ORIGINAL INVESTIGATION

Genotype-dependent effects of adolescent nicotine exposureon dopamine functional dynamics in the nucleusaccumbens shell in male and female mice: a potentialmechanism underlying the gateway effect of nicotine

Price E. Dickson & Tiffany D. Rogers & Deranda B. Lester & Mellessa M. Miller &

Shannon G. Matta & Elissa J. Chesler & Dan Goldowitz & Charles D. Blaha &

Guy Mittleman

Received: 2 August 2010 /Accepted: 18 December 2010# Springer-Verlag 2011

AbstractRationale The tendency to use cocaine is determined bygenetic and environmental effects across the lifespan. Onecritical environmental effect is early drug exposure, whichis both driven by and interacts with genetic background.The mesoaccumbens dopamine system, which is criticallyinvolved in the rewarding properties of drugs of abuse,undergoes significant development during adolescence, andthus may be at particular risk to repeated nicotine exposureduring this period, thereby establishing vulnerability forsubsequent adult psychostimulant use.Objectives We tested the hypotheses that adolescent nicotineexposure results in attenuation of the enhancing effects ofcocaine on medial forebrain bundle (MFB) electricalstimulation-evoked dopamine release in the nucleus accumbens

shell (AcbSh) in adulthood and that this effect is significantlyinfluenced by genotype.Methods Mice from the progenitor strains C57BL/6J andDBA/2J and those from the BXD20/TyJ and BXD86/RwwJrecombinant inbred lines were exposed to nicotine viaosmotic minipumps from postnatal day (P) 28 to P56.When mice reached P70, dopamine functional dynamics inAcbSh was evaluated by means of in vivo fixed potentialamperometry in combination with electrical stimulation ofmesoaccumbens dopaminergic axons in the MFB.Results Adolescent exposure to nicotine in all strains dose-dependently reduced the ability of a fixed-dose challengeinjection of cocaine (10 mg/kg, i.p.) to enhance MFBelectrical stimulation-evoked dopamine release in AcbSh inadults. The magnitude of this effect was genotype-dependent.Conclusions These results suggest a genotype-dependentmechanism by which nicotine exposure during adolescencecauses persistent changes in the sensitivity to “hard”stimulants such as cocaine.

Keywords Nicotine . Dopamine . BXD . C57BL/6J . DBA/2J .Minipump . Electrochemistry . Amperometry . Bodyweight . Cocaine

Introduction

The tendency to use cocaine is determined by genetic andenvironmental effects across the lifespan. One criticalenvironmental effect is early drug exposure, which is bothdriven by and interacts with genetic background (Goldmanet al. 2005). The gateway hypothesis is that the use of oneclass of drug affects the propensity to use a second class of

P. E. Dickson : T. D. Rogers :D. B. Lester :M. M. Miller :C. D. Blaha :G. Mittleman (*)Department of Psychology, University of Memphis,Memphis, TN 38152, USAe-mail: g.mittleman@mail.psyc.memphis.edu

S. G. MattaDepartment of Anatomy and Neurobiology,University of Tennessee Health Science Center,Memphis, TN 38163, USA

E. J. CheslerThe Jackson Laboratory,Bar Harbor, ME 04609, USA

D. GoldowitzCentre for Molecular Medicine and Therapeutics,Department of Medical Genetics, University of British Columbia,Vancouver, BC, Canada

PsychopharmacologyDOI 10.1007/s00213-010-2159-2

drug later in life (Kandel 1975; Kandel et al. 1992). Thishypothesis has been investigated mostly with respect to theuse of cannabis during adolescence and its effect on thesubsequent use of other illicit drugs (Fergusson et al. 2006;Fergusson and Horwood 2000; Golub and Johnson 2002;Kandel and Yamaguchi 2002; Kandel et al. 1992).However, the possibility exists that other drugs which arefrequently used during adolescence such as nicotine andalcohol may also predispose individuals to the later use ofillicit drugs.

With respect to nicotine, there are two lines of evidencesuggesting the possibility of a nicotine gateway effect. First,epidemiological studies indicate that adolescents who usetobacco are significantly more likely to use illicit drugs incomparison to non-tobacco-using teens (Hanna et al. 2001;Lai et al. 2000; Merrill et al. 1999; Torabi et al. 1993;Wagner and Anthony 2002). This effect has been found tobe particularly pronounced in adolescents diagnosed withattention deficit hyperactivity disorder, suggesting thatcertain subpopulations may be genetically predisposed toa nicotine gateway effect (Biederman et al. 2006). Second,there is substantial evidence from human and animalstudies that nicotine exposure during adolescence disruptsthe normal process of development in multiple areas of thebrain (reviewed in Dwyer et al. 2009), suggesting possibleneurobiological mechanisms for the observed relationshipbetween nicotine use and illicit drug use in humanpopulations. Thus, although the precise biological mecha-nisms remain unclear, there is accumulating evidence tosupport the hypothesis for a gateway effect of nicotine.

Adolescence is a time period during which major devel-opmental changes are occurring in the brain (Andersen 2003).These changes include linear increases in white matter (Pauset al. 1999), an inverted U-shaped change in gray mattervolume (Giedd 2004; Giedd et al. 1999), and rapid pruningof synaptic connections. It is through this process that thebrain becomes wired to adapt to the demands of theenvironment (Andersen 2003). The mesoaccumbens dopa-mine (DA) system, in addition to its involvement in motorand cognitive function, is a central component of the brainreward system (Girault and Greengard 2004) and undergoessignificant adaptation during adolescence. These changesinclude increased DA synthesis and turnover (Andersen et al.1997; Spear 2000), progressive increases in presynaptic DAreuptake transporter (DAT) binding sites (Coulter et al. 1996;Tarazi et al. 1998), progressive pruning of D1, D2, and D4receptors (Tarazi and Baldessarini 2000), and developmentof presynaptic DA autoreceptors (Hedner and Lundborg1985). Therefore, repeated nicotine exposure during thisperiod may alter the normal developmental course of theneural substrates involved in the processing of rewardingstimuli. Indeed, multiple studies in rodents have shownthat even brief periods of adolescent nicotine exposure

can cause persistent changes in the mesoaccumbens DAsystem (Abreu-Villaca et al. 2003; Collins and Izenwasser2004; Trauth et al. 1999, 2001). Additionally, adolescentnicotine exposure has been shown to affect later cocainereward and sensitivity in adult mice (Collins andIzenwasser 2004; Kelley and Middaugh 1999; McQuownet al. 2007). Adolescence, therefore, is a unique point ofvulnerability during which exposure to nicotine may causepermanent changes in neural structures which are respon-sible for the processing of rewarding stimuli, therebyestablishing vulnerability for subsequent adult psychosti-mulant use.

An emerging area of biomedical research is to under-stand the interaction of genes with the environment (Cabibet al. 2000; Haile et al. 2007; Miller 2010; Philip et al.2010; van der Veen et al. 2007). Despite a breadth ofstudies on the behavioral and neuroanatomical effects ofadolescent nicotine exposure (Abreu-Villaca et al. 2003;Collins and Izenwasser 2004; Collins et al. 2004; Kelleyand Middaugh 1999; McQuown et al. 2007; Trauth et al.1999, 2001), there is a paucity of information about howgenetic variation influences neuroadaptive changes in thefunctional dynamics of the DA neuronal systems inresponse to adolescent exposure to nicotine. This informa-tion is critical for understanding the interaction of geneticsand the adolescent environment on adult drug addiction andfor development of early interventions.

In the current study, we have addressed this gap in theliterature by investigating the extent to which adolescentnicotine exposure affects DA functional dynamics in thenucleus accumbens shell (AcbSh) during early adulthood,and whether this effect is significantly influenced bygenotype. We chose to measure DA in the AcbSh becauseAcbSh DA is strongly related to the rewarding properties ofdrugs of abuse (Di Chiara et al. 2004). Using subcutane-ously implanted mini-osmotic pumps, we exposed mice tovehicle or one of three doses of nicotine (24, 36, or 48 mg/kg/day) over the entire course of adolescence (postnataldays 28–56). Approximately 1 week after nicotine exposureended, we measured functional DA dynamics in the AcbShusing fixed potential amperometry, a technique allowingreal-time monitoring of DA oxidation current evoked bybrief electrical stimulation of ascending mesoaccumbensdopaminergic projections (Dugast et al. 1994).

As subjects, we used four different strains of inbredmice: C57BL/6J (B6), DBA/2J (D2), BXD20, and BXD86mice. We chose these strains for several reasons. First, theB6 and D2 inbred mouse strains have been shown topossess approximately three million single-nucleotide poly-morphisms (SNP), comprising 16% of mouse diversityamong genotyped strains (Roberts et al. 2007). Many moreSNPs are likely to be discovered under multiple existingresequencing efforts under way. Second, the DA system in

Psychopharmacology

B6 and D2 mice has been extensively studied, and thesestrains have been found to differ on multiple parametersincluding DA metabolism, release, receptor densities,receptor distribution, and the activity of second messengers(see, for review, Puglisi-Allegra and Cabib 1997). Third,behavioral phenotypes of B6 and D2 mice have beenextensively studied and have been found to differ pro-foundly in their propensity to self administer cocaine(Grahame and Cunningham 1995; Kuzmin and Johansson2000; Rocha et al. 1998), cocaine-induced hyperlocomotion(Rocha et al. 1998), and cocaine-induced conditioned placepreference (Cunningham et al. 1999; Orsini et al. 2005;Seale and Carney 1991). In addition to the differentialperformance of B6 and D2 mice on tasks assessing therewarding properties of psychostimulants, studies usingthese strains have shown gene-by-environment interactionson the propensity to self administer cocaine as a function ofhousing condition and maternal environment (van der Veenet al. 2007, 2008) and amphetamine-induced conditionedplace preference as a function of a brief period of foodshortage (Cabib et al. 2000). Finally, the B6 and the D2strains are the progenitor strains for the BXD recombinantinbred (RI) panel, of which the BXD20 and BXD86 strainsare part (Peirce et al. 2004; Taylor et al. 1977, 1999;Williams et al. 2001). The BXD RI panel has frequentlybeen used to map complex polygenic traits (Chesler et al.2003; Philip et al. 2010) including those directly related tothe DA system (Boone et al. 2008; Janowsky et al. 2001;Jones et al. 1999).

Materials and methods

The following experiments were approved by the Institu-tional Animal Care and Use Committee at the University ofMemphis and conducted in accordance with the NationalInstitutes of Health Guidelines for the Care and Use ofLaboratory Animals. Efforts were made to reduce thenumber of animals used and to minimize animal pain anddiscomfort.

Animals

Male and female C57BL/6J (B6), DBA/2J (D2), BXD20/TyJ (BXD20), and BXD86/RwwJ (BXD86) mice werebred at Oak Ridge National Laboratory (ORNL). Damswith litters of male and female pups were shipped fromORNL to the University of Memphis (UM) when pupswere approximately 3 weeks of age. Upon arriving atUM, mice were housed in a temperature-controlledenvironment (21±1°C) with a 12-h light/12-h dark cycle(lights on at 0600 hours). Food and water were availablead libitum.

Nicotine pretreatment

Male and female mice from each litter were randomlyassigned to one of four conditions (0, 24, 36, or 48 mg/kg/day of nicotine) with the stipulation that only one mousefrom each litter was assigned to a condition. Although weattempted to balance the sample size of all subgroups, thefinal subgroup sample size became unequal due tovariability in litter size and sex ratio as well as somedegree of mortality and attrition of experimental subjects.Final sample sizes were as follows, with the number ofmales (M) and females (F) in each subgroup listedseparately: C57BL/6J (n=15)—0 mg/kg/day 3(M) 1(F),24 mg/kg/day 2(M) 2(F), 36 mg/kg/day 2(M) 2(F), and48 mg/kg/day 1(M) 2(F); DBA/2J (n=13)—0 mg/kg/day 2(M) 2(F), 24 mg/kg/day 1(M) 2(F), 36 mg/kg/day 1(M) 2(F),and 48 mg/kg/day 2(M) 1(F); BXD20 (n=17)—0 mg/kg/day2(M) 2(F), 24 mg/kg/day 4(M) 2(F), 36 mg/kg/day 1(M) 2(F), and 48 mg/kg/day 1(M) 3(F); and BXD86 (n=15)—0 mg/kg/day 2(M) 3(F), 24 mg/kg/day 3(M) 1(F), 36 mg/kg/day 1(M) 2(F), and 48 mg/kg/day 3(M) 0(F).

On P28 (the beginning of adolescence), all nicotine-exposed mice were singly housed and implanted with anAlzet (Cupertino, CA, USA) mini-osmotic pump (model1004) which provided 28 days of continuous subcutaneousdelivery of 24, 36, or 48 mg/kg/day of nicotine (free base)dissolved in sterile water. Sterile water was used as thenicotine vehicle because when nicotine solution is neutral-ized to a pH of around 7, it degrades rapidly (approximately50% degradation over 10 days). Nicotine dissolved in waterhas a pH closer to 4 and is much more stable, with less than10% degradation over 10 days (Benwell et al. 1995; Mattaet al. 2007). Nicotine doses were chosen based onpreviously published guidelines of nicotine dose selectionin mice (Matta et al. 2007) and were calculated based onthe average weight of B6, D2, BXD20, and BXD86 mice atP42, the midpoint of nicotine exposure. In this regard, itshould be noted that nicotine has an extremely short half-life in mice (plasma and brain t1/2=5.9–6.9 min after i.p.injection) compared to the half-life in humans or rats (Mattaet al. 2007). The rapid metabolism of this drug makes itextremely difficult to attain sustained nicotine levels inmice unless mini-osmotic pumps are used. Control micewere subjected to the same housing and surgical proceduresas nicotine-exposed mice but were implanted with sterilewater-filled pumps. The slow release rate of solution fromthe pumps (~74 μl of vehicle or nicotine solution over theentire 28 days of exposure) made it unlikely that adifference in pH between the vehicle and nicotine solutionswould, by itself, result in either a localized or systemiceffect. Therefore, we did not attempt to adjust for differ-ences in pH between vehicle and nicotine solution. On P56,pumps were explanted. Pump implantation and explantation

Psychopharmacology

were performed under isoflurane anesthesia using sterilesurgical techniques. Between P28 and P56, all mice wereweighed at the same time daily in order to evaluate theeffects of chronic nicotine exposure on body weight.

DA functional dynamics

The effect of adolescent nicotine exposure on DA functionaldynamics following a cocaine challenge (10 mg/kg, i.p.) wasexamined as follows. Seven to 10 days after pumps wereexplanted, mice were anesthetized with urethane (1.5 mg/kg,i.p.) and mounted in a stereotaxic frame (David KopfInstruments, Tujunga, CA, USA) within a mouse head-holder adaptor (Stoelting, Wood Dale, IL, USA), ensuringthe skull was flat. Body temperature was maintained at 36±0.5°C with a temperature-regulated heating pad (TC-1000;CWE Inc., New York, NY, USA). In each mouse, a concentricbipolar stimulating electrode (SNE-100; Rhodes Medical Co.,CA, USA) was implanted into the left medial forebrain bundle(MFB) (coordinates: AP −2.1 mm from bregma,ML +1.1mmfrom midline and DV −4.8 mm from dura; Paxinos andFranklin 2001), and a stainless steel auxiliary and Ag/AgClreference electrode combination was placed in surfacecontact with contralateral cortical tissue approximately2.0 mm posterior to bregma. A carbon fiber microelectrodewith an active recording surface of 250 μm (length) by10 μm (o.d.) (Thornel Type P, Union Carbide, Pittsburgh,PA, USA) was then implanted into the left AcbSh (coordinates:AP +1.5 mm from bregma, ML +1.0 mm from midline, andDV −4.0 mm from dura; Paxinos and Franklin 2001).

Fixed potential amperometry coupled with carbon fibermicroelectrodes has been confirmed as a valid technique forreal-time monitoring of DA oxidation current evoked bybrief electrical stimulation of ascending mesoaccumbensdopaminergic projections (Benoit-Marand et al. 2000;Dugast et al. 1994; Lester et al. 2008, 2009; Schönfuß etal. 2001; Suaud-Chagny 2004). Confirmation of thechemical specificity of our amperometric measurementshas been provided by previous studies (e.g., Lee et al. 2006;Mittleman et al. 2008), showing that systemic administra-tion of the selective DA reuptake inhibitor nomifensinesignificantly increases electrical stimulation-evoked DAoxidation current and delays recovery to prestimulationbaseline levels.

All amperometric recordings were made within aFaraday cage to increase the signal to noise ratio (Forsterand Blaha 2003). A fixed potential (+0.8 V) was applied tothe recording electrode and oxidation current was moni-tored continuously (10K samples/s) with an electrometer(ED401 e-corder 401 and EA162 Picostat, eDAQ Inc.,Colorado Springs, CO, USA), filtered at 10 Hz. Approxi-mately 20 min following implantation of the recordingelectrode, a series of cathodal monophasic current pulses

(800 μA) was delivered to the stimulating electrode viaan optical isolator and programmable pulse generator(Iso-Flex/Master-8; AMPI, Jerusalem, Israel). The stimu-lation protocol consisted of fifteen 0.5-ms duration pulsesat 50 Hz delivered every 30 s over a 30-min testingperiod. Baseline levels of MFB stimulation-evoked DArelease in the AcbSh were monitored for 10 min in eachmouse prior to cocaine administration.

Data collation and statistical analysis

In order to quantify MFB stimulation-evoked DA release(efflux) via the recorded oxidation current, prestimulationcurrent values were normalized to zero current values anddata points occurring within the range of 0.25 s pre- and2.0 s post-onset of the stimulation were extracted from thecontinuous record at 30-s intervals over the course of20 min cocaine post-injection. For each animal, changes instimulation-evoked DA oxidation current occurring at thepeak of each extracted response after a challenge injectionof cocaine were expressed as mean percent changes withrespect to the 10-min average pre-cocaine baseline responsepeak (100%). These percentages were then used tostatistically compare differences between strain and doseof nicotine pretreatment across time using repeated meas-ures analysis of variance (RANOVA). In these analyses, thedependent variable was always the percentage of baselineMFB-evoked DA response at 30 s intervals post-stimulation, henceforth referred to as DA response. Addi-tionally, daily body weights between P28 and P56 wereanalyzed using RANOVA. Wilks' Lambda was used whenreporting results of multivariate tests. For all statistical tests,the alpha level was set at 0.05. When significant inter-actions were detected, Bonferroni-corrected post hoc testswere used to further identify significant differences betweengroups.

Histology

Upon the completion of each experimental session, an irondeposit was made in the MFB stimulation site by passingdirect anodic current (100 μA for 10 s) through thestimulating electrode. Direct current (1 mA for 10 s) wasapplied to the recording electrode to mark its implantationsite in the AcbSh. Mice were then euthanized with a 0.25-ml intracardial injection of urethane (0.345 g/ml). Brainswere removed, immersed overnight in 10% bufferedformalin containing 0.1% potassium ferricyanide, and thenstored in 30% sucrose/10% formalin solution until section-ing. After fixation, 30-μm coronal sections were sliced in acryostat at −30°C, with a Prussian blue spot resulting froma redox reaction of the ferricyanide marking the stimulationsite. Placements of stimulating and recording electrodes

Psychopharmacology

were determined under a light microscope and recorded onrepresentative coronal diagrams (Paxinos and Franklin 2001).

Chemicals

Nicotine hydrogen tartrate salt, cocaine hydrochloride, andurethane were obtained from Sigma-Aldrich Chemical Co.(St. Louis, MO, USA). Nicotine hydrogen tartrate salt andurethane were dissolved in distilled water. Cocaine hydro-chloride was dissolved in 0.9% saline. Reported nicotinedoses are in free base form. The appropriate amount ofnicotine hydrogen tartrate salt necessary to achieve the freebase concentrations of 24, 36, or 48 mg/kg/day wasachieved using calculations reported in Matta et al. (2007).

Mortality and attrition of experimental subjects

Over the 28-day course of nicotine exposure, 12 micewere dropped from the study due to mortality, infectionat the incision site, or because the implanted pumps hadbecome externalized. Additionally, two mice died duringthe collection of electrochemical data, and duringhistology, it was revealed that in four mice the DArecording electrode had been placed in the accumbenscore, not in the shell. These six mice were also excludedfrom statistical analyses.

Results

Effect of adolescent nicotine exposure on body weight

Body weights of mice were recorded daily beginningimmediately prior to pump implantation on P28 andending just prior to pump explantation on P56. In orderto investigate mouse weights over the period of pumpimplantation, we performed RANOVA using day as thewithin-subjects factor and strain and dose as between-subjects factors. The initial RANOVA conducted overall strains indicated that regardless of strain, no nicotinedose significantly affected average weight gain over the28-day period (day × dose: F(81, 54.7)=1.213, p = ns).However, depending on the strain, nicotine dose didsignificantly affect the pattern of weight gain (day ×strain × dose: F(243, 174.9)=1.377, p<0.05), and thiseffect was related to strain differences in body weightover the 4-week pump implantation period (strain: F(3,44)=8.161, p<0.01). To assess the extent of weight gainacross the 28-day period of nicotine exposure, wecollapsed across the strain and dose variables. At thebeginning of nicotine exposure (P28), mean weight of allmice was 12.45 g. By the end of nicotine exposure (P56),mean weight of all mice had increased to 19.65 g.

Stereotaxic placements of electrodes

As illustrated in Fig. 1a–d, the DA recording electrodes(n=60) were localized within the AcbSh (ranging from1.34 to 1.54 mm anterior to bregma, 0.35 to 0.5 mm lateralto midline, and 3.3 to 3.8 mm ventral to dura), and the tipsof the stimulating electrodes (n=60) were positionedwithin the anatomical boundaries of the MFB (rangingfrom 1.94 to 2.18 mm posterior to bregma, 0.80 to 1.2 mmlateral to midline, and 4.25 to 4.85 mm ventral to dura)across all mouse strains examined. Placements of theseelectrodes did not differ significantly among strains orbetween genders.

Effect of body weight on evoked DA release

To examine the possibility that the DA response wassignificantly affected by small differences in the amount ofnicotine to which mice were exposed, we performed acorrelational analysis using (1) weight during the 28 daysof nicotine exposure and (2) DA response. Both variableswere collapsed across time. There was no statisticallysignificant relationship between these two variables,r(58)=0.184, p = ns, indicating that the small differencesin weight between mice (and the resulting small differences inthe amount of nicotine to which mice were exposed duringadolescence) did not significantly account for variability inthe DA response following cocaine challenge.

Effect of adolescent nicotine exposure on evoked DArelease

In order to examine the effect of adolescent nicotineexposure on DA response following cocaine challenge,we performed RANOVA using DA response as thedependent factor, time as the within-subjects factor andstrain and dose as between-subjects factors. The RANOVAindicated significant interactions between strain × dose ×time (F(351, 75.8)=8.16, p<0.001), dose × time (F(117,18.88)=87.27, p<0.001), and strain × time (F(117, 18.88)=58.95, p<0.001). Therefore, Bonferroni-corrected post hoctests were used to examine subgroup differences whichrevealed the following patterns in the data. First, as shownin Fig. 2, all doses of nicotine significantly reduced theability of a challenge injection of cocaine (10 mg/kg, i.p.) toenhance the MFB stimulation-evoked DA response in theAcbSh relative to vehicle-treated controls. This was true forall strains of mice (p<0.05 for all comparisons). Second,post hoc tests indicated a dose–response effect of nicotine.Specifically, in all strains, the 36-mg/kg/day dose ofnicotine significantly reduced the DA response relative tothe 24-mg/kg/day dose, while the 48-mg/kg/day dosesignificantly reduced the DA response relative to the 36-

Psychopharmacology

mg/kg/day dosage (p<0.05 for all comparisons). Finally, asshown in Fig. 3, although the overall effect of adolescentnicotine exposure was to reduce the ability of a challengeinjection of cocaine to enhance MFB stimulation-evokedDA release in the AcbSh, the magnitude of this effectvaried significantly as a function of genetic background.Adolescent nicotine exposure had the least impact on B6mice, with the 24-, 36-, and 48-mg/kg/day nicotine dosesreducing the DA response relative to controls by 20.2%,31.4%, and 45.6%, respectively. D2 mice, however, showedthe largest effect of adolescent nicotine exposure, with the24-, 36-, and 48-mg/kg/day nicotine doses reducing the DAresponse relative to controls by 54.6%, 65.3%, and 71.4%,

respectively. As shown in Fig. 3, BXD20 and BXD86 micefell between these two extremes. At each dose, thebetween-strain differences in cocaine-enhanced MFBstimulation-evoked DA release were statistically significant(p<0.05) in most cases (see Fig. 3).

Discussion

The current study shows, in mice, that daily exposure tonicotine over the entire course of adolescence (P28 to P56)markedly affects DA functional dynamics in the AcbShduring early adulthood and that this effect is significantly

Fig. 1 Representative coronalsections of the mouse brain(adapted from the atlas ofPaxinos and Franklin 2001)indicating the placements(shaded gray ovals for malesand dashed-line ovals forfemales) of amperometricrecording electrodes in thenucleus accumbens shell(AcbSh) and stimulatingelectrodes in the medialforebrain bundle (MFB) fora C57BL/6J, b DBA/2J, cBXD20, and d BXD86 mousestrains. Insets (center, top andbottom) show an expandedview of the placements ofrecording and stimulatingelectrodes in these animalsat the stereotaxic coordinatesof +1.42 and −2.06 mm frombregma

Psychopharmacology

influenced by genotype. With respect to the effects ofnicotine, three main conclusions resulted from this study.First, in all strains of mice (B6, D2, BXD20, and BXD86)and at all doses, adolescent nicotine exposure significantlydecreased the ability of a single challenge injection ofcocaine to enhance the MFB stimulation-evoked DAresponse in the AcbSh. Second, this effect was nicotinedose-dependent. Specifically, exposure to higher doses ofnicotine during adolescence resulted in significantly greaterreductions in the enhancement of the MFB stimulation-evoked DA response following cocaine challenge. Finally,and perhaps most importantly, the ability of adolescentnicotine exposure to reduce the MFB stimulation-evokedDA response following cocaine challenge in the AcbShvaried significantly as a function of genetic background,with D2 mice being most affected, B6 mice being leastaffected, and BXD20 and BXD86 mice falling betweenthese two extremes.

In addition to the nicotine-related findings, the differ-ences in the DA response magnitude between the B6 andD2 strains at the control dose (0 mg/kg/day) support thenotion of a significantly higher number of active DAT sitesin B6 mice equating to a lower responsivity to DAT

C57BL/6J

MF

B-E

voke

d D

A R

elea

se(%

of P

re-C

ocai

ne R

espo

nse)

0

100

200

300

400

500

600

7000 mg/kg24 mg/kg36 mg/kg48 mg/kg

DBA/2J

0

100

200

300

400

500

600

7000 mg/kg24 mg/kg36 mg/kg48 mg/kg

BXD20

0 2 4 6 8 10 12 14 16 18 20

0 2 4 6 8 10 12 14 16 18 20

0 2 4 6 8 10 12 14 16 18 20

0 2 4 6 8 10 12 14 16 18 20

0

100

200

300

400

500

600

7000 mg/kg24 mg/kg36 mg/kg48 mg/kg

BXD86

Time (min) Post-Cocaine (10 mg/kg)

0

100

200

300

400

500

600

7000 mg/kg24 mg/kg36 mg/kg48 mg/kg

A B

C D

Fig. 2 MFB stimulation-evoked dopamine release within the nucleusaccumbens shell following a fixed-dose challenge injection of cocaine(10 mg/kg, i.p.) in young adult a C57BL/6J, b DBA/2J, c BXD20, and

d BXD86 mice exposed to one of three doses of nicotine (24, 36, or48 mg/kg/day) or vehicle during adolescence (P28 to P56). Error barsrepresent the standard error of the mean

Nicotine Dose (mg/kg/day)

0 24 36 48Ave

rage

Incr

ease

in M

FB

-Evo

ked

DA

Rel

ease

(% o

f Pre

-Coc

aine

Res

pons

e)

0

100

200

300

400

500C57BL/6JDBA/2JBXD20 BXD86

n.s.

n.s.

n.s.

n.s.

n.s.

Fig. 3 Average percent increase, relative to the pre-cocaine response,in MFB stimulation-evoked dopamine release within the nucleusaccumbens shell over the first 20 min following a fixed-dosechallenge injection of cocaine (10 mg/kg, i.p.) in young adultC57BL/6J, DBA/2J, BXD20, and BXD86 mice exposed to one ofthree doses of nicotine (24, 36, or 48 mg/kg/day) or vehicle duringadolescence (P28 to P56). At each dose, the between-strain differencesin MFB stimulation-evoked dopamine release were statisticallysignificant (p<0.05), unless indicated. Error bars represent thestandard error of the mean

Psychopharmacology

blockers, such as cocaine. Together with DAT having anequal affinity (Kd) for cocaine in these two strains (Bosyand Ruth 1989), these findings are highly consistent withrelatively higher DAT density (Bmax) in the AcbShassociated with lower levels of cocaine-induced locomotion(Tolliver et al. 1994; Tolliver and Carney 1994; Womer etal. 1994) and lower rates of acquisition, but higher totalintake of cocaine (lower reinforcing potency; Caine et al.1993) in adult B6 relative to D2 mice during cocaineintravenous self-administration (Kuzmin and Johansson2000; Rocha et al. 1998; van der Veen et al. 2007).

It is important to exclude the possibility that factors otherthan genetic background and nicotine dose were responsi-ble for the subgroup differences seen in the DA response tococaine challenge. In vivo nicotine metabolism in male andfemale B6 and D2 mice has been investigated anddetermined to be similar between strains and sexes (Siuand Tyndale 2007). This suggests that differences in theelectrochemical response following cocaine challenge be-tween male and female B6 and D2 mice observed in thisstudy were not due to different rates of nicotine metabo-lism. To our knowledge, nicotine metabolism in BXD lineshas not been investigated. Therefore, it remains possiblethat rates of nicotine metabolism vary in the two BXD linesused in this study. However, in the current study, the mostdivergent effects of adolescent nicotine exposure were seenin B6 and D2 mice, which supports the conclusion thatgenotype-dependent effects of adolescent nicotine exposureand not strain differences in the rate of nicotine metabolismwere responsible for the significant differences in the DAresponse following cocaine challenge in B6 and D2animals.

Although there was some variability in the daily nicotinedose due to differences in weights between sexes andstrains, the data suggest that it is unlikely that these smalldifferences in nicotine exposure accounted for the largebetween-strain differences in the DA response followingthe cocaine challenge. First, as shown in Fig. 3, thevariability around the mean of the DA response in eachstrain or RI line was extremely low. This would not beexpected if weight differences between sexes played aprominent role. Second, there was no statistically signifi-cant relationship between weight during the 28 days ofnicotine exposure and DA response, indicating that thesmall differences in weight between mice (and the resultingsmall differences in the amount of nicotine to which micewere exposed during adolescence) did not significantlyaccount for variability in the DA response followingcocaine challenge. This supports the hypothesis thatunderlying genetic differences in response to adolescentnicotine exposure, and not small differences in nicotinedosage, accounted for the between-group differences in DAresponse following cocaine challenge. Nonetheless, it remains

possible that differences in nicotine dose between sexes andstrains made a small contribution to the observed results.

Neurobiological mechanisms

Although the current study shows genotype-dependent alter-ations in DA functional dynamics of the AcbSh as a result ofadolescent nicotine exposure, the proximal neurobiologicalmechanisms responsible for these alterations remain to bedetermined. One reasonable possibility is that in all strains,adolescent nicotine exposure resulted in an increase in DATdensity in the AcbSh, and that this effect was particularlypronounced in susceptible strains (e.g., D2 mice). Thishypothesis is supported by findings of increased DATdensities in the nucleus accumbens core and shell ofnicotine-exposed periadolescent male rats (Collins et al.2004). An increase in DAT densities in the AcbSh followingadolescent nicotine exposure would correspond to, due to therelatively greater amount of cocaine required to block theincreased number of DAT sites, a higher total intake ofcocaine in drug self-administration paradigms and a reduc-tion in cocaine-conditioned place preference. With respect tonicotine-exposed mice and rats, this hypothesis is supportedby findings of a reduction in cocaine-conditioned placepreference in adult mice treated with nicotine duringadolescence (Kelley and Middaugh 1999; Kelley and Rowan2004) and an increase in cocaine self-administration in adultrats treated with nicotine during adolescence (McQuown etal. 2007).

It is also possible that adolescent nicotine exposure actsthrough other mechanisms, perhaps in concert with increasedDAT densities, to change DA dynamics in the AcbSh. Thishypothesis is supported by, among others, studies indicatingthat adolescent nicotine exposure results in persistent changesin the AMPA GluR2/3 subunit in the striatum (thought to beinvolved in the control of addictive behaviors; Adriani et al.2004), cortical cholinergic hypoactivity (Slotkin et al. 2007),and long-lasting increments in depolarization-induced DArelease in the medial prefrontal cortex following adolescentexposure to nicotine (Counotte et al. 2009). Furtherinvestigation is needed to reveal and untangle the mecha-nisms underlying the observed changes in (1) AcbSh DAdynamics (this study) and (2) the rewarding properties ofdrugs of abuse following adolescent nicotine exposure(Kelley and Middaugh 1999; Kelley and Rowan 2004;McQuown et al. 2007).

It should be noted that a variety of factors unrelated toadolescent drug exposure have been shown to mediate theeffects of or propensity to initiate the use of addictivedrugs. Among these are sex (Bobzean et al. 2010; Vansickelet al. 2010), environmental enrichment (Chauvet et al.2009; Smith et al. 2009; Thiel et al. 2010), group housing(van der Veen et al. 2007), maternal environment (van der

Psychopharmacology

Veen et al. 2008), social status (Morgan et al. 2002), andsocioeconomic status in humans (Humensky 2010; Lemstra etal. 2008). In some cases, these factors, like adolescent nicotineexposure in the current study, have been shown to interactwith genetic background (van der Veen et al. 2008, 2007).

Translational significance

As it relates to human addiction, the study of the B6 and D2inbred mouse strains is informative for two reasons. First, itallows for the discovery of general principles pertaining togene-by-environment relationships, as observed in thisstudy and others (e.g., Cabib et al. 2000; van der Veen etal. 2007, 2008). These findings in mice are directly relevantto humans because they suggest the existence of abiological mechanism by which individuals with a suscep-tible genetic background may be more vulnerable to thegateway effect of nicotine exposure (through smoking orsmokeless tobacco use during adolescence). Specifically,such exposure may increase the likelihood of adult abuse ofcocaine and potentially other drugs. This mechanism maywork independently of any psychosocial effects in humanadolescents. Second, the B6 and D2 strains are theprogenitors of the BXD recombinant inbred lines (Peirceet al. 2004; Taylor et al. 1999), which have been used forseveral decades to map complex polygenic traits, includingthose related to the rewarding effects of cocaine (Philip etal. 2010). The observed differences in the effects ofnicotine on DA functional dynamics between the progenitorstrains and the two BXD lines used in this study suggestthat the full panel of 79 BXD RI lines could be used toidentify provisional chromosomal locations of genes influ-encing the biological mechanism of gateway effects.Studies in BXD lines have direct relevance to humandisease because the great extent of the linkage homologybetween mice and humans enables extrapolation from themouse genome to the human genome (Crabbe et al. 1994;Plomin et al. 1991).

Conclusion

To our knowledge, this is the first study showing (1) thatadolescent nicotine exposure causes persistent changes inDA function in the AcbSh and (2) that this effect isdependent on genotype. These results suggest a genotype-dependent mechanism by which nicotine exposure duringadolescence predisposes individuals to the later use ofcocaine, a potential mechanism underlying the gatewayeffect of nicotine observed in humans. Additionally, theseresults indicate that the BXD recombinant inbred lines canbe used to examine the underlying genetic and neurochem-ical mechanisms by which adolescent exposure to chronic

nicotine influences the predisposition to use cocaine andother drugs in adulthood.

Acknowledgments This project was made possible by a DunavantProfessorship awarded to GM. For their assistance with datacollection, the authors thank Erin Clardy and Tom Schneider.

Conflicts of interest None.

References

Abreu-Villaca Y, Seidler FJ, Slotkin TA (2003) Impact of adolescentnicotine exposure on adenylyl cyclase-mediated cell signaling:enzyme induction, neurotransmitter-specific effects, regionalselectivities, and the role of withdrawal. Brain Res 988:164–172. doi:10.1016/S0006-8993(03)03368-7

Adriani W, Granstrem O, Macri S, Izykenova G, Dambinova S,Laviola G (2004) Behavioral and neurochemical vulnerabilityduring adolescence in mice: studies with nicotine. Neuropsycho-pharmacology 29:869–878. doi:10.1038/sj.npp.1300366

Andersen SL (2003) Trajectories of brain development: point ofvulnerability or window of opportunity? Neurosci Biobehav Rev27:3–18. doi:10.1016/S0149-7634(03)00005-8

Andersen SL, Dumont NL, Teicher MH (1997) Developmentaldifferences in dopamine synthesis inhibition by (+/–)-7-OH-DPAT. Naunyn-Schmiedebergs Arch Pharmacol 356:173–181.doi:10.1007/PL00005038

Benoit-Marand M, Jaber M, Gonon F (2000) Release and eliminationof dopamine in vivo in mice lacking the dopamine transporter:functional consequences. Eur J Neurosci 12:2985–2992.doi:10.1046/j.1460-9568.2000.00155.x

Benwell ME, Balfour DJ, Birrell CE (1995) Desensitization of thenicotine-induced mesolimbic dopamine responses during con-stant infusion with nicotine. Br J Pharmacol 114:454–460

Biederman J, Monuteaux MC, Mick E, Wilens TE, Fontanella JA,Poetzl KM, Kirk T, Masse J, Faraone SV (2006) Is cigarettesmoking a gateway to alcohol and illicit drug use disorders? Astudy of youths with and without attention deficit hyperactivitydisorder. Biol Psychiatry 59:258–264. doi:10.1016/j.biopsych.2005.07.009

Bobzean SA, Dennis TS, Addison BD, Perrotti LI (2010) Influence ofsex on reinstatement of cocaine-conditioned place preference.Brain Res Bull. doi:10.1016/j.brainresbull.2010.09.003

Boone EM, Hawks BW, Li W, Garlow SJ (2008) Genetic regulation ofhypothalamic cocaine and amphetamine-regulated transcript(CART) in BxD inbred mice. Brain Res 1194:1–7. doi:10.1016/j.brainres.2007.11.074

Bosy TZ, Ruth JA (1989) Differential inhibition of synaptosomalaccumulation of [3H]-monoamines by cocaine, tropacocaine andamphetamine in four inbred strains of mice. Pharmacol BiochemBehav 34:165–172. doi:10.1016/0091-3057(89)90368-7

Cabib S, Orsini C, Le Moal M, Piazza PV (2000) Abolition andreversal of strain differences in behavioral responses to drugs ofabuse after a brief experience. Science 289:463–465.doi:10.1126/science.289.5478.463

Caine BS, Lintz R, Koob GF (1993) Intravenous drug self-administration techniques in animals. In: Sahgal A (ed)Behavioural neuroscience: a practical approach. Oxford Uni-versity Press, Oxford, pp 117–143

Chauvet C, Lardeux V, Goldberg SR, Jaber M, Solinas M (2009)Environmental enrichment reduces cocaine seeking and reinstatementinduced by cues and stress but not by cocaine. Neuropsychopharma-cology 34:2767–2778. doi:10.1038/npp.2009.127

Psychopharmacology

Chesler EJ, Wang J, Lu L, Qu Y, Manly KF, Williams RW (2003)Genetic correlates of gene expression in recombinant inbredstrains: a relational model system to explore neurobehavioralphenotypes. Neuroinformatics 1:343–357. doi:10.1385/NI:1:4:343

Collins SL, Izenwasser S (2004) Chronic nicotine differentially alterscocaine-induced locomotor activity in adolescent vs. adult maleand female rats. Neuropharmacology 46:349–362. doi:10.1016/j.neuropharm.2003.09.024

Collins SL, Wade D, Ledon J, Izenwasser S (2004) Neurochemicalalterations produced by daily nicotine exposure in periadolescentvs. adult male rats. Eur J Pharmacol 502:75–85. doi:10.1016/j.ejphar.2004.08.039

Coulter CL, Happe HK, Murrin LC (1996) Postnatal development ofthe dopamine transporter: a quantitative autoradiographic study.Brain Res Dev Brain Res 92:172–181. doi:10.1016/0165-3806(96)00004-1

Counotte DS, Spijker S, Van de Burgwal LH, Hogenboom F,Schoffelmeer AN, De Vries TJ, Smit AB, Pattij T (2009) Long-lasting cognitive deficits resulting from adolescent nicotineexposure in rats. Neuropsychopharmacology 34:299–306.doi:10.1038/npp.2008.96

Crabbe JC, Belknap JK, Buck KJ (1994) Genetic animal models ofalcohol and drug abuse. Science 264:1715–1723. doi:10.1126/science.8209252

Cunningham CL, Dickinson SD, Grahame NJ, Okorn DM, McMullinCS (1999) Genetic differences in cocaine-induced conditionedplace preference in mice depend on conditioning trial duration.Psychopharmacology (Berl) 146:73–80. doi:10.1007/s002130051090

Di Chiara G, Bassareo V, Fenu S, De Luca MA, Spina L, Cadoni C,Acquas E, Carboni E, Valentini V, Lecca D (2004) Dopamine anddrug addiction: the nucleus accumbens shell connection. Neuro-pharmacology 47(Suppl 1):227–241. doi:10.1016/j.neuropharm.2004.06.032

Dugast C, Suaud-Chagny MF, Gonon F (1994) Continuous in vivomonitoring of evoked dopamine release in the rat nucleusaccumbens by amperometry. Neuroscience 62:647–654.doi:10.1016/0306-4522(94)90466-9

Dwyer JB, McQuown SC, Leslie FM (2009) The dynamic effects ofnicotine on the developing brain. Pharmacol Ther 122:125–139.doi:10.1016/j.pharmthera.2009.02.003

Fergusson DM, Horwood LJ (2000) Does cannabis use encourageother forms of illicit drug use? Addiction 95:505–520.doi:10.1046/j.1360-0443.2000.9545053.x

Fergusson DM, Boden JM, Horwood LJ (2006) Cannabis use andother illicit drug use: testing the cannabis gateway hypoth-es i s . Addic t ion 101:556–569. doi :10 .1111/ j .1360-0443.2005.01322.x

Forster GL, Blaha CD (2003) Pedunculopontine tegmental stimulationevokes striatal dopamine efflux by activation of acetylcholineand glutamate receptors in the midbrain and pons of the rat. Eur JNeurosci 17:751–762. doi:10.1046/j.1460-9568.2003.02511.x

Giedd JN (2004) Structural magnetic resonance imaging of theadolescent brain. Ann N Y Acad Sci 1021:77–85. doi:10.1196/annals.1308.009

Giedd JN, Blumenthal J, Jeffries NO, Castellanos FX, Liu H,Zijdenbos A, Paus T, Evans AC, Rapoport JL (1999) Braindevelopment during childhood and adolescence: a longitudinalMRI study. Nat Neurosci 2:861–863. doi:10.1038/13158

Girault JA, Greengard P (2004) The neurobiology of dopaminesignaling. Arch Neurol 61:641–644. doi:10.1001/archneur.61.5.641

Goldman D, Oroszi G, Ducci F (2005) The genetics of addictions:uncovering the genes. Nat Rev Genet 6:521–532. doi:10.1038/nrg1635

Golub A, Johnson BD (2002) Substance use progression and harddrug use in inner-city New York. In: Kandel DB (ed) Stages andpathways of drug involvement: examining the gateway hypoth-esis. Cambridge University Press, New York, pp 90–112

Grahame NJ, Cunningham CL (1995) Genetic differences in intrave-nous cocaine self-administration between C57BL/6J and DBA/2Jmice. Psychopharmacology 122:281–291. doi:10.1007/BF02246549

Haile CN, Kosten TR, Kosten TA (2007) Genetics of dopamine andits contribution to cocaine addiction. Behav Genet 37:119–145.doi:10.1007/s10519-006-9115-2

Hanna EZ, Yi HY, Dufour MC, Whitmore CC (2001) The relationshipof early-onset regular smoking to alcohol use, depression, illicitdrug use, and other risky behaviors during early adolescence:results from the youth supplement to the Third National Healthand Nutrition Examination Survey. J Subst Abuse 13:265–282.doi:10.1016/S0899-3289(01)00077-3

Hedner T, Lundborg P (1985) Development of dopamine autorecep-tors in the postnatal rat brain. J Neural Transm 62:53–63.doi:10.1007/BF01260415

Humensky JL (2010) Are adolescents with high socioeconomic statusmore likely to engage in alcohol and illicit drug use in earlyadulthood? Subst Abuse Treat Prev Policy 5:19. doi:10.1186/1747-597X-5-19

Janowsky A, Mah C, Johnson RA, Cunningham CL, Phillips TJ,Crabbe JC, Eshleman AJ, Belknap JK (2001) Mapping genes thatregulate density of dopamine transporters and correlated behav-iors in recombinant inbred mice. J Pharmacol Exp Ther 298:634–643

Jones BC, Tarantino LM, Rodriguez LA, Reed CL, McClearn GE,Plomin R, Erwin VG (1999) Quantitative-trait loci analysis ofcocaine-related behaviours and neurochemistry. Pharmacoge-netics 9:607–617

Kandel D (1975) Stages in adolescent involvement in drug use.Science 190:912–914. doi:10.1126/science.1188374

Kandel DB, Yamaguchi K (2002) Stages of drug involvement in theUS population. In: Kandel DB (ed) Stages and pathways of druginvolvement: examining the gateway hypothesis. CambridgeUniversity Press, New York, pp 65–89

Kandel DB, Yamaguchi K, Chen K (1992) Stages of progression indrug involvement from adolescence to adulthood: furtherevidence for the gateway theory. J Stud Alcohol 53:447–457

Kelley BM, Middaugh LD (1999) Periadolescent nicotine exposurereduces cocaine reward in adult mice. J Addict Dis 18:27–39.doi:10.1300/J069v18n03_04

Kelley BM, Rowan JD (2004) Long-term, low-level adolescentnicotine exposure produces dose-dependent changes in cocainesensitivity and reward in adult mice. Int J Dev Neurosci 22:339–348. doi:10.1016/j.ijdevneu.2004.04.002

Kuzmin A, Johansson B (2000) Reinforcing and neurochemicaleffects of cocaine: differences among C57, DBA, and 129 mice.Pharmacol Biochem Behav 65:399–406. doi:10.1016/S0091-3057(99)00211-7

Lai S, Lai H, Page JB, McCoy CB (2000) The association betweencigarette smoking and drug abuse in the United States. J AddictDis 19:11–24. doi:10.1300/J069v19n04_02

Lee KH, Blaha CD, Harris BT, Cooper S, Hitti FL, Leiter JC, RobertsDW, Kim U (2006) Dopamine efflux in the rat striatum evokedby electrical stimulation of the subthalamic nucleus: potentialmechanism of action in Parkinson's disease. Eur J Neurosci23:1005–1014. doi:10.1111/j.1460-9568.2006.04638.x

Lemstra M, Bennett NR, Neudorf C, Kunst A, Nannapaneni U,Warren LM, Kershaw T, Scott CR (2008) A meta-analysis ofmarijuana and alcohol use by socio-economic status inadolescents aged 10–15 years. Can J Public Health 99:172–177

Psychopharmacology

Lester DB, Miller AD, Pate TD, Blaha CD (2008) Midbrainacetylcholine and glutamate receptors modulate accumbal dopa-mine release. Neuroreport 19:991–995. doi:10.1097/WNR.0b013e3283036e5e

Lester DB, Miller AD, Blaha CD (2009) Muscarinic receptor blockade inthe ventral tegmental area attenuates cocaine enhancement oflaterodorsal tegmentum stimulation-evoked accumbens dopamineefflux in the mouse. Synapse 64:216–223. doi:10.1002/syn.20717

Matta SG, Balfour DJ, Benowitz NL, Boyd RT, Buccafusco JJ, CaggiulaAR, Craig CR, Collins AC, Damaj MI, Donny EC, Gardiner PS,Grady SR, Heberlein U, Leonard SS, Levin ED, Lukas RJ, MarkouA, Marks MJ, McCallum SE, Parameswaran N, Perkins KA,Picciotto MR, Quik M, Rose JE, Rothenfluh A, Schafer WR,Stolerman IP, Tyndale RF,Wehner JM, Zirger JM (2007) Guidelineson nicotine dose selection for in vivo research. Psychopharmacol-ogy 190:269–319. doi:10.1007/s00213-006-0441-0

McQuown SC, Belluzzi JD, Leslie FM (2007) Low dose nicotinetreatment during early adolescence increases subsequent cocainereward. Neurotoxicol Teratol 29:66–73. doi:10.1016/j.ntt.2006.10.012

Merrill JC, Kleber HD, Shwartz M, Liu H, Lewis SR (1999)Cigarettes, alcohol, marijuana, other risk behaviors, and Amer-ican youth. Drug Alcohol Depend 56:205–212. doi:10.1016/S0376-8716(99)00034-4

Miller G (2010) The seductive allure of behavioral epigenetics.Science 329:24–27. doi:10.1126/science.329.5987.24

Mittleman G, Goldowitz D, Heck DH, Blaha CD (2008) Cerebellarmodulation of frontal cortex dopamine efflux in mice: relevanceto autism and schizophrenia. Synapse 62:544–550. doi:10.1002/syn.20525

Morgan D, Grant KA, Gage HD, Mach RH, Kaplan JR, Prioleau O,Nader SH, Buchheimer N, Ehrenkaufer RL, Nader MA (2002)Social dominance in monkeys: dopamine D2 receptors andcocaine self-administration. Nat Neurosci 5:169–174.doi:10.1038/nn798

Orsini C, Bonito-Oliva A, Conversi D, Cabib S (2005) Susceptibilityto conditioned place preference induced by addictive drugs inmice of the C57BL/6 and DBA/2 inbred strains. Psychopharma-cology 181:327–336. doi:10.1007/s00213-005-2259-6

Paus T, Zijdenbos A, Worsley K, Collins DL, Blumenthal J, Giedd JN,Rapoport JL, Evans AC (1999) Structural maturation of neuralpathways in children and adolescents: in vivo study. Science283:1908–1911. doi:10.1126/science.283.5409.1908

Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxiccoordinates. Academic Press, San Diego

Peirce JL, Lu L, Gu J, Silver LM, Williams RW (2004) A new set ofBXD recombinant inbred lines from advanced intercross pop-ulations in mice. BMC Genet 5:7. doi:10.1186/1471-2156-5-7

Philip VM, Duvvuru S, Gomero B, Ansah TA, Blaha CD, Cook MN,Hamre KM, Lariviere WR, Matthews DB, Mittleman G, Gold-owitz D, Chesler EJ (2010) High-throughput behavioral pheno-typing in the expanded panel of BXD recombinant inbred strains.Genes Brain Behav 9:129–159. doi:10.1111/j.1601-183X.2009.00540.x

Plomin R, McClearn GE, Gora-Maslak G, Neiderhiser JM (1991) Useof recombinant inbred strains to detect quantitative trait lociassociated with behavior. Behav Genet 21:99–116. doi:10.1007/BF01066330

Puglisi-Allegra S, Cabib S (1997) Psychopharmacology of dopamine: thecontribution of comparative studies in inbred strains of mice. ProgNeurobiol 51:637–661. doi:10.1016/S0301-0082(97)00008-7

Roberts A, Pardo-Manuel de Villena F, Wang W, McMillan L,Threadgill DW (2007) The polymorphism architecture of mousegenetic resources elucidated using genome-wide resequencingdata: implications for QTL discovery and systems genetics.Mamm Genome 18:473–481. doi:10.1007/s00335-007-9045-1

Rocha BA, Odom LA, Barron BA, Ator R, Wild SA, Forster MJ(1998) Differential responsiveness to cocaine in C57BL/6J andDBA/2J mice. Psychopharmacology 138:82–88. doi:10.1007/s002130050648

Schönfuß D, Reum T, Olshausen P, Fischer T, Morgenstern R (2001)Modelling constant potential amperometry for investigations ofdopaminergic neurotransmission kinetics in vivo. J NeurosciMethods 112:163–172. doi:10.1016/S0165-0270(01)00465-4

Seale TW, Carney JM (1991) Genetic determinants of susceptibility tothe rewarding and other behavioral actions of cocaine. J AddictDis 10:141–162. doi:10.1300/J069v10n01_10

Siu EC, Tyndale RF (2007) Characterization and comparison ofnicotine and cotinine metabolism in vitro and in vivo in DBA/2and C57BL/6 mice. Mol Pharmacol 71:826–834. doi:10.1124/mol.106.032086

Slotkin TA, MacKillop EA, Rudder CL, Ryde IT, Tate CA, Seidler FJ(2007) Permanent, sex-selective effects of prenatal or adolescentnicotine exposure, separately or sequentially, in rat brain regions:indices of cholinergic and serotonergic synaptic function, cellsignaling, and neural cell number and size at 6 months of age.Neuropsychopharmacology 32:1082–1097. doi:10.1038/sj.npp.1301231

Smith MA, Iordanou JC, Cohen MB, Cole KT, Gergans SR, Lyle MA,Schmidt KT (2009) Effects of environmental enrichment onsensitivity to cocaine in female rats: importance of control ratesof behavior. Behav Pharmacol 20:312–321. doi:10.1097/FBP.0b013e32832ec568

Spear LP (2000) The adolescent brain and age-related behavioralmanifestations. Neurosci Biobehav Rev 24:417–463.doi:10.1016/S0149-7634(00)00014-2

Suaud-Chagny MF (2004) In vivo monitoring of dopamine overflowin the central nervous system by amperometric techniquescombined with carbon fibre electrodes. Methods 33:322–329.doi:10.1016/j.ymeth.2004.01.009

Tarazi FI, Baldessarini RJ (2000) Comparative postnatal developmentof dopamine D(1), D(2) and D(4) receptors in rat forebrain. Int JDev Neurosci 18:29–37. doi:10.1016/S0736-5748(99)00108-2

Tarazi FI, Tomasini EC, Baldessarini RJ (1998) Postnatal developmentof dopamine and serotonin transporters in rat caudate–putamenand nucleus accumbens septi. Neurosci Lett 254:21–24.doi:10.1016/S0304-3940(98)00644-2

Taylor BA, Bedigian HG, Meier H (1977) Genetic studies of the Fv-1locus of mice: linkage with Gpd-1 in recombinant inbred lines. JVirol 23:106–109

Taylor BA, Wnek C, Kotlus BS, Roemer N, MacTaggart T, Phillips SJ(1999) Genotyping new BXD recombinant inbred mouse strainsand comparison of BXD and consensus maps. Mamm Genome10:335–348. doi:10.1007/s003359900998

Thiel KJ, Engelhardt B, Hood LE, Peartree NA, Neisewander JL(2010) The interactive effects of environmental enrichment andextinction interventions in attenuating cue-elicited cocaine-seeking behavior in rats. Pharmacol Biochem Behav 97:595–602. doi:10.1016/j.pbb.2010.09.014

Tolliver BK, Carney JM (1994) Comparison of cocaine and GBR12935: effects on locomotor activity and stereotypy in two inbredmouse strains. Pharmacol Biochem Behav 48:733–739.doi:10.1016/0091-3057(94)90340-9

Tolliver BK, Belknap JK, Woods WE, Carney JM (1994) Geneticanalysis of sensitization and tolerance to cocaine. J PharmacolExp Ther 270:1230–1238

Torabi MR, Bailey WJ, Majd-Jabbari M (1993) Cigarette smoking asa predictor of alcohol and other drug use by children andadolescents: evidence of the "gateway drug effect". J Sch Health63:302–306. doi:10.1111/j.1746-1561.1993.tb06150.x

Trauth JA, Seidler FJ, McCook EC, Slotkin TA (1999) Adolescentnicotine exposure causes persistent upregulation of nicotinic

Psychopharmacology

cholinergic receptors in rat brain regions. Brain Res 851:9–19.doi:10.1016/S0006-8993(99)01994-0

Trauth JA, Seidler FJ, Ali SF, Slotkin TA (2001) Adolescent nicotineexposure produces immediate and long-term changes in CNSnoradrenergic and dopaminergic function. Brain Res 892:269–280. doi:10.1016/S0006-8993(00)03227-3

van der Veen R, Piazza PV, Deroche-Gamonet V (2007) Gene–environment interactions in vulnerability to cocaine intravenousself-administration: a brief social experience affects intake in DBA/2J but not in C57BL/6J mice. Psychopharmacology 193:179–186.doi:10.1007/s00213-007-0777-0

van der Veen R, Koehl M, Abrous DN, de Kloet ER, Piazza PV,Deroche-Gamonet V (2008) Maternal environment influencescocaine intake in adulthood in a genotype-dependent manner.PLoS One 3:e2245. doi:10.1371/journal.pone.0002245

Vansickel AR, Stoops WW, Rush CR (2010) Human sex differencesin d-amphetamine self-administration. Addiction 105:727–731.doi:10.1111/j.1360-0443.2009.02858.x

Wagner FA, Anthony JC (2002) Into the world of illegal drug use:exposure opportunity and other mechanisms linking the use ofalcohol, tobacco, marijuana, and cocaine. Am J Epidemiol155:918–925. doi:10.1093/aje/155.10.918

Williams RW, Gu J, Qi S, Lu L (2001) The genetic structure ofrecombinant inbred mice: high-resolution consensus maps forcomplex trait analysis. Genome Biol 2(11):RESEARCH0046.doi:10.1186/gb-2001-2-11-research0046

Womer DE, Jones BC, Erwin VG (1994) Characterization of dopaminetransporter and locomotor effects of cocaine, GBR 12909, epidepr-ide, and SCH 23390 in C57BL and DBAmice. Pharmacol BiochemBehav 48:327–335. doi:10.1016/0091-3057(94)90534-7

Psychopharmacology