, with individuals initiating use prior to age 14 four times as likely to experience drug abuse or dependence related problems (Robins and Przybeck 1985; Anthony and Petronis 1995; DeWit et al. 2000) This relationship begs the ques tion: do those predisposed to substance abuse and addiction initiate use earlier in life, or does the initiation of drug use earlier in life result in an increased propensity to drug addiction? While a few behavioral studies have attempted to address this in the human population, it is likely that if early drug use predisposes one to addiction, the biological mechanism for this process is drug induced alterations in the development of neural systems involved in motivated behavior. A preponderance of eviden ce implicates the mesolimbic DA system as directly involved in a variety of basic motivational processes and appears to be key in the initiation and maintenance of drug administration behavior (Phillips et al. 1983 ; Hoebel

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94 1985; Koob 1992a, 1992b; Koob et al. 1994; Koob 1996b, 1999; Leshner and Koob 1999; Koob 2000) Further, developmental studies have indicated that this system is undergoing substantial changes during the adolescent period (Seeman et al. 1987; Levy 1991; Teicher et al. 1993; Andersen and Gazzara 1994; Teicher et al. 1995; Coulter et al. 1996; Andersen et al. 1997; Anderson et al. 1997; Coulter et al. 1997; Teicher et al. 1998; Andersen and Teicher 1999; Seeman 1999a; Seeman 1999b; Andersen and Teicher 2000; Andersen et al. 2002) This relationship has lead investigators to suggest that some observed behavioral differences in adul ts and adolescents are mediated by alterations in the functionality of the DAergic system in the NAcc, as well as by the changing strength of various modulatory components within and innervating the NAcc (Spear 2000b; Chambers et al. 2003; Smith 2003) These developmental transitions mediate the observed impulsiveness and risk taking behaviors common to the adolescent period. As such, they are likely to mediate the initiation and maintenance of drug use, a set of behaviors that are clearly risky and often impulsive in nature (Zuckerman 1983, 1986, 1994) The present results suggest that the mesolimbic projection to the NAcc septi is functionally different across adolescence. Basal DAergic tone differs across this period (Figure Two: A) and basal tone has previously been shown to be inversely related to phasic DAergic activity, sugg esting that DAergic responsivity to stimuli may vary with age. These DAergic differences may be mediated in part by differences in metabolism as basal DOPAC levels also vary during the adolescent period (Figure One: A). These differences are most dramati cally represented by transitional changes in DOPAC/DA ratio, indicative of changes in turnover processes across development (Figure Three: A).

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95 In each case (DA, DOPAC or DOPAC/DA ratio) PND 45 animals (the equivalent of late adolescence) reveal themselves as neurochemically unique in NAcc responses. PND 45 animals exhibit elevated basal DA levels in the NAcc relative to all other ages while exhibiting lower DOPAC values than other adolescents or adults. These factors combine to produce an animal with ver y low turnover ratios relative to other ages (Figure Three: A), indicating a decreased ability at this age to effectively regulate extracellular DA concentrations. This increase in basal DAergic tone, in conjunction with the influence of tonic DA levels o n phasic activity suggests that PND 45 animals may exhibit a decrease in DAergic responsivity to DAergic agonists relative to other ages. Alternatively, reduced turnover rates may indicate an inability to effectively metabolize DA that would result in ele vated NAcc DA in response to an appropriate stimulus. All ages exhibited a change in basal DA and metabolites with repeated ethanol exposure. Basal DOPAC levels increased in a dose dependent fashion (Figure One: B) while all doses of EtOH appeared to prod uce elevated basal DA levels (Figure Two: B). Consequently, DOPAC/DA ratios also increased in a dose dependent fashion (Figure Three: C) a tiered effect carried by the dose dependent influence of repeated EtOH on NAcc DOPAC concentrations. Interestingly, when examined in ratio form (DOPAC/DA) PND 45 animals were again unique in their response to ethanol. While all other ages exhibited an increase in DOPAC/DA ratio with repeated ethanol, PND 45 animals exhibited no change in basal DOPAC/DA turnover (Figur e Three: B). This result suggests that, while in most ages repeated EtOH and subsequent potentiation of DA release results in an increased rate of DA metabolism, in PND 45 animals no compensatory change in DAergic metabolic rate exists to blunt the neuroc hemical effects

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96 of repeated EtOH on NAcc DAergic projections. This failure to alter basal NAcc DAergic metabolism in response to repeated treatment suggests that PND 45 may be more susceptible to the incentive motivational properties of EtOH with multiple exposures. This outcome has been observed using an EtOH conditioned place preference paradigm (see Experiment Two). For all ages, the administration of EtOH produced significant increases in NAcc DOPAC in a dose dependent fashion regardless of treatment regimen (Figure Four). Further, for PND 25 and 35 animals specifically, Acute EtOH elevated DOPAC levels to a greater degree than PND 45 and 60 animals (Figure Five). This increase is indicative of an increase in DA levels following EtOH injection and su ggests that younger animals exhibit a greater acute response to EtOH, either through increased DA release or an acute increase in metabolism, than older animals. For all ages, EtOH administration produced an elevation in DA concentration regardless of trea tment regimen (Figure Six). Acute administration produced dose dependent increases in NAcc DA following EtOH challenge. Repeated treatment also produced dose dependent increases in NAcc DA, however the repeated administration of high doses (1.0 and 2.0 g /kg EtOH) blunted the DAergic response to EtOH challenge when compared to Acute doses of EtOH. Further, doses of 1.0 and 2.0 g/kg administered repeatedly resulted in significant elevations of NAcc DA in animals given a Saline challenge on test (Expectancy animals). These responses suggest, in general, that EtOH effectively elevates NAcc DA across ages, that repeated administration of high doses blunts acute responses to EtOH and that cues associated with high doses of EtOH can

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97 effectively alter NAcc DA fo llowing repeated EtOH exposure in the same context. These results are critical for understanding the process of addiction in general. Contemporary theory regarding the role of NAcc DA in the process of addiction (and in motivated behavior in general) is that enhanced DAergic activity in the NAcc facilitates the salience (perceptual significance) of stimuli, increasing the ability of secondary stimuli to acquire incentive motivational properties or to facilitate the inherent motivational capacity of prima ry reinforcers (Berridge and Robinson 1998; Di Chiara 1999; Di Chiara et al. 1999; Robinson and Berridge 2000; Berridge and Robinson 2003; McClure et al. 2003; Rob inson and Berridge 2003) The present data indicates that repeated high doses of EtOH results in DAergic responses to cues indicative of EtOH administration, suggesting Pavlovian conditioning of a drug induced neurochemical response. As such, according to incentive salience models, perception of secondary stimuli with associations to EtOH administration exhibit enhanced stimulus salience and therefore are more likely to influence behavior choices than stimuli lacking salience enhancing properties. In ot her words, the classically conditioned neurochemical response to drug related cues produces enhanced perceptual awareness of these cues relative to other information in the environment, biasing thoughts and subsequent behavior toward drug use. Although, in general repeated ethanol exposure blunts subsequent responding to ethanol administration, late adolescent animals (PND 45) are unique in their pattern of response to ethanol administration at moderate doses (1.0 g/kg/i.p.). These animals exhibit a potent iated NAcc DA response on subsequent challenge, rather than a reduced response. According to the conditioned incentive salience model discussed above, the

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98 potentiation of NAcc DA release to repeated ethanol in late adolescent animals allows for the streng thening of contextual associations to a greater degree than in animals whose later ethanol exposures produce reduced responsivity. These successive Pavlovian pairings may imbue context and cues with greater motivational strength, promoting drug related st imulus salience and creating associations with greater resistance to extinction or remodeling. This enhanced conditionability is evidenced in PND 45 animals using an ethanol CPP paradigm. The present results clearly implicate ethanol exposure during adole scence as a critical risk factor in the development of alcohol abuse and addiction. Although not a necessary factor for later dependence, exposure to ethanol during adolescence does result in significant neurobiological changes in systems intimately invol ved in motivated behaviors. These changes do not influence addiction in a causal sense, but rather probabilistically. By enhancing the establishment and maintenance of drug stimuli associations, the likelihood of drug related thoughts and behaviors becom es enhanced within the appropriate stimulus context. Further, each subsequent exposure within this context strengthens the association further and increases the ability of the environment to drive behavior choice. The behavioral patterns classified as ad diction are revealed over successive experiences, as possibility shifts towards certainty and choice behavior comes primarily under stimulus control.

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99 References Andersen, S. L. and R. A. Gazzara (1994). The development of d2 autorece ptor mediated modulation of k(+) evoked dopamine release in the neostriatum. Brain Res Dev Brain Res 78 (1): 123 30. Andersen, S. L., M. Rutstein, J. M. Benzo, J. C. Hostetter and M. H. Teicher (1997). Sex differences in dopamine receptor overproduction and elimination. Neuroreport 8 (6): 1495 8. Andersen, S. L. and M. H. Teicher (1999). Cyclic adenosine monophosphate (camp) changes dramatically across periadolescence and region. Society for Neuroscience Abstracts 25 : 1471. Andersen, S. L. and M. H. Teicher ( 2000). Sex differences in dopamine receptors and their relevance to adhd. Neurosci Biobehav Rev 24 (1): 137 41. Andersen, S. L., A. P. Thompson, E. Krenzel and M. H. Teicher (2002). Pubertal changes in gonadal hormones do not underlie adolescent dopamine re ceptor overproduction. Psychoneuroendocrinology 27 (6): 683 91. Anderson, S. L., N. L. Dumont and M. H. Teicher (1997). Developmental differences in dopamine synthesis inhibition by (+/ ) 7 oh dpat. Naunyn Schmiedebergs Arch Pharmacol 356 (2): 173 81. Anlez ark, G. M., G. W. Arbuthnott, J. E. Christie, T. J. Crow and P. J. Spear (1972). Electrical self stimulation with brain stem electrodes. J Physiol (Lond) 227 (2): 6P 7P. Anlezark, G. M., G. W. Arbuthnott, J. E. Christie, T. J. Crow and P. J. Spear (1974). E lectrical self stimulation in relation to cells of origin of catecholamine containing neural systems ascending from the brain stem. J Physiol (Lond) 237 (2): 31P 32P. Anthony, J. C. and K. R. Petronis (1995). Early onset drug use and risk of later drug prob lems. Drug Alcohol Depend 40 (1): 9 15. Berridge, K. C. and T. E. Robinson (1998). What is the role of dopamine in reward: Hedonic impact, reward learning, or incentive salience? Brain Res Brain Res Rev 28 (3): 309 69. Berridge, K. C. and T. E. Robinson (200 3). Parsing reward. Trends Neurosci 26 (9): 507 13. Chambers, R. A., J. R. Taylor and M. N. Potenza (2003). Developmental neurocircuitry of motivation in adolescence: A critical period of addiction vulnerability. Am J Psychiatry 160 (6): 1041 52. Coulter, C. L., H. K. Happe and L. C. Murrin (1996). Postnatal development of the dopamine transporter: A quantitative autoradiographic study [published erratum appears in brain res dev brain res 1996 jan 2;98(1):150]. Brain Res Dev Brain Res 92 (2): 172 81.

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100 Coulter, C. L., H. K. Happe and L. C. Murrin (1997). Dopamine transporter development in postnatal rat striatum: An autoradiographic study with [3h]win 35,428. Brain Res Dev Brain Res 104 (1 2): 55 62. Crow, T. J. (1971). The relation between electrical self stimula tion sites and catecholamine containing neurons in the rat mesencephalon. Experientia 27 (6): 662. Crow, T. J. (1972). A map of the rat mesencephalon for electrical self stimulation. Brain Res 36 (2): 265 73. Crow, T. J. (1973). Catecholamine containing neur ones and electrical self stimulation. 2. A theoretical interpretation and some psychiatric implications. Psychol Med 3 (1): 66 73. Demotes Mainard, J., C. Henry, Y. Jeantet, J. Arsaut and E. Arnauld (1996). Postnatal ontogeny of dopamine d3 receptors in the mouse brain: Autoradiographic evidence for a transient cortical expression. Brain Res Dev Brain Res 94 (2): 166 74. DeWit, D. J., E. M. Adlaf, D. R. Offord and A. C. Ogborne (2000). Age at first alcohol use: A risk factor for the development of alcohol dis orders. Am J Psychiatry 157 (5): 745 50. Di Chiara, G. (1999). Drug addiction as dopamine dependent associative learning disorder. Eur J Pharmacol 375 (1 3): 13 30. Di Chiara, G., G. Tanda, V. Bassareo, F. Pontieri, E. Acquas, S. Fenu, C. Cadoni and E. Carbo ni (1999). Drug addiction as a disorder of associative learning. Role of nucleus accumbens shell/extended amygdala dopamine. Ann N Y Acad Sci 877 : 461 85. Hoebel, B. G. (1985). Brain neurotransmitters in food and drug reward. Am J Clin Nutr 42 (5 Suppl): 11 33 50. Hoebel, B. G., A. P. Monaco, L. Hernandez, E. F. Aulisi, B. G. Stanley and L. Lenard (1983). Self injection of amphetamine directly into the brain. Psychopharmacology (Berl) 81 (2): 158 63. Koob, G. F. (1992a). Drugs of abuse: Anatomy, pharmacology a nd function of reward pathways. Trends Pharmacol Sci 13 (5): 177 84. Koob, G. F. (1992b). Neural mechanisms of drug reinforcement. Ann N Y Acad Sci 654 : 171 91. Koob, G. F. (1992c). Neurobiological mechanisms in cocaine and opiate dependence. Res Publ Assoc Res Nerv Ment Dis 70 : 79 92. Koob, G. F. (1996a). Drug addiction: The yin and yang of hedonic homeostasis. Neuron 16 (5): 893 6. Koob, G. F. (1996b). Hedonic valence, dopamine and motivation. Mol Psychiatry 1 (3): 186 9.

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101 Koob, G. F. (1999). The role of the striatopallidal and extended amygdala systems in drug addiction. Ann N Y Acad Sci 877 : 445 60. Koob, G. F. (2000). Neurobiology of addiction. Toward the development of new therapies. Ann N Y Acad Sci 909 : 170 85. Koob, G. F., B. Caine, A. Markou, L. Pulvir enti and F. Weiss (1994). Role for the mesocortical dopamine system in the motivating effects of cocaine. NIDA Res Monogr 145 : 1 18. Leshner, A. I. and G. F. Koob (1999). Drugs of abuse and the brain. Proc Assoc Am Physicians 111 (2): 99 108. Levy, F. (1991 ). The dopamine theory of attention deficit hyperactivity disorder (adhd). Aust N Z J Psychiatry 25 (2): 277 83. McClure, S. M., N. D. Daw and P. R. Montague (2003). A computational substrate for incentive salience. Trends Neurosci 26 (8): 423 8. Moghaddam, B. and B. S. Bunney (1989). Differential effect of cocaine on extracellular dopamine levels in rat medial prefrontal cortex and nucleus accumbens: Comparison to amphetamine. Synapse 4 (2): 156 61. Odell, W. D. (1990). Sexual maturation in the rat. Control o f onset of puberty. M. M. Grumbach, P. C. Sizonenko and M. L. Aubert. Baltimore, Williams and Wilkins : 183 210. Olds, M. E. and J. L. Fobes (1981). The central basis of motivation: Intracranial self stimulation studies. Annu Rev Psychol 32 : 523 74. Phillip s, A. G., C. L. Broekkamp and H. C. Fibiger (1983). Strategies for studying the neurochemical substrates of drug reinforcement in rodents. Prog Neuropsychopharmacol Biol Psychiatry 7 (4 6): 585 90. Phillips, T. J. and E. H. Shen (1996). Neurochemical bases of locomotion and ethanol stimulant effects. Int Rev Neurobiol 39 : 243 82. Philpot, R. M., S. McQuown and C. L. Kirstein (2001). Stereotaxic localization of the developing nucleus accumbens septi. Brain Res Dev Brain Res 130 (1): 149 53. Roberts, D. C. (198 9). Breaking points on a progressive ratio schedule reinforced by intravenous apomorphine increase daily following 6 hydroxydopamine lesions of the nucleus accumbens. Pharmacol Biochem Behav 32 (1): 43 7. Robins, L. N. and T. R. Przybeck (1985). Age of onse t of drug use as a factor in drug and other disorders. NIDA Res Monogr 56 : 178 92. Robinson, T. E. and K. C. Berridge (2000). The psychology and neurobiology of addiction: An incentive sensitization view. Addiction 95 Suppl 2 : S91 117. Robinson, T. E. and K. C. Berridge (2003). Addiction. Annu Rev Psychol 54 : 25 53. SAMHSA (2003a). Alcohol use by persons under the legal drinking age of 21, Substance Abuse and Mental Health Services Administration.

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102 SAMHSA (2003b). Overview of findings from the 2002 nasional survey on drug use and health. Rockville, Office of Applied Studies. SAMHSA (2003c). Results from the 2002 national survey on drug use and health: National findings. Rockville, Substance Abuse and Mental Health Services Administration. Seeman, P. (1999a). Images in neuroscience. Brain development, x: Pruning during development. Am J Psychiatry 156 (2): 168. Seeman, P., N. H. Bzowej, H. C. Guan, C. Bergeron, L. E. Becker, G. P. Reynolds, E. D. Bird, P. Riederer, K. Jellinger, S. Watanabe and et al. (1987). Hu man brain dopamine receptors in children and aging adults. Synapse 1 (5): 399 404. Seeman, T. (1999b). Developmental influences across the life span. Ann N Y Acad Sci 896 : 64 5. Smith, R. F. (2003). Animal models of periadolescent substance abuse. Neurotoxi col Teratol 25 (3): 291 301. Spear, L. (2000a). Modeling adolescent development and alcohol use in animals. Alcohol Res Health 24 (2): 115 23. Spear, L. P. (2000b). The adolescent brain and age related behavioral manifestations. Neurosci Biobehav Rev 24 (4): 417 63. Stanwood, G. D., S. McElligot, L. Lu and P. McGonigle (1997). Ontogeny of dopamine d3 receptors in the nucleus accumbens of the rat. Neurosci Lett 223 (1): 13 6. Stellar, J. R., A. E. Kelley and D. Corbett (1983). Effects of peripheral and central d opamine blockade on lateral hypothalamic self stimulation: Evidence for both reward and motor deficits. Pharmacol Biochem Behav 18 (3): 433 42. Tarazi, F. I. and R. J. Baldessarini (2000). Comparative postnatal development of dopamine d(1), d(2) and d(4) re ceptors in rat forebrain. Int J Dev Neurosci 18 (1): 29 37. Teicher, M. H., S. L. Andersen and J. C. Hostetter, Jr. (1995). Evidence for dopamine receptor pruning between adolescence and adulthood in striatum but not nucleus accumbens. Brain Res Dev Brain R es 89 (2): 167 72. Teicher, M. H., N. I. Barber, H. A. Gelbard, A. L. Gallitano, A. Campbell, E. Marsh and R. J. Baldessarini (1993). Developmental differences in acute nigrostriatal and mesocorticolimbic system response to haloperidol. Neuropsychopharmacol ogy 9 (2): 147 56. Teicher, M. H., N. L. Dumont and S. L. Andersen (1998). The developing prefrontal cortex: Is there a transient interneuron that stimulates catecholamine terminals? Synapse 29 (1): 89 91. Zito, K. A., G. Vickers and D. C. Roberts (1985). Di sruption of cocaine and heroin self administration following kainic acid lesions of the nucleus accumbens. Pharmacol Biochem Behav 23 (6): 1029 36.

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103 Zuckerman, M. (1974). The sensation seeking motive. Prog Exp Pers Res 7 : 79 148. Zuckerman, M. (1983). Biolog ical bases of sensation seeking, impulsivity, and anxiety. Hillsdale, Erlbaum. Zuckerman, M. (1986). Sensation seeking and the endogenous deficit theory of drug abuse. NIDA Res Monogr 74 : 59 70. Zuckerman, M. (1994). Behavioral expressions and biosocial ba ses of sensation seeking. New York, Cambridge University Press.

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104 GENERAL DISCUSSION The demonstration of reduced NP during adolescence, even an aversion to novelty in late adolescence, was an unanticipated outcome regarding the ontogeny of NP. Al though under examined in rodent models, there are a few reports indicating that responsiveness to novel stimuli peaks during adolescence in mice and rats (Adriani et al. 1998; Spear 2000; Laviola et al. 2003) This is the anticipated profile in adolescents given more extensive reports of sensation seeking, risk taking and harm avoidance in humans (Kandel 1982; Zu ckerman 1994; Laviola et al. 1999; Spear 2000; Martin et al. 2002; Chambers et al. 2003; Crawford et al. 2003) The lack of agreement of this study with previous reports in rodents and humans is likely a function of the paradigm utilized. The playground m aze is a true measure of NP, while other procedures tend to measure behavioral sensitivity to novelty, usually in the form of changes in locomotor activity. Methods utilizing locomotor activity in the presence of novelty as a measure, fail to identify the hedonic aspects of the novel stimulus and are incorrectly identified as NP procedures and correlates to sensation seeking. In fact, increased behavioral activation in the presence of novelty has been considered a measure of anxiety in humans and an indicat or of introversion or shyness (Bell et al. 1995) As such, it may be more suitable to characterize locomotion in the presence of novelty as sensation responding rather than sensation seeking.

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105 It is not unreasonable to observe a positive relationship between novelty induced activity and responsivness to drugs of abuse. Given the DAergic commonality of the motor systems and the systems mediating drug motivation some relationship would be expected using activity as a dependent me asure. Alternately, numerous studies link anxiety and perceived stress to an increased risk of substance abuse and addiction (Baer et al. 1987; Deykin et al. 1987; Johnson and Pandina 1993; Baer and Bray 1999; DeWit et al. 1999) therefore stress induced locomotion in the presence of novelty would also be anticipated to predict substance use effectively. Because the playground maze is a measure of NP, a nd the exploration of the unknown is inherently risky, this result is better characterized of as an indicator of curiosity, risk taking and harm avoidance. When considered in this frame the data presented here indicate a developmental reduction in these be haviors through the adolescent period. Because increasing habituation trials attenuates a novelty aversion in late adolescents and induces a NP in adults it is most reasonable to assume that the reduction in NP through adolescence represents a transitional increase in neophobia or harm avoidance, and not reduced curiosity or attraction to novelty. In essence, this developmental pattern supports the notion of the transition to contextual regulation of behavior that Spear (2000) suggested occurs during adoles cence. In adults ethanol induce CPP outcomes are consistent with the literature in rats (S tewart and Grupp 1986, 1989; Gauvin and Holloway 1992; Holloway et al. 1992; Schechter and Krimmer 1992) This consistency lends support to the interesting patterns observed in adolescence. Late adolescent animals exhibit an ethanol induced CPP at doses o f 0.5 and 1.0 g/kg i.p. indicating that late adolescent animals find the effects of

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106 ethanol at moderate doses rewarding. In contrast, adults exhibit a progressive increase in avoidance of the ethanol paired environment while pre and early adolescent anima ls do not demonstrate conditioning at 0.5 g/kg and exhibit a strong aversion at 1.0 g/kg. Prior data from our laboratory has shown that PND 25 animals exhibit significant increases in NAcc DA when given 1.0 g/kg of ethanol i.p. (Philpot and Kirstein 1998) Viewing dopaminergic activity in the NAcc as st rictly mediating reward, these data would appear to be in conflict with the alcohol place aversion observed at this age. However, DAergic activity may be mediating reinforcement by way of increasing environmental salience of both appetitive and aversive s timuli. Viewing NAcc DA as mediating stimulus salience suggests that the magnitude, but not the direction, of drug conditioning is related to NAcc DA levels (for discussion see Appendicies). Factors mediating the direction of the effect may be related to a nxiety observed in the NP paradigm. Late adolescent animals were the only age to exhibit a novelty related aversion and it may be an underlying increase in anxiety that renders the sedative/anxiolytic effects of ethanol rewarding in this age. As previously mentioned there is a strong relationship between increased stress and increased risk of alcohol use in the adolescent specifically (Baer et al. 1987; Deykin et al. 1987; Johnson and Pandina 1993; Baer and Bray 1999; DeWit et al. 1999) Further, this is substantial evidence indicating elevated responsiveness to stressors during adolescence in both humans and rodents (for discussion see Spear, 2000). The refore, it is possible that a developmental transition in sensitivity to stressors makes the late adolescent animal particularly vulnerable to the effects of ethanol. The neurochemical data clearly indicate developmental differences in both the basal and e thanol mediated output of accumbal DA, a factor that may influence both the

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107 age dependent sensitivity to ethanol induced place preference and the developmental transitions in response to novelty. Again late adolescent animals demonstrate a unique profile, exhibiting higher basal concentrations of DA and exhibiting resistance to turnover increases (DOPAC/DA) produced by repeated ethanol treatment in other ages. The ability of ethanol to induce a CPP in PND 45 animals may result from the large, sustained DA r esponse across repeated exposures that is not observed in other ages. The continued elevated response across exposures allows for more effective conditioning, be it through sustained reward and/or enhanced salience as proposed in the attentional model (see Appendix). Enhanced stimulus salience, through elevated NAcc DA output may be at the heart of the observed NP profile. Although the neurochemical response to novelty was not tested, the pattern of basal DA levels in the NAcc is a near mirror image of the developmental pattern for NP. Given the relationship between drugs of abuse, sensation seeking and the mesolimbic DA system, this coincidence warrants further investigation. Conclusions This project characterized the ontogenic profiles of ethanols rewardi ng effects and underlying neurochemical actions as well as examined the transitional development of behaviors associated with addiction in the adolescent animal. These studies revealed: 1) developmental differences in behavioral responding to novel stimuli ; 2) ontogenic differences in the rewarding efficacy of ethanol; 3) basal differences, as a function of development, in the tonic activity of DA in the NAcc; 4) differences in the DAergic response to acute ethanol administration across adolescence; 5) age dependent differences in the development of cellular tolerance to the DAergic effects of ethanol; and 6)

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108 developmental differences in the ability of alcohol to establish neurochemical expectancies of drug, i.e. conditioned neurochemical responses to drug a ssociated cues. The present project provides clear behavioral evidence for a distinct biological transition in novelty seeking through the adolescent period. The observed pattern, given the relationship between sensation seeking, NP and substance abuse li ability in humans (Zuckerman 1986; McCourt et al. 1993) suggests a developme ntal transition in the rewarding efficacy if drugs of abuse. The ethanol CPP paradigm confirms that prediction, with PND 45 animals exhibiting a unique response profile clearly indicating that adolescence represents a unique risk period for alcohol addicti on. The present project confirms observations made in the human population and further, because this is an animal model of addiction, suggests the distinct possibility that basic biological factors may be fundamental in the manifestation of adolescent alco hol use and subsequent addiction. The results suggest the possibility that the biological transition that occurs during adolescence may manifest itself, in part, as increased preference for the appetitive aspects of alcohol. Given the substantial amount of data that suggests higher risk of alcoholism in those initiating alcohol use in adolescence (Hawkins et al. 1992; Hawkins et al. 1997; Grant and Dawson 1998; DeWit et al. 1999; DeWit et al. 2000; Guo et al. 2000) ; it is importa nt to examine the mechanisms which underlie these behavioral tendencies. The present project demonstrates that reward mechanisms do not function identically in late adolescent and young adult animals and that exposure to ethanol during adolescence results in significant neurobiological changes in systems intimately involved in motivated behaviors. The attentional model of addiction (see Appendix) indicates that modified DA

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109 activity contributes to addiction by enhancing the establishment and maintanence of drug stimuli associations, increasing the likelihood of drug related thoughts and behaviors within the drug associated context. Further, each subsequent exposure within these context strengthens the association further and increases the ability of the env ironment to drive behavior choice. The behavioral patterns classified as addiction are revealed over successive experiences, as possibility shifts towards certainty and choice behavior comes primarily under stimulus control. The functional neurochemistry of the developing adolescent leaves them particularly vulnerable to this process.

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126 APPENDICES

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Appendix A 127 STEREOTAXIC LOCALIZA TION OF THE DEVELOPI N G NUCLEUS ACCUMBENS SEPTI Abstract The nucleus accumbens septi (NAcc) has been implicated as a mediator of a variety of disorders, most notably substance abuse. The development of this system is a critical area for investigation, and has been largely ov erlooked. Specifically, few studies have focussed on dopamine (DA), its neurochemical pathways and the long term consequences of manipulating the dopaminergic (DAergic) system in the developing animal. Important insight into the establishment of addictio n, its development and time course, may be found by examining the development of the periadolescent DA system, specifically the mesocorticolimbic system. Recent developmental studies demonstrate dramatic changes in DAergic levels, receptor concentrations a nd transporter levels during periadolescent development. These ontogenetic changes, as well as drug exposure during development, may predispose the adolescent animal to addiction. Given that humans typically experiment with and initiate drug use during the adolescent period it is proposed that developmental alterations in the mesolimbic DA projection areas, specifically the NAcc, are an essential area for investigation in drug addiction. The present paper presents formulas for the weight based calculation o f stereotaxic coordinates for the NAcc in rats across development to facilitate further research in the area.

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Appendix A (Continued) 128 Introduction Recent studies have shown a unique profile for the development of the NAcc and its DAergic systems [3,5,6,10,12, 33,35,38] This profile of transformation suggests a potential for unique susceptibility of this transitional system to the effects of chronic or repeated exposure to DAergic agonists during adolescent development. Specifically, exposure to drugs of abu se, which almost universally act as DAergic agonists in the NAcc, can dramatically affect not only receptor and transporter systems but neurochemical profiles as well [25,26] The NAcc has been strongly implicated as a critical brain regi on in the mediation of drug use and potentially drug addiction [16 18,22] A variety of hypotheses have been presented as to how the structure specifically mediates the addictive process, however no clear cut theory has been established. Regardless, a variety of converging lines of evidence point to this structure as being of primary importance in the establishment and/or maintenance of drug use [16 18,22] Adolescence is a period of experimentation and risk taking in bo th human and non human animals, and drug use is often initiated during adolescence (for review see Spear, 2000) [31] Given these findings it is important to investigate the nature of the relationship of drug use and abuse during the adoles cent period and ongoing neurochemical activity in the NAcc. Considering the importance of the NAcc in substance abuse, the ontogeny and/or events that occur during development of the NAcc demand attention, with particular focus on these ongoing processes through birth, preadolescence, periadolescence and into adulthood Specifically defining these age periods is a complex issue that has recently been addressed and the periadolescent period of rats defined as approximately between

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Appendix A (Continued) 129 the ages of postnatal day s (PND) 21 and PND 50 [31] Research conducted in young developing animals, during the periadolescent period, has examined the behavioral responsiveness of adolescent rats and mice to DA agonists [1,2,19,27,32] the devel opmental patterns of DA receptors [3,5,13,29,36] as well as DA transporters numbers [8,9] through adolescence (PND 21 50). Behavioral data indicate that adolescent rats show a reduced sensitivity to amphetamine and cocaine [7,21] an increased response to apomorphine [30] an increased sensitivity to haloperidol relative to younger and older animals [32] and an increased sensitivity to reward [20] Neuroche mically, basal DA synthesis in the NAcc is lower in postnatal day 30 (PND 30) than PND 40 rats and turnover rates for PND 30 animals are less than those reported in adult animals [6] Research in our laboratory [25,26] ha s found that early adolescent rats (PND 25) have basal DA levels similar to those reported in adult animals [24] Importantly, response profiles to alcohol [25] and cocaine [26] at this age are similar th ose observed in adult animals [11,14,23] as well. Our current research focuses on drug or drug expectancy induced changes at later stages in adolescence and into adulthood, a time period that more closely parallels human abuse profiles. As mentioned previously, it is also a time period of numerous changes in the mesolimbic DA system. Research on DA receptor populations indicates a pattern of overproduction and pruning that occurs across adolescence in a sex specific manner [3,5,36] with males exhibiting greater levels across age and greater over production of D1 and D2 receptor types. Similar patterns have been reported in humans as well [29] In rats, the density of D1, D2, and D4 receptor populations in the NAcc increase and peak at PND 28, and then decline significantly to

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Appendix A (Continued) 130 adult levels by PND 60 [34] Furthermore, D3 receptor numbers appear to increase monotonically, with some reports finding adult levels at weaning (i.e., PND 21) [10] while others find D3 levels in weanlings far lower than those observed in adult animals [33] In conjunction with receptor density changes, D1 stimulatory and D2 inhibitory effects on adenylyl cyclase production are less a pparent in adolescence than in adult animals [4] Parallel with these changes, DA transporter levels are increasing in concentration in the NAcc to adult levels through adolescence [8,9] These data indicate that there is a distinct likelihood that the adolescent NAcc is unique in relation to adult and young animals. Therefore, it is necessary to examine this possibility extensively within the behaving organism. The present study has established formulas for the accurate calculation of stereotaxic coordinates of the NAcc in the developing rat. These results allow for the application of a variety of in vivo neurochemical techniques to permit the quantification and evaluation of critical drug related systems in alive, awak e and freely moving adolescent animals. These neurochemical procedures ( in vivo microdialysis, in vivo voltametry, in vivo chronoamperometometry) would allow for a complementary analysis of ongoing neurochemical changes in the NAcc in response to drugs, d rug related stimuli, chronic drug exposure during development, stress and a variety of other interesting relationships. Information derived from using these techniques across age would determine transitional changes in DA and its effects in the NAcc. Addi tionally, examining these effects in conjunction with drug abuse during the periadolescent period

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Appendix A (Continued) 131 would provide critical insight into the mechanisms that underlie the establishment and maintenance of addiction Methods Subjects : Seventy five Sprague Dawley (Zivic Miller Laboratories) rats of postnatal day (PND) 25, 35, 45 and 60 were used as subjects in this experiment. Pups were sexed and culled to 10 pups per litter on PND 1. On PND 21 pups were weaned and group housed until surgery. Animals were maintain ed in a temperature/humidity controlled vivarium on a 12 hour light dark cycle. Intracranial Implantation : Rats were anesthetized on either PND 25, 35, 45 or 60 using a xylazine/ketamine cocktail (0.15 and 1.0 mg/kg respectively). Rats were then placed in a stereotaxic frame. An incision was made over the skull and a hole drilled above the right hemisphere. A guide cannula was affixed with cyanoacrylate to the skull surface. A probe was lowered to the nucleus accumbens septi (NAcc) and cresyl violet (1 l) was injected to enhance the drop site. Following the procedure, animals were sacrificed with an anesthetic overdose of xylazine/ketamine cocktail. Brains were removed, frozen in methyl butane ( 40 o C), and sliced in 40 m sections for histological verifica tion. Results A total of 24 (6 @ PND 25, 7 @ PND 35, 5 @ PND 45 and 6 @ PND 60) surgeries were determined to be correctly positioned in the shell of the NAcc. Significant differences in weight were identified for SEX, F(1,67)=140.426, p<0.05, AGE, F (3,67) =563.615, p<0.05 and SEX by AGE interaction, F (3,67)=22.552, p<0.05 with male exhibiting greater increases in weight across age (See Figure One). A regression

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Appendix A (Continued) 132 analysis of the effective dropsites across ages generated weight derived formulas for probe pla cement (x = the animals weight at the time of surgery; y = the distance in mm for the particular dimension). Weight explained 94% of the variability in location on the anterior posterior axis (See Figure Two), 82% of the variability in location on the med ial lateral axis (See Figure Three) and 87% of the variability in location on the dorsal ventral axis (See Figure Four). Using the average weights of each age/sex category the formulas yield the following effective coordinates at each age (See Table One). Discussion The present study determined formulas for future investigations to target the NAcc in the developing rat. An interesting note for consideration given the present data involves the average weights determined across age and comparison to weights frequently reported in the literature for 'adult' animals. Numerous studies report using rats with weights ranging from 200 300g and these animals are considered adults within the framework of the study. Our analysis of age and weight, however, would ind icate that although females weighing 250g are adult animals, male rats at similar weights are not adults but rather late adolescent animals with ages somewhere between 50 and 55 days (see Figure Two). Therefore, studies utilizing both male and female anima ls should exercise caution in using weights alone to establish chronological age. The present data provide a foundation for potential studies of the ongoing neurochemical processes in the rat NAcc at different stages of adolescence. The examination of this structure during adolescence may provide critical insight into important issues including drug abuse. Specifically, systematic study of the accumbens

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Appendix A (Continued) 133 during development may help clarify the neurochemical processes involved in the establishment and mainten ance of substance abuse. Studies on the influence of repeated substance use during adolescence on the developing neurochemistry of the NAcc are ongoing in our laboratory. Specifically, focusing on the integration of opponent process and attentional/associ ative learning models of addiction within an adolescent framework. No other stimuli have been shown to elevate accumbal DA more effectively and reliably than drugs of abuse [15,28,37] Given this fact, a critical question for developmental research and addiction is to determine the long term effects of repeated administration of DA agonists on the developing adolescent DAergic system. Following such stimulation it is important to determine if the system will develop normally or if proper de velopment is dependent upon the status and feedback of the internal milieu. If proper development of the DA system is dependent on a self regulation process (i.e. receptor concentrations/sensitivity, transporter levels, enzyme activity and DA levels change s are symbiotic) substance use during this critical developmental period may lead to an alteration in basal DA activity in the adult. It is our hypothesis that the adolescent system is self regulatory in its development and that chronic introduction of ex ternally imposed elevations in DA (e.g., drugs of abuse) may lead to a basal hypoactive. Subsequently, the system may be stimulus hyperresponsive in adulthood, a result that may manifest itself behaviorally as anhedonia, sensation seeking and an increased likelihood of addiction. Future studies using these coordinates during this critical developmental period will provide insight into the unique mechanisms that may underlie the establishment of addiction in adolescence.

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Appendix A (Continued) 135 [14] Hoebel, B.G. and Hernandez, L., Microdialysis studies of psychostimulants, NIDA Res Monogr 95 (1989) 343 4. [15] Joseph, M.H. and Hodges, H., Lever pressing for food reward and changes in dopamine turnover and uric acid in rat caudate and nucleus accumbens studied chronically by in vivo voltammetry, J Neurosci Methods 34 (1990) 143 9. [16] Koob, G.F., Circuits, drugs, and drug addiction, Adv Pharmacol 42 (1998) 978 82. [17] Koob, G.F., Drug abuse and alcoholism. Overview, Adv Pharmacol 42 (1998) 969 77. [18] Koob, G.F., Sanna, P.P. and Bloom, F.E., Neuroscience of addiction, N euron 21 (1998) 467 76. [19] Laviola, G., Adriani, W., Terranova, M.L. and Gerra, G., Psychobiological risk factors for vulnerability to psychostimulants in human adolescents and animal models, Neurosci Biobehav Rev 23 (1999) 993 1010. [20] Laviola, G., Dell'Omo, G., Alleva, E. and Bignami, G., Ontogeny of cocaine hyperactivity and conditioned place preference in mice, Psychopharmacology 107 (1992) 221 8. [21] Laviola, G., Wood, R.D., Kuhn, C., Francis, R. and Spear, L.P., Cocaine sensitization in periad olescent and adult rats, J Pharmacol Exp Ther 275 (1995) 345 57. [22] Leshner, A.I. and Koob, G.F., Drugs of abuse and the brain, Proc Assoc Am Physicians 111 (1999) 99 108. [23] Nurmi, M., Ashizawa, T., Sinclair, J.D. and Kiianmaa, K., Effect of prior e thanol experience on dopamine overflow in accumbens of AA and ANA rats, Eur J Pharmacol 315 (1996) 277 83. [24] Parsons, L.H. and Justice, J.B., Jr., Extracellular concentration and in vivo recovery of dopamine in the nucleus accumbens using microdialysis J Neurochem 58 (1992) 212 8. [25] Philpot, R.M. and Kirstein, C.L., The effects of repeated alcohol exposure on the neurochemistry of the periadolescent nucleus accumbens septi, Neuroreport 9 (1998) 1359 63. [26] Philpot, R.M. and Kirstein, C.L., Repea ted cocaine exposure: effects on catecholamines in the nucleus accumbens septi of periadolescent animals, Pharmacol Biochem Behav 62 (1999) 465 72. [27] Reinstein, D.K., McClearn, D. and Isaacson, R.L., The development of responsiveness to dopaminergic ag onists, Brain Res 150 (1978) 216 23. [28] Salamone, J.D., Cousins, M.S. and Snyder, B.J., Behavioral functions of nucleus accumbens dopamine: empirical and conceptual problems with the anhedonia

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Appendix A (Continued) 136 hypothesis, Neurosci Biobehav Rev 21 (1997) 341 59. [29] Se eman, P., Bzowej, N.H., Guan, H.C., Bergeron, C., Becker, L.E., Reynolds, G.P., Bird, E.D., Riederer, P., Jellinger, K., Watanabe, S. and et al., Human brain dopamine receptors in children and aging adults, Synapse 1 (1987) 399 404. [30] Shalaby, I.A. and Spear, L.P., Psychopharmacological effects of low and high doses of apomorphine during ontogeny, Eur J Pharmacol 67 (1980) 451 9. [31] Spear, L.P., The adolescent brain and age related behavioral manifestations [In Process Citation], Neurosci Biobehav Re v 24 (2000) 417 63. [32] Spear, L.P. and Brake, S.C., Periadolescence: age dependent behavior and psychopharmacological responsivity in rats, Dev Psychobiol 16 (1983) 83 109. [33] Stanwood, G.D., McElligot, S., Lu, L. and McGonigle, P., Ontogeny of dopam ine D3 receptors in the nucleus accumbens of the rat, Neurosci Lett 223 (1997) 13 6. [34] Tarazi, F.I. and Baldessarini, R.J., Comparative postnatal development of dopamine D(1), D(2) and D(4) receptors in rat forebrain, Int J Dev Neurosci 18 (2000) 29 3 7. [35] Tarazi, F.I., Tomasini, E.C. and Baldessarini, R.J., Postnatal development of dopamine and serotonin transporters in rat caudate putamen and nucleus accumbens septi, Neurosci Lett 254 (1998) 21 4. [36] Teicher, M.H., Andersen, S.L. and Hostetter, J.C., Jr., Evidence for dopamine receptor pruning between adolescence and adulthood in striatum but not nucleus accumbens, Brain Res Dev Brain Res 89 (1995) 167 72. [37] Tzschentke, T.M., Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues, Prog Neurobiol 56 (1998) 613 72. [38] Wang, L. and Pitts, D.K., Postnatal development of mesoaccumbens dopamine neurons in the rat: electrophysiological studies, Brain Res Dev Brain Res 79 (1994) 19 28.

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Appendix A (Continued) 137 Figure One Interaction of Gender and Age on Weight 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 Age (PND) Male Female Male 65.47 140.16 213.67 293.13 Female 61.70 115.58 159.68 215.28 25 35 45 60

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Appendix A (Continued) 138 Figure Two Anterior Coordinates by Weight y= .001x + 2.05, r 2 =.94 2.05 2.1 2.15 2.2 2.25 2.3 2.35 0 50 100 150 200 250 300 350 Weight (g) Anterior Coordinate (mm)

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Appendix A (Continued) 139 Figure Three Lateral Coordinates by Weight y= .001x + .40, r 2 =.82 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0 50 100 150 200 250 300 350 Weight (g) Lateral Coordinates (mm)

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Appendix A (Continued) 140 Figure Four Ventral Coordinates by Weight y= .006x + 6.22, r 2 =.87 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 0 100 200 300 400 Weight (g) Ventral Coordinates (mm)

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Appendix A (Continued) 141 Table One : Optimal Coordinates by Mean Weight Postnatal Day Sex Weight Anterior Posterior Medial Lateral Dorsal Ventral 25 F 61.70g 2.11 +/ 0.01 0.46 +/ 0.01 6.60 +/ 0.03 M 65.47g 2.11 +/ 0.01 0.46 +/ 0.01 6.60 +/ 0.03 35 F 115.58g 2.17 +/ 0.01 0.52 +/ 0.01 6.91 +/ 0.07 M 140.16g 2.19 +/ 0.01 0.54 +/ 0.01 7.06 +/ 0.04 45 F 159.68g 2.21 +/ 0.01 0.56 +/ 0.01 7.18 +/ 0.07 M 213.67g 2.26 +/ 0.02 0.61 +/ 0 .02 7.50 +/ 0.11 60 F 215.28g 2.27 +/ 0.02 0.62 +/ 0.02 7.51 +/ 0.11 M 293.28g 2.34 +/ 0.03 0.69 +/ 0.03 7.98 +/ 0.15

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Appendix A (Continued) 142 Figure Captions FIGURE ONE: Interaction of Sex and Age with Weight. During periadolescence male rats exhibit significantly la rger increases in weight (g) as they age. FIGURE TWO: Regression line for anterior/posterior coordinates calculated from successful probe placements across weight (g). y = 0.001x + 2.05mm, r 2 = 0.94. FIGURE THREE: Regression line for medial/lateral coor dinates calculated from successful probe placements across weight (g). y = 0.001x + 0.40mm, r 2 = 0.82. FIGURE FOUR: Regression line for dorsal/ventral coordinates calculated from successful probe placements across weight (g). y = 0.006x 6.22mm, r 2 = 0. 87. TABLE ONE: Summary table providing mean weights (g) at PND 25, 35, 45 and 60 and the stereotaxic coordinates derived from these means.

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Appendix B 1 43 THE ATTENTIONAL MODE L OF THE NUCLEUS ACC UMBENS SEPTI AND ADDICTION Introduction The attentional model o f addiction states that addiction is a process resulting from the facilitation of both classical conditioning and associative learning processes by the natural neurochemical effects of drugs of abuse. A universal quality of addictive substances is the abil ity to enhance dopaminergic output in the nucleus accumbens septi (Robinson and Berridge 1993; Sarnyai and Kovacs 1994; Phillips and Shen 1996; Herz 1997; Tzschentke 1998; Di Chiara 1999; Di C hiara et al. 1999; Koob 1999; Leshner and Koob 1999; Nehlig 1999; Koob 2000) a limbic structure that has been shown repeatedly to mediate the initiation and maintenance of conditioned behaviors (Robinson and Berridge 1993; Sarnyai and Kovacs 1994; Phillips and Shen 1996; Di Chiara 1999; Di Chiara et al. 1999; Koob 1999; Leshner and Koob 1999; Nestler et al. 2001) For example, animals will rapidly learn to lever press for electrical stimula tion of the ventral tegmental area, a mesencephalic nucleus which provides DAergic innervation to the NAcc (Kornetsky and Por rino 1992; Fiorino et al. 1993) Further, the application of DA antagonists to the NAcc attenuate the intracranial self stimulation process (Vaccarino and Vaccarino 1989) indic ating that the DAergic processes in the NAcc were critical to the maintenance of VTA self stimulation.

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Appendix B (Continued) 144 Similar processes have been identified with a range of abused substances with infusions that produce elevations of DA in the NAcc resulting in the estab lishment and maintenance of self administration behavior (Vaccarino and Vaccarino 1989; Singh et al. 1997) Additionally, systematic manipulations of the DAergic response in the NAcc results in predictable changes in drug SA rates, with potentiations decreasing SA (Pulvirenti and Koob 1994; Rothman and Glowa 1995) attenuation increasing SA (Corrigall and Coen 1991a; Weissenborn et al. 1996; Izzo et al. 2001) and blockade terminating SA (Corrigall and Coen 1991b; Rothman and Glowa 1995; Weissenborn et al. 1996) These data indicate a well established role for the NAcc in the process of drug taking behavior. The exact role of the NAcc DA response in the addictive process has been the subject of debate since the di scovery that specific brain regions could support behavior. The initial assumptions centered around the concepts of reward areas or pleasure centers (Wise 1978; Wise and Bozarth 1985; Wi se and Rompre 1989) In essence, DA response in the NAcc produced pleasure and therefore reinforced and maintained behavior. However, this hypothesis failed to explain the marked negative affect reported by many addicts and required modification. Later hy pothesis centered on the concept of dysphoria, that repeated drug use resulted in a negative affective state that was alleviated by later drug use (Koob and Le Moal 1997, 2001) This too fell by the way side as more complex hypothesis were developed. The hedonic homeostasis hypothesis was a hybrid of the positive reinforcement model and the dysphoria model and proposed that addiction was

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Appendix B (Continued) 145 the result of a dysregulation of mechanisms which maintained hedonic tone (Koob and Le Moal 1997) Two pro minent models currently are the associative learning model (Di Chiara 1998, 1999) and the incentive sensitization theory (Robinson and Berridge 1993, 2000, 2001, 2003) The associative learning model has its foundation in the conce pt of NAcc DA mediating positive reinforcement and suggests that addiction is the result of overpowering cues associated with drug use that drive future drug using behavior. The incentive sensitization model suggests that repeated drug use produces a facil itated DA response in the NAcc that results in increased salience of drugs and drug related stimuli, thus driving behavior. These two perspectives serve as the backbone for the attentional model of addiction which takes a slightly different view on the fun ctional role of accumbal DA. Functional Roles of the NAcc Many studies have implicated the NAcc in a diverse array of behaviors, each of which might be well explained by influences on attentional processes, rather than reward or reinforcement. For example the NAcc responds to novelty and novel stimuli, exhibiting elevations of DA, and these elevations are not seen in response to familiar stimuli that have no direct biological significance or associations with biologically significant stimuli (Rebec et al. 1997; Young et al. 1998) Given t his profile, it is possible that accumbal DA elevation is simply gating attention, increasing sensory awareness, a response important both for appetitive stimuli and unfamiliar stimuli as well. New stimuli command attention. They may be dangerous, they may have some health benefit, they

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Appendix B (Continued) 146 may prove useless, but until explored they are an unknown quantity. This necessitates attentional responses in the presence of novelty. As previously discussed the predominant view of the functional role of the NAcc in the p ast 20 years has been as a mediator of reward (Hoebel 1985; Bozarth 1986; Hoebel et al. 1989; White 1989; Koob 1992a, 1992b; Willner et al. 1992; Berridge 1996; Koob 1996; Herz 1997; Salamone et al. 1997; Bardo 1998; Berridge and Robinson 1998; Tzschentke 1998; Di Chiara 1999; Ikemoto and Panksep p 1999; Koob 1999; Leshner and Koob 1999; Koob 2000; Schultz et al. 2000) Natural reinforcers such as food, water or sex elevate DA in the NAcc which has lead to the hypothesis that this structure mediates reward and reinforcement (Tzschentke 1998) Although the NAcc may be critically involved in reward and reinf orcement, it is but a subset of the currently identified reinforcement circuit (which is comprised of the anterior bed nuclei of the medial forebrain bundle, the ventral tegmental area, the NAcc and the ventral pallidum) (White 1989; Fibiger et al. 1992; Koob 1992a; Bard o 1998; Leshner and Koob 1999) and the response profile of the NAcc specifically to novelty and punishers suggests that the role of the NAcc is more than just reinforcement related. The critical evidence for NAcc DA as mediating attentional processes is d erived from the latent inhibition (LI) literature (Feldon and Weiner 1992; Young et al. 1993) There is increasing evidence that the accumbens is much more than a reward structure, or a mediator of reinforcement, but rather an integral part of a circuit that mediates attentiona l processes and associative learning. Specifically, increased accumbal DA may be a necessary response to emotionally salient stimuli (Weiner et al. 1996; Gray et al.

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Appendix B (Continued) 147 1997; Gray 1998b; Murphy et al. 2000) Significant evidence for the role of the NAcc in attentional processes comes from research using latent inhibit ion (LI) procedures and learned helplessness models. In LI subjects are exposed to a non contingent stimulus repeatedly (Preexposure Phase), following which a contingency is applied (Conditioning). For example, a rat may be repeatedly exposed to a light st imulus within a Skinner box. Following this non contingent exposure to the light, the rat begins conditioning sessions in which the light is paired with shock. The CS preexposure results in delayed acquisition of classically conditioned responding when c ompared to animals that received no pretreatment. LI is the reduction in ability to learn this later contingency as a result of the prior non contingent experience and, presumably, decreased attention to the stimulus. Manipulations of accumbal DA influen ce the ability to establish LI in a fashion suggestive of attentional gating, in that DA agonists block LI (Young et al. 1993; Thornton et al. 1996; Weiner et al. 1996; Broersen et al. 1999; Di Chiara 2000) (i.e. subjects do not habituate to the non contingent stimulus presentations) while DA antagonists facilitate LI (Young et al. 1993; Thornton et al. 1996; Weiner et al. 1996; Di Chiara 2000) (subjects take longe r to learn the newly applied contingency when under the influence of DA antagonists). Studies of the involvement of the mesolimbic DA system in learned helplessness paradigms clearly indicate that mesolimbic DA responses cannot be directly indicative of re ward or reinforcement but must instead mediate a more complex process (Anisman and Zacharko 1986; Abercrombie et al. 1989; Deutch et al. 1990; Puglisi Allegra et al. 1991)

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Appendix B (Continued) 148 In learned helplessness type procedures, laboratory animals are exposed to inescapable negative stimuli and changes in behavioral profiles are measured. Typically, initial exposure prompts attempts at escape, but as the inevitability of the situat ion is established the animal becomes passive in response to the stimulus, even when avenues for escape are provided. Neurochemical analysis of the DA response in the NAcc to escapable vs. inescapable stress reveals two unique profiles. Research examining the effects of electric shock on accumbal DA levels indicates that aversive stimuli produce DA elevations in the NAcc (Blake and Stein 1987; Kali vas and Abhold 1987; Abercrombie et al. 1989; Deutch et al. 1990; Puglisi Allegra et al. 1991) Exposure to shock produces a biphasic response in the NAcc, an initial elevation in DA, followed by a depression in extracellular DA. Examination of mesolimbic DAergic responses to restraint stress indicates an interesting profile. Restrained animals exhibit an initial elevation in DA that is then reduced to basal values. Following release from restraint, DA levels are again elevated above basal values. Further, chronic inescapable stress results in an attenuated DA response in the NAcc. This pattern of response is not easily explained by strict novelty or reward/punishment based theories. However, an attentional model (based on learned helplessness research) can explain this outcome. Immediately following restraint environmental attention is critical to attempt escape, hence elevated accumbal DA. However, following a period of struggle it become clear that escape is not possible at which time enhanced environment al awareness is non functional and in fact a reduced

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Appendix B (Continued) 149 awareness may be adaptive. Finally, upon release environmental attention should be elevated, either to further escape or to learn the contingencies that produced escape. In general, these profiles do not correspond well to a reinforcement/reward model of NAcc DA but could be explained under an attentional theory. New stressors or sustained avoidable or escapable stressors would require an alert attentive response to cues of the stressor or the stressor it self in order to maximize safety and minimize stress. An attentional model of NAcc DA is supported by NAcc DA profiles identified under these conditions with DA elevating in response to escape/avoidance associated cues. However, in the case of unavoidable stress, continued elevated attention in response to the stressor is not adaptive and potentially dangerous. Under such conditions the most adaptive response would be a passive reduction of sensory stimuli, or decreased environmental attention, since active avoidance is ineffective at removing the stressor. A depression of DA activity in the NAcc of humans may be protective against psychotic decompensation resulting from repeated insult by decreasing attention to ongoing stressors (O'Donnell and Grace 1998) Attention is clearly important in associative learning processes and the NAcc again exhibits intere sting profiles in associative learning procedures. Importantly, it has been repeatedly shown that the pairing of a neutral stimulus with a reinforcer or punisher results in an elevation in accumbal DA in response to the previously neutral stimulus alone. T he neutral stimulus has acquired some of the neurochemical properties of the unconditioned stimulus. Clearly, in the case of a natural reinforcer like food, the neutral stimulus has no satiating properties and therefore cannot of itself acquire the reinfor cing

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Appendix B (Continued) 150 properties of food. Therefore it seems unreasonable to suggest a transfer of reinforcement. By the same argument, although the case cannot be as clearly made, the transference of punishing properties seems unlikely, although the survival benefit in th is case is obvious. It seems more likely that the system acts similarly for associations to rewards and punishers and that it serves to facilitate attention to the environment when a cue signals an upcoming event of biological importance, positive or negat ive. This concept is similar to Hullian or Spences r g s g expectancy mediation of instrumental responding that proposes that, with repeated associations, environmental stimuli acquire the ability to induce unconditional stimulus (US) like responses (r g ) and corresponding internal sensations (s g ) which constitute expectations that motivate behavior (Spence et al. 1950) In this instance, the ability of a natural reinforcer or punisher to attract attention becomes transferred to congruent environmental stimuli, making them cues which attract attention and motivate US related responses. Of considerable significa nce in this idea of NAcc DA mediating attention is recent research examining the NAcc role in associative learning between two neutral stimuli. Research by Young et al. (1998) has shown that the pairing of two neutral stimuli produces elevations in accumbal DA, while the two stimuli presented individually produce no response. Sti mulus Neurochemical Response Light Stable DA Tone Stable DA Light + Tone Elevated DA

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Appendix B (Continued) 151 This elevation may be explained by the novel pair of stimuli, however further investigation suggests that the response could be more than just the result of n ovelty. Pairing one of the two neutral stimuli with shock results in a conditioned elevation in DA to the previously neutral stimulus. Interestingly, the neutral stimulus that has had no pairing with shock, but had been previously paired the shock conditio ned stimulus, also acquires the ability to elevate accumbal DA when presented alone. This demonstrates second order conditioning on a neurochemical level. Conditioning Test Stimulus Neurochemical Response Light Stable DA Tone Stable DA Shock Elevated DA Light + Tone Light + Shock Light Elevated DA Tone Elevated DA Although novelty or pairing could explain the initial elevation of DA upon presentation of the two neutral stimuli, it fails to explain the latter c onditioned response of DAergic elevation to a neutral stimulus never paired with shock. An attentional model of accumbal DA activity can explain each of the patterns of elevation more clearly. The elevation that occurs as the consequence of the novel pairi ng is merely that, a novelty response. As previously discussed novelty should require attention because a new item, or new

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Appendix B (Continued) 152 situation, requires study to learn about potential rewards and pitfalls. Following conditioning the CS induced elevation signals an e xpectancy of an upcoming punisher, this anticipation facilitates environmental attention to aid in avoidance or escape. The second order conditioning represents the application of acquired associations to facilitate environmental awareness to aid in avoida nce or escape. Attention can reasonably explain each aspect of this procedure, while other prominent views of the NAcc and DA fall short at one point or another in this structure. The Role of the NAcc in Addiction Research by Gray (1998b) has proposed circuitry involving the NAcc as mediating attentional processes and some aspects of schizophrenia, specifically positive symptoms. Gray has suggested that complex interconnections between limbic, motor, sensory and cortical systems are involve d in feedback loops for the purpose of sensory regulation. In this model, accumbal DA is involved in the gating of sensory information through a multiple component system termed the striato thalamo cortico limbic loop. Although this system does not work in isolation from other inputs, the primary interconnections are as follows. Through GABAergic interconnections with the ventral pallidum, which in turn sends a GABAergic projection to the nucleus reticularis thalami, alterations in NAcc DA are capable of re gulating sensory flow to the cortex (see Diagram One). The nucleus reticularis thalami is a thalamic structure that appears capable of regulating the flow of information from sensory thalamo cortical afferents through an array of inhibitory connections wit h these nuclei. In theory, DA elevations in the NAcc would inhibit GABAergic projections to the ventral pallidum, in turn disinhibiting the

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Appendix B (Continued) 153 GABAergic projections from the ventral pallidum to the nucleus reticularis thalami. This process would result in the inhibition of the nucleus reticularis thalami and the disinhibition of sensory flow to the cortex. Furthermore, cortical regions in turn modulate flow from the thalamus through reciprocal projections, as well as modulate limbic influences through feedback loops, providing multiple levels of top down regulatory processing. This circuit as a whole has been proposed to regulate attention (Gray 1998a) By this model, attention is defined by an increased, but regulated flow of information from the thalamus to the cortex, and regulation occurs via cortical feedback and limbic inputs. Therefore, when a stimulus elevates accumb al DA the ventral pallidum is disinhibited. This disinhibition results in less inhibition of sensory flow to the cortex by the nucleus reticularis thalami, resulting in greater sensory input. This sensory input, however, must be specific to stimuli that ca n effectively increase accumbal DA (i.e., the sensory systems must be directed to stimuli that can sustain and/or facilitate the activity of this circuit) or the process of elevated sensory flow will be interrupted. Therefore, STIMULI S' Thalamus Cortex mPFC NAcc Diag ram One : Specific environmental stimuli (S') are processed for significance by the cortex. If S' is significant, NAcc DA is facilitated, increasing cortical flow of sensory information related to the ongoing stimulus, S'.

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Appendix B (Continued) 154 when attention is directed t o 'significant' stimuli, which elevate NAcc DA, there is increased cortical activity in relation to the sensory input of that stimulus. However, if attention shifts to a non significant stimulus or the significance of the object is lost (e.g. habituation) the result is a decrease in cortical sensory information, decreased sensory information about the relevant stimulus and therefore decreased attention or focus. Only stimuli that can effectively increase the cortical sensory flow (i.e. novelty, natural rein forcers, environmental cues for reward or punishment, expectation, drugs of abuse, etc.) can sustain focused attention (Diagram One). Attentional Model of Addiction Under normal circumstances the circuit acts to facilitate entry of sensory information to the cortex of ongoing stimuli, or focused attention. However, in the case STIMULI S' Thalamus Cortex mPFC NAcc Diagram Two : Drug administration facilitates sensory flow to the cortex (Red) of environmental stimuli, regardless of species significance, by elevating NAcc DA Drug Administration

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Appendix B (Continued) 155 of drugs of abuse, the gate which regulates the cortical flow of sensory information is forced open. Rather than an elegant circuit which elevates or reduces cortical sensory flow ( salience) depending on the importance of the current stimulus, the flow of sensory information in universally facilitated (Diagram Two). This serves two functions: 1) it increases the range of stimuli which can be associated with drug use and 2) it increas es the likelihood that any given stimuli in the environment will be associated with drug use. Additionally, with repeated stimulus/drug pairings, environmental stimuli acquire the innate salience enhancing properties of drugs, the ability to elevate NAcc DA (Diagram Three). It is important to note here that Young et. al. demonstrated that NAcc conditioning can occur simply via co occurrence, without contingent reinforcement or punishment. Further, the potency of each individual classical conditioning tri al is STIMULI S A Thalamus Cortex mPFC NAcc UR Diagram Three : Repeated drug administration sets the foundation for classical conditioning (Blue) between specific environmental stimuli (S A ) and the drug induced elevation in NAcc DA. Drug Administration US

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Appendix B (Continued) 156 enhanced by the salience enhancing effects of the drug, resulting in fairly rapid and powerful conditioning. Therefore, after a series of stimulus/drug pairings, specific environmental stimuli acquire the ability to elevate NAcc DA and thus enhance t heir salience by facilitating stimulus related cortical flow. Consequently, drug associated environmental stimuli become highly salient in the environment and since they are associated with drugs and drug using behavior they drive drug related thoughts and ultimately further drug using behavior. This in turn facilitates the strength of current associations and provides for other associations to become drug related incentives (Diagrams Four and Five, following pages).

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Appendix B (Continued) 157 STIMULI S A CS Thalamus Cortex mPFC NAcc CR STIMULI S' S A Thalamus Cortex mPFC NAcc Drug Administration Diagram Four : Chronic use of a drug in the presence of specific environmental stimuli (S A ) results in the classical conditioning of the NAcc DA response to the environmental stimuli. Therefore, these stimuli now facilitate their own increased sensory overflow to the cortex, causing ongoing thoughts to be predominantly about drug related stimuli and drugs, overshadowing sensory processes of other competing stimuli (S'). These ongoing thoughts increase the probability that ongoing behavior will be related to drug use and the response of drug use may alleviate tension produced by obsessive thoughts of drugs. Use sets up reinforcement of current conditioning as well as conditioning of new environmental stimuli. S' Thalamus Cortex

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Appendix B (Continued) 158 STIMULI I S A CS Thalamus Cortex mPFC NAcc CR S' S A Thalamus Cortex mPFC NAcc Drug Administration Diagram Five : The ability of drug administration to relieve focus on drug related environmental stimuli serves as a negative reinforcer for drug administration. This results in the establishment of drug related stimuli as incentives ( I ) for drug using behavior. Specifically, the presence of drug related stimuli set s the occasion for negative events that can be alleviated by drug use. Therefore, drug administration in the presence of drug related stimuli becomes an automatic process and this process of use serves to facilitate already established associations and pro duce new associations that can drive drug using behavior. S' Thalamus Cortex

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Appendix B (Continued) 159 References Abercrombie, E. D., K. A. Keefe, D. S. DiFrischia and M. J. Zigmond (1989). Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex. J Neurochem 52 (5): 1655 8. Anisman, H. and R. M. Zacharko (1986 ). Behavioral and neurochemical consequences associated with stressors. Ann N Y Acad Sci 467 : 205 25. Bardo, M. T. (1998). Neuropharmacological mechanisms of drug reward: Beyond dopamine in the nucleus accumbens. Crit Rev Neurobiol 12 (1 2): 37 67. Berridge K. C. (1996). Food reward: Brain substrates of wanting and liking. Neurosci Biobehav Rev 20 (1): 1 25. Berridge, K. C. and T. E. Robinson (1998). What is the role of dopamine in reward: Hedonic impact, reward learning, or incentive salience? Brain Res Bra in Res Rev 28 (3): 309 69. Blake, M. J. and E. A. Stein (1987). Brain stimulation of the ventral tegmental area attenuates footshock escape: An in vivo autoradiographic analysis of opiate receptors. Brain Res 435 (1 2): 181 94. Bozarth, M. A. (1986). Neural basis of psychomotor stimulant and opiate reward: Evidence suggesting the involvement of a common dopaminergic system. Behav Brain Res 22 (2): 107 16. Broersen, L. M., J. Feldon and I. Weiner (1999). Dissociative effects of apomorphine infusions into the me dial prefrontal cortex of rats on latent inhibition, prepulse inhibition and amphetamine induced locomotion. Neuroscience 94 (1): 39 46. Corrigall, W. A. and K. M. Coen (1991a). Cocaine self administration is increased by both d1 and d2 dopamine antagonists Pharmacol Biochem Behav 39 (3): 799 802. Corrigall, W. A. and K. M. Coen (1991b). Selective dopamine antagonists reduce nicotine self administration. Psychopharmacology 104 (2): 171 6. Deutch, A. Y., W. A. Clark and R. H. Roth (1990). Prefrontal cortical d opamine depletion enhances the responsiveness of mesolimbic dopamine neurons to stress. Brain Res 521 (1 2): 311 5. Di Chiara, G. (1998). A motivational learning hypothesis of the role of mesolimbic dopamine in compulsive drug use. J Psychopharmacol 12 (1): 54 67. Di Chiara, G. (1999). Drug addiction as dopamine dependent associative learning disorder. Eur J Pharmacol 375 (1 3): 13 30.

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Appendix B (Continued) 160 Di Chiara, G. (2000). Role of dopamine in the behavioural actions of nicotine related to addiction. Eur J Pharmacol 393 (1 3): 295 314. Di Chiara, G., G. Tanda, V. Bassareo, F. Pontieri, E. Acquas, S. Fenu, C. Cadoni and E. Carboni (1999). Drug addiction as a disorder of associative learning. Role of nucleus accumbens shell/extended amygdala dopamine. Ann N Y Acad Sci 877 : 461 85. Feldon, J. and I. Weiner (1992). From an animal model of an attentional deficit towards new insights into the pathophysiology of schizophrenia. J Psychiatr Res 26 (4): 345 66. Fibiger, H. C., A. G. Phillips and E. E. Brown (1992). The neurobiology of cocai ne induced reinforcement. Ciba Found Symp 166 : 96 111. Fiorino, D. F., A. Coury, H. C. Fibiger and A. G. Phillips (1993). Electrical stimulation of reward sites in the ventral tegmental area increases dopamine transmission in the nucleus accumbens of the r at. Behav Brain Res 55 (2): 131 41. Gray, J. A. (1998a). Abnormal contents of consciousness: The transition from automatic to controlled processing. Adv Neurol 77 : 195 208. Gray, J. A. (1998b). Integrating schizophrenia. Schizophr Bull 24 (2): 249 66. Gray, J. A., P. M. Moran, G. Grigoryan, S. L. Peters, A. M. Young and M. H. Joseph (1997). Latent inhibition: The nucleus accumbens connection revisited. Behav Brain Res 88 (1): 27 34. Herz, A. (1997). Endogenous opioid systems and alcohol addiction. Psychopharma cology (Berl) 129 (2): 99 111. Hoebel, B. G. (1985). Brain neurotransmitters in food and drug reward. Am J Clin Nutr 42 (5 Suppl): 1133 50. Hoebel, B. G., L. Hernandez, D. H. Schwartz, G. P. Mark and G. A. Hunter (1989). Microdialysis studies of brain norepi nephrine, serotonin, and dopamine release during ingestive behavior. Theoretical and clinical implications. Ann N Y Acad Sci 575 : 171 91. Ikemoto, S. and J. Panksepp (1999). The role of nucleus accumbens dopamine in motivated behavior: A unifying interpret ation with special reference to reward seeking. Brain Res Brain Res Rev 31 (1): 6 41. Izzo, E., C. Orsini, G. F. Koob and L. Pulvirenti (2001). A dopamine partial agonist and antagonist block amphetamine self administration in a progressive ratio schedule. Pharmacol Biochem Behav 68 (4): 701 8. Kalivas, P. W. and R. Abhold (1987). Enkephalin release into the ventral tegmental area in response to stress: Modulation of mesocorticolimbic dopamine. Brain Res 414 (2): 339 48.

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Appendix B (Continued) 161 Koob, G. F. (1992a). Drugs of abuse: A natomy, pharmacology and function of reward pathways. Trends Pharmacol Sci 13 (5): 177 84. Koob, G. F. (1992b). Neural mechanisms of drug reinforcement. Ann N Y Acad Sci 654 : 171 91. Koob, G. F. (1996). Hedonic valence, dopamine and motivation. Mol Psychiat ry 1 (3): 186 9. Koob, G. F. (1999). The role of the striatopallidal and extended amygdala systems in drug addiction. Ann N Y Acad Sci 877 : 445 60. Koob, G. F. (2000). Neurobiology of addiction. Toward the development of new therapies. Ann N Y Acad Sci 909 : 170 85. Koob, G. F. and M. Le Moal (1997). Drug abuse: Hedonic homeostatic dysregulation. Science 278 (5335): 52 8. Koob, G. F. and M. Le Moal (2001). Drug addiction, dysregulation of reward, and allostasis. Neuropsychopharmacology 24 (2): 97 129. Kornetsky C. and L. J. Porrino (1992). Brain mechanisms of drug induced reinforcement. Res Publ Assoc Res Nerv Ment Dis 70 : 59 77. Leshner, A. I. and G. F. Koob (1999). Drugs of abuse and the brain. Proc Assoc Am Physicians 111 (2): 99 108. Murphy, C. A., M. Pezze, J. Feldon and C. Heidbreder (2000). Differential involvement of dopamine in the shell and core of the nucleus accumbens in the expression of latent inhibition to an aversively conditioned stimulus. Neuroscience 97 (3): 469 477. Nehlig, A. (1999). Are we de pendent upon coffee and caffeine? A review on human and animal data. Neurosci Biobehav Rev 23 (4): 563 76. Nestler, E. J., M. Barrot and D. W. Self (2001). Deltafosb: A sustained molecular switch for addiction. Proc Natl Acad Sci U S A 98 (20): 11042 6. O'Do nnell, P. and A. A. Grace (1998). Dysfunctions in multiple interrelated systems as the neurobiological bases of schizophrenic symptom clusters. Schizophr Bull 24 (2): 267 83. Phillips, T. J. and E. H. Shen (1996). Neurochemical bases of locomotion and ethan ol stimulant effects. Int Rev Neurobiol 39 : 243 82. Puglisi Allegra, S., A. Imperato, L. Angelucci and S. Cabib (1991). Acute stress induces time dependent responses in dopamine mesolimbic system. Brain Res 554 (1 2): 217 22. Pulvirenti, L. and G. F. Koob ( 1994). Dopamine receptor agonists, partial agonists and psychostimulant addiction. Trends Pharmacol Sci 15 (10): 374 9.

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Appendix B (Continued) 162 Rebec, G. V., J. R. Christensen, C. Guerra and M. T. Bardo (1997). Regional and temporal differences in real time dopamine efflux in the nucleus accumbens during free choice novelty. Brain Res 776 (1 2): 61 7. Robinson, T. E. and K. C. Berridge (1993). The neural basis of drug craving: An incentive sensitization theory of addiction. Brain Res Brain Res Rev 18 (3): 247 91. Robinson, T. E. and K. C. Berridge (2000). The psychology and neurobiology of addiction: An incentive sensitization view. Addiction 95 Suppl 2 : S91 117. Robinson, T. E. and K. C. Berridge (2001). Incentive sensitization and addiction. Addiction 96 (1): 103 14. Robinson, T. E. and K. C. Berridge (2003). Addiction. Annu Rev Psychol 54 : 25 53. Rothman, R. B. and J. R. Glowa (1995). A review of the effects of dopaminergic agents on humans, animals, and drug seeking behavior, and its implications for medication development.Focus on gbr 12909. Mol Neurobiol 11 (1 3): 1 19. Salamone, J. D., M. S. Cousins and B. J. Snyder (1997). Behavioral functions of nucleus accumbens dopamine: Empirical and conceptual problems with the anhedonia hypothesis. Neurosci Biobehav Rev 21 (3): 341 59. Sarnya i, Z. and G. L. Kovacs (1994). Role of oxytocin in the neuroadaptation to drugs of abuse. Psychoneuroendocrinology 19 (1): 85 117. Schultz, W., L. Tremblay and J. R. Hollerman (2000). Reward processing in primate orbitofrontal cortex and basal ganglia. Cere b Cortex 10 (3): 272 84. Singh, J., T. Desiraju and T. R. Raju (1997). Dopamine receptor sub types involvement in nucleus accumbens and ventral tegmentum but not in medial prefrontal cortex: On self stimulation of lateral hypothalamus and ventral mesenceph alon. Behav Brain Res 86 (2): 171 9. Spence, K. W., G. Bergmann and R. Lippitt (1950). A study of simple learning under irrelevant motivational reward conditions. J Exp Psychol 40 (5): 539 51. Thornton, J. C., S. Dawe, C. Lee, C. Capstick, P. J. Corr, P. Cot ter, S. Frangou, N. S. Gray, M. A. Russell and J. A. Gray (1996). Effects of nicotine and amphetamine on latent inhibition in human subjects. Psychopharmacology (Berl) 127 (2): 164 73. Tzschentke, T. M. (1998). Measuring reward with the conditioned place pr eference paradigm: A comprehensive review of drug effects, recent progress and new issues. Prog Neurobiol 56 (6): 613 72. Vaccarino, F. J. and A. L. Vaccarino (1989). Antagonism of cholecystokinin function in the rostral and caudal nucleus accumbens: Differ ential effects on brain stimulation reward. Neurosci Lett 97 (1 2): 151 6.

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Appendix B (Continued) 163 Weiner, I., G. Gal, J. N. Rawlins and J. Feldon (1996). Differential involvement of the shell and core subterritories of the nucleus accumbens in latent inhibition and amphetamine in duced activity. Behav Brain Res 81 (1 2): 123 33. Weissenborn, R., V. Deroche, G. F. Koob and F. Weiss (1996). Effects of dopamine agonists and antagonists on cocaine induced operant responding for a cocaine associated stimulus. Psychopharmacology (Berl) 12 6 (4): 311 22. White, N. M. (1989). Reward or reinforcement: What's the difference? Neurosci Biobehav Rev 13 (2 3): 181 6. Willner, P., R. Muscat and M. Papp (1992). Chronic mild stress induced anhedonia: A realistic animal model of depression. Neurosci Biob ehav Rev 16 (4): 525 34. Wise, R. A. (1978). Catecholamine theories of reward: A critical review. Brain Res 152 (2): 215 47. Wise, R. A. and M. A. Bozarth (1985). Brain mechanisms of drug reward and euphoria. Psychiatr Med 3 (4): 445 60. Wise, R. A. and P. P. Rompre (1989). Brain dopamine and reward. Annu Rev Psychol 40 : 191 225. Young, A. M., R. G. Ahier, R. L. Upton, M. H. Joseph and J. A. Gray (1998). Increased extracellular dopamine in the nucleus accumbens of the rat during associative learning of neutral stimuli. Neuroscience 83 (4): 1175 83. Young, A. M., M. H. Joseph and J. A. Gray (1993). Latent inhibition of conditioned dopamine release in rat nucleus accumbens. Neuroscience 54 (1): 5 9.

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ABOUT THE AUTHOR R ex Montgomery Philpot was bor n September 14 th 1970 in the city of Rockledge, on the east coast of Florida. He began his formal education one score and eight years ago, a process that has continued, unceasingly, until the publication of this body of work. Duri ng this journey, three events worthy of mention have occurred his marriage of thirteen years to his childhood love, Elizabeth Ann Laughlin, and the birth of his two children. Rebecca Ann Philpot was born June 22 nd 1991 in Rockledge, Florida and Grace Taylor Philpot was born Ma rch, 26 th 1998 in the city of Tampa on the west coast of Florida. This dissertation marks the end of one journey and the eager anticipation of a new future, in which many new memories will be formed and memorialized elsewhere, in time and place.


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ABSTRACT: The mesolimbic dopamine (DA) system has been implicated in providing the basis of pleasure, guiding the general mechanism of reinforcement as well as motivation. Support for these roles have grown from neurochemical research in the field of addiction. It is now well known that DA activity increases in the nucleus accumbens septi (NAcc) with exposure to addictive substances. Moreover, pharmacological manipulation of this system produces predictable changes in the administration of drugs of abuse, as well as natural reinforcers. This system is responsive to natural reinforcers and addiction may be the transference of routine mesolimbic function to environmental stimuli predictive of drug administration. The role of the NAcc in addiction specifically appears to be the facilitation of attention to drug-paired stimuli and addiction may be the behavioral manifestation of conditioned NAcc DA reactivity to the presence of drug-related stimuli. Although these findings have been reported in adults, few studies have focused on adolescence, the time when drug use/abuse begins. Adolescents may be particularly susceptible to addiction when considered in the light of this hypothesis. Recent research has revealed that the mesolimbic system of periadolescent animals is undergoing dramatic transition in functional tone. DA receptor and transporter levels are up regulated, synthesis rates are altered, and innervation from prefrontal cortex (PFC), involved in regulating tonic and phasic DA activity, is increasing. Consequently, during adolescence there is a dramatic change in tonic DA levels, variations in phasic responses to acute drug administration and alterations in how the system adapts to repeated drug exposure. The present study utilizes the procedures of conditioned place preference, Novelty preference and in vivo microdialysis to determine how this conditioning process changes during the period of adolescence. The results indicate that adolescents are different from adults not only on behavioral measures associated with drug abuse, but in their neurochemical responsiveness to alcohol, and that these differences are related to a general developmental aspect of adolescence that renders them susceptible to addiction.
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