Effects of acute tyrosine/phenylalanine depletion on ... - UGA Psychology

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Brian Hitsman & James MacKillop &. Anne Lingford-Hughes & Tim ... Montgomery et al. 2003). The acute .... (HAM-D); Hamilton 1967] were completed. Eligible.
Psychopharmacology DOI 10.1007/s00213-007-0995-5

ORIGINAL INVESTIGATION

Effects of acute tyrosine/phenylalanine depletion on the selective processing of smoking-related cues and the relative value of cigarettes in smokers Brian Hitsman & James MacKillop & Anne Lingford-Hughes & Tim M. Williams & Faheem Ahmad & Sally Adams & David J. Nutt & Marcus R. Munafò

Received: 16 May 2007 / Accepted: 18 October 2007 # Springer-Verlag 2007

Abstract Rationale Acute tyrosine/phenylalanine depletion (ATPD) is a validated neurobiological challenge that results in reduced dopaminergic neurotransmission, allowing examination of the effects of a hypodopaminergic state on craving-related processes. Objectives We studied 16 nonabstaining smokers (>10 cigarettes/day; 9 males; age 20–33 years) to whom was administered a tyrosine/phenylalanine-free mixture (TYR/ PHE-free) and a balanced amino acid mixture (BAL) in a double-blind, counterbalanced, crossover design. Methods Subjective cigarette craving, attentional bias to smoking-related word cues, relative value of cigarettes, nega-

B. Hitsman (*) Centers for Behavioral and Preventive Medicine, The Miriam Hospital and Brown Medical School, Coro Building, Suite 500, 1 Hoppin Street, Providence, RI 02903, USA e-mail: [email protected] J. MacKillop Center for Alcohol and Addiction Studies, Brown University, Providence, RI, USA A. Lingford-Hughes : T. M. Williams : F. Ahmad : D. J. Nutt Academic Unit of Psychiatry, University of Bristol, Bristol, UK S. Adams : M. R. Munafò (*) Department of Experimental Psychology, University of Bristol, 12a Priory Road, Bristol BS8 1TU, UK e-mail: [email protected]

tive mood, and expired carbon monoxide (CO) levels were measured at various timepoints through 300 min. Participants smoked at hourly intervals to prevent acute nicotine withdrawal during testing. Results The TYR/PHE-free mixture, as compared to the BAL mixture, was associated with a greater increase in CO levels from baseline ( p=0.01). Adjusting for the potential confounding influence of between-condition differences in CO levels across time, TYR/PHE-free mixture was associated with increased demand for cigarettes ( p=0.01) and decreased attentional bias toward smoking-related words ( p=0.003). There were no significant differences between conditions in either subjective craving or depressed or anxious mood ( p values>0.05). Conclusion Among nonabstaining daily smokers, acute dopaminergic depletion via ATPD may influence smoking behavior and indices of smoking-related motivation, such as attentional bias to smoking cues and relative cigarette value, which are not readily captured by subjective craving. Keywords Cigarette smoking . Attentional bias . Relative value . Cigarette craving . Dopamine

Introduction Converging evidence from animal and human studies indicates that cigarette smoking behavior is under partial influence of dopamine-dependent mesolimbic and mesocortical brain systems. Nicotine stimulates burst firing of dopaminergic neurons in animals when binding to nicotinic acetylcholine receptors in the midbrain tegmentum (Di

Psychopharmacology

Chiara 2000), which in turn enhances release of dopamine in the outer shell of the nucleus accumbens and prefrontal cortex (Benwell and Balfour 1992; Corrigall et al. 1994). The cascade of events, including changes in postsynaptic signaling, is theorized to be positively reinforcing with strong hedonic and appetitive motivational properties (Epping-Jordan et al. 1998; Koob and Le Moal 1997). The synthesis of brain dopamine is dependent on the availability of its amino acid precursor, tyrosine, in plasma. Administration of an amino acid mixture that lacks tyrosine, as well as its precursor phenylalanine, decreases availability of tyrosine and phenylalanine in plasma through processes of increased protein synthesis. This results in increased competition for transport across the blood–brain barrier (Oldendorf and Szabo 1976; Pardridge 1977). In animals, administration of the tyrosine/phenylalaninefree (TYR/PHE-free) mixture decreases the availability of tyrosine in the brain, resulting in direct action on dopamine (McTavish et al. 1999b). Noradrenergic response to either amphetamine or idazoxan is unaffected (McTavish et al. 1999a), suggesting a selective effect on dopamine. Administration of the TYR/PHE-free mixture in humans increases prolactin levels (Harmer et al. 2001) and decreases extracellular dopamine levels, as indexed by changes in [11C]raclopride binding (Leyton et al. 2004; Montgomery et al. 2003). The acute tyrosine/phenylalanine depletion (ATPD) technique represents a valuable paradigm for examining the effects of reduced dopamine availability on addictive behaviors. Despite the promise of this approach for identifying core biobehavioral processes underlying chronic addiction, to our knowledge only one study has applied it to nicotine dependence. A recent study of 15 male smokers (Casey et al. 2006) found no effect of ATPD on selfreported craving or smoking topography. Subjective craving may be distinct from other craving-related mechanisms that contribute to relapse following acute smoking abstinence (Munafò and Hitsman 2006; Perkins et al. 1997). The incentive-sensitization model (Robinson and Berridge 1993, 2000) asserts that repeated use of substances that enhance mesolimbic dopaminergic neurotransmission results in permanent or semipermanent changes in the system. This process results in stimuli associated with substance use being assigned higher levels of incentive salience (i.e., subjective “wanting” of the substance). One hypothesized consequence is an attentional bias towards associated stimuli, which is not necessarily closely coupled with subjective craving. Numerous studies have employed the modified Stroop to investigate attentional biases towards smoking-related cues in daily smokers (Munafò and Hitsman 2006). In most cases, only a weak correlation has been observed between

degree of attentional bias and level of subjective cigarette craving. Consequently, cognitive constructs such as attentional bias to smoking cues may index facets of reward and motivational processes not necessarily entirely captured by self-report measures of craving (Perkins et al. 1997; Tiffany and Conklin 2000). A similar distinction has been observed in studies of the relative value of addictive substances vs money. Relative value has been measured by eliciting selfreport of the desired level of consumption of a substance under multiple (and increasing) levels of price. In general, the more a person is willing to spend to obtain a substance, such as a cigarette, the greater its relative value. These studies have found moderate correlations between relative value and subjective craving, but far from collinear relationships (e.g., Mackillop et al. 2007). More objective cognitive and behavioral indicators of motivational drive to smoke, such as attentional bias to smoking-related cues and relative value of cigarettes, may be especially sensitive to acute changes in dopaminergic neurotransmission. In the current study, we investigated the influence of ATPD via administration of a TYR/PHE-free mixture, as compared with a BAL mixture, on motivation to smoke, as indexed by attentional bias for smoking-related cues, relative value of cigarettes, and subjective cigarette craving, and mood among non-nicotine deprived male and female smokers. Based on the role of dopamine in mediating nicotine’s rewarding and motivational effects, we hypothesized that administration of the TYR/PHE-free mixture would be associated with increased attentional bias for smoking-related cues and increased value of cigarettes. Based in part on Casey and colleagues’ (2006) results, we did not hypothesize an effect on subjective craving. We also did not predict effects on negative mood as ATPD has been found to influence mood only in healthy female social drinkers and only after exposure to a psychological stress challenge (Leyton et al. 2000). The TYR/PHE-free and BAL challenges were administered 1 week apart using a double-blind, counterbalanced, crossover design.

Methods Participants Participants were 16 healthy volunteers who reported smoking 10 to 25 cigarettes per day. Exclusion criteria were the following: current intention to quit smoking, defined by a score >5 on a 10-point scale of readiness to quit (with 10=strong desire); any current illicit drug use; current or past psychiatric disorder (DSM-based), including substance abuse or dependence other than nicotine depen-

Psychopharmacology

Candidates who responded to recruitment flyers placed on the University of Bristol precinct attended an initial session for eligibility screening, which included a medical and psychiatric history and physical examination, electrocardiogram, and routine blood testing. A urine specimen was collected for illicit drug use screening and a breath test to screen for recent alcohol consumption. Finally, assessments of nicotine dependence [Fagerstrom test for nicotine dependence (FTND)] (Heatherton et al. 1991) and depressive symptoms [21-item Hamilton depression rating scale (HAM-D); Hamilton 1967] were completed. Eligible participants then completed the two 7-h test sessions conducted at the Bristol Royal Infirmary. The study was approved by the Bath Research Ethics Committee (UK) and the Institutional Review Board of The Miriam Hospital (USA). Participants were reimbursed £60 plus travel expenses.

nausea and sedation. The composition for males was isoleucine 15 g, leucine 22.5 g, lysine 17.5 g, methionine 5 g, valine 17.5 g, threonine 10 g, and tryptophan 2.5 g. The BAL mixture for males additionally contained tyrosine 12.5 g and phenylalanine 12.5 g. Females received 20% less by weight of each amino acid than males. The amino acids were suspended in tap water, which was flavored with lemon and lime or cherry vanilla (participant’s preference) to disguise the unpalatable taste of the mixture. To minimize any possibility of nicotine withdrawal during testing, participants took monitored cigarette breaks at 60-, 120-, 180-, 240-, 300-, and 360-min intervals. An exhaled CO breath sample was taken 10 min after each cigarette. Subjective craving and mood ratings, along with a second blood sample for amino acid analysis, were taken again at 300 min. The Minnesota Withdrawal ScaleRevised (MWS-R) (Hughes and Hatsukami 1986) was completed to verify that participants were not in withdrawal. Finally, participants completed the reassessment of depressive symptoms and the assessments of relative cigarette value and attentional bias to smoking-related cues. Upon completion of the 300-min assessments, they were offered a balanced meal and then allowed to leave when subjectively and objectively stable. The second session was identical to the first, except that the alternate mixture was given. Eight participants completed the BAL condition first and eight completed the TYR/PHE-free condition first.

Experimental sessions

Measures

Participants came to the laboratory at 08:00 hour on the morning of each test session after having followed a lowprotein diet (total protein content less than 20 g) for the preceding 24 h and after having fasted from midnight. They were instructed to smoke normally between awakening and arriving at the laboratory. To minimize the possibility of caffeine withdrawal during testing, participants who were daily coffee or tea drinkers were instructed to have their usual morning amount. Female participants were tested during the follicular phase of their menstrual cycle. For the baseline assessment, participants provided a blood sample for amino acid analysis and an exhaled CO breath sample to quantify recent smoking rate. They also completed assessments of mood, craving, and depressive symptoms. Immediately following the baseline assessment, participants consumed either the TYR/PHE-free or BAL amino acid mixture in a randomized, double-blind, counterbalanced manner. We used the 90-g TYR/PHE-free mixture developed by McTavish and colleagues (McTavish et al. 1999b) to sufficiently challenge the dopaminergic system yet minimize potential physical side effects, especially

The primary outcomes were attentional bias to smokingrelated cues and relative value of cigarettes in response to ATPD vs balanced amino acid challenge. The modified Stroop task was identical to that used in prior research (Mogg and Bradley 2002; Munafò et al. 2003) and consisted of 12 practice trials followed by 12 blocks, each consisting of 2 buffer and 12 experimental trials (i.e., 144 experimental trials in total). The stimulus words were (1) 12 smoking-related words (cigarette, fags, lighter, matches, inhale, ashtray, smoke, nicotine, cigar, tobacco, puff, filter) and (2) 12 categorized neutral words (blanket, garage, shampoo, handle, sofa, curtain, switch, bathroom, vase, hallway, duster, lounge). Each block presented one set of stimulus words (smoking or neutral) under either masked or unmasked exposure conditions. The order of the 12 blocks was randomized across participants. Sets were matched for length and frequency using published norms (Caroll et al. 1971). Uncategorized neutral words (e.g., lucky, donkey, cultural, calliper) were used for the practice and buffer trials. An awareness check was given to participants immediately after completing the Stroop to confirm the

dence; history of neurological illness; significant current medical illness; pregnancy or lactation; current or recent (past 3 months) use of psychotropic medications (antidepressants, anxiolytics, or neuroleptics) or herbal supplements known to affect mood (e.g., St. John’s Wort); current use of smoking cessation pharmacotherapy; or uncorrected impaired vision or color blindness. All participants gave their written informed consent prior to screening. Eligibility screening

Psychopharmacology

masked exposure condition. This was a lexical decision task to identify the presence of a word or nonword and consisted of 12 practice and 24 test trials as described by Munafo and colleagues (2003). Relative value of cigarettes vs money was assessed using a cigarette purchase task (CPT) (Jacobs and Bickel 1999). The CPT is a self-report measure that is an analog of a progressive-ratio operant task (Hodos 1961) using self-reported consumption under various levels of price. Relative value is assessed by generating a cigarette demand curve or a quantitative representation of economic demand for cigarettes at multiple levels of price. In turn, the demand curve is used to characterize several facets of relative value. Prior studies have used the CPT as a traittype measure (i.e., typical smoking during a typical day) (Jacobs and Bickel 1999; Mackillop and Murphy 2007; Murphy and MacKillop 2006), but we used it as a state measure, assessing demand for cigarettes over a 3-h period. Participants were assessed at the following 16 costs: zero (free), £0.01, £0.05, £0.13, £0.25, £0.50, £1, £3, £6, £11, £35, £70, £140, £280, £560, and £1,120 (Jacobs and Bickel 1999; Murphy and MacKillop 2006). Secondary outcomes were subjective craving, mood, and depressive symptoms as measured using the brief questionnaire of smoking urges (QSU-Brief) (Cox et al. 2001), state-trait anxiety inventory—state subscale (STAI) (Spielberger et al. 1983), profile of mood states (POMS) (McNair 1971), and visual analog scale (VAS) ratings of the following symptoms: nausea, tremor of fingers, difficulty concentrating, restlessness, depression, anxiety, irritability, drowsiness, headache, nervousness, fatigue, hunger, excess energy, and euphoria. Depressive symptoms were measured using a modified version of the Hamilton depression rating scale (MHAMD). The M-HAMD included only those nine items that could change meaningfully over the course of an experimental session: low energy, depressed mood, feelings of guilt, decreased concentration and motor activity, anxious mood, decreased appetite, loss of pleasure, feelings of worthlessness, and loss of interest. Plasma levels of tyrosine and phenylalanine were measured at baseline and 300 min (approximate time of peak depletion) to confirm that the ATPD challenge procedure produced the predicted decline in levels. The ratio of tyrosine + phenylalanine to other large neutral amino acids (LNAAs; valine, tryptophan, isoleucine, leucine) was also measured. Plasma was separated by centrifugation and stored at −30°C. Total concentrations of tyrosine, phenylalanine, valine, tryptophan, isoleucine, and leucine were measured using automated high-performance liquid chromatography with fluorescence end-point detection and precolumn sample derivatization adapted from methods described by Furst and colleagues (1990). Norvaline

was used as an internal standard. The detection limit was 1.3 pg/ml and inter- and intra-assay coefficients of variation were 13 and 8%, respectively. Statistical analysis We first examined the changes in plasma levels of tyrosine and phenylalanine, as well as in the ratio of tyrosine + phenylalanine/LNAAs, from baseline to 300 min during the TYR/PHE-free condition compared with the BAL condition. Data were analyzed using repeated measures 2×2×2 multivariate analysis of variance (MANOVAs), with mixture (TYR/PHE-free, BAL) and time (baseline, 300 min) as within-subjects factors and condition order (TYR/PHE-free first, BAL first) as a between-subjects factor. Next, we examined change in exhaled CO values across the two conditions, as well as nicotine withdrawal scores at 300 min between conditions because any difference in smoking or withdrawal would confound the analyses of attentional bias and cigarette reward value. CO data were analyzed using repeated measures 3×2×2 MANOVA, with time (baseline, 180 min, 300 min) and mixture (TYR/PHE-free, BAL) as within-subjects factors and condition order (TYR/PHE-free first, BAL first) as a between-subjects factor. The intermediate (180 min) timepoint was chosen as the midpoint between baseline and likely peak depletion (300 min). MWS-R data were analyzed using repeated measures 2×2 MANOVA with mixture (TYR/PHE-free, BAL) as a withinsubjects factor and condition order (TYR/PHE-free first, BAL first) as a between-subjects factor. After these important experimental checks, we analyzed the primary outcomes variables. Modified Stroop data were analyzed using a repeated measures 2×2×2×2 MANOVA of color-naming response times with word type (smokingrelated, control), exposure (masked, unmasked), and mixture (TYR/PHE-free, BAL) as within-subjects factors and condition order (TYR/PHE-free first, BAL first) as a between-subjects factor. CPT data were analyzed using methods similar to those of prior studies involving purchase tasks to generate demand curves (Jacobs and Bickel 1999; Murphy and MacKillop 2006), including the application of nonlinear demand curve equation (Hursh et al. 1988). Five indices of relative value were derived: (1) intensity of demand (i.e., cigarette consumption at the lowest level of cost); (2) maximum cigarette expenditure (Omax; i.e., maximum amount of money a participant was willing to spend across various increments of price); (3) price at maximum consumption (Pmax; the price corresponding to Omax, reflecting the point at which demand becomes elastic); (4) elasticity of demand (sensitivity to escalating cost); and (5) breakpoint (i.e., first price at which consumption was fully suppressed). These indices reflect different topographical facets of the demand curve. Data

Psychopharmacology

were analyzed using 2×2 repeated-measures MANOVA, with mixture (TYR/PHE-free, BAL) as a within-subjects factor and condition order (TYR/PHE-free first, BAL first) as a between-subjects factor. The secondary outcomes of mood (anxiety, total mood disturbance), craving, and depressive symptoms were analyzed using repeated-measures 2×2×2 MANOVAs, with mixture (TYR/PHE-free, BAL) and time (baseline, 300 min) as within-subjects factors and condition order (TYR/PHE-free first, BAL first) as a between-subjects factor. We conducted correlational analyses of the plasma amino acid data to evaluate whether change in tyrosine or the ratio of tyrosine + phenylalanine to LNAAs were associated with change on any dependent measure demonstrating a significant effect of ATPD. All statistically significant interaction effects among mixture condition and time were decomposed with simple main effects analyses. Where no significant condition order effect was evident, the order factor was removed from the analysis. Where an order effect was present, it was retained and the direction of the effect was probed using simple interaction effects analyses stratifying on order. An alpha of 0.05 was used for all outcomes.

Results

in levels from baseline to 300 min in the TYR/PHE-free condition (tyrosine F [1, 10]=33.3, p0.40). Among participants who received the TYR/ PHE-free mixture first, there were no significant main effects or interactions ( p values>0.13). These data are presented graphically in Fig. 2. A comparison of RT data from the first session only, with mixture (TYR/PHE-free/BAL) as a between-subjects factor, indicated a significant main effect of word type (F [1, 11]=11.6, p=0.006). This was qualified by a marginal word type × mixture interaction (F [1, 5]=4.4, p=0.06). A similar analysis of data from the second test session indicated no main effects or interactions ( p values>0.65). During the TYR/PHE-free condition, the difference in RTs for smoking stimuli compared to control stimuli was not significantly correlated with reduction in either plasma tyrosine or the ratio of tyrosine + phenylalanine to other LNAAs (p values>0.26).

Table 1 Means and standard deviations (ms) for reaction times to unmasked and masked smoking-related and control stimuli by challenge condition and condition order Order

TYR/PHE-free condition BAL first TYR/PHE-free first Combined BAL condition BAL first TYR/PHE-free first Combined

Smoking-related

Control

Unmasked

Masked

Combined

Unmasked

Masked

Combined

577.89 (186.59) 585.00 (90.53) 581.44 (140.95)

572.80 (103.87) 560.88 (75.40) 566.84 (87.42)

575.35 (139.02) 572.94 (82.59) 574.14 (109.86)

591.17 (160.43) 562.75 (87.34) 576.96 (124.97)

556.65 (146.39) 551.48 (77.06) 554.06 (112.42)

573.91 (152.30) 557.11 (81.48) 565.51 (117.67)

573.18 (120.74) 551.66 (77.28) 562.42 (98.03)

538.83 (106.42) 538.68 (65.25) 538.75 (84.81)

556.00 (112.03) 545.17 (69.01) 550.59 (89.57)

531.45 (115.64) 567.66 (88.04) 549.56 (100.51)

512.86 (113.70) 534.01 (83.50) 523.43 (96.46)

522.15 (111.54) 550.84 (84.20) 536.50 (96.11)

Psychopharmacology

Fig. 2 Mean color-naming interference for smoking-related stimuli by challenge condition and condition order. Interference scores were calculated for each participant by subtracting the mean RTs for control stimuli from those for smoking-related stimuli. This index reflects the word type effect in the RT data, with positive values indicating an interference effect of smoking-related stimuli. Errors bars represent SEM

The percentage of awareness check trials with correct responses was calculated for all participants. The overall mean percentage correct was 52% in the BAL condition and 51% in the TYR/PHE-free condition, neither of which was significantly above chance performance of 50% (BAL, t[13]=1.32, p=0.21; TYR/PHE-free, t[13]=0.46, p=0.65). Relative value of cigarettes Performance on the CPT topographically conformed to expectations: demand for cigarettes exhibited a decelerating curve in response to escalating price and expenditure exhibited the characteristic inverted U-shaped curve (see Fig. 3). Hursh and colleagues’ (1988) demand equation provided a very good fit to the aggregated data for both the BAL and TYR/PHE-free conditions (R2 =0.94 and R2 = 0.93, respectively). In addition, the equation provided a very good fit to the individual participant data under both conditions (BAL, median R2 =0.94 [IQR=0.93– 0.99]; TYR/PHE-free: median R 2 = 0.93 [IQR = 0.91– 0.96]). Initial CPT analyses revealed no significant order effects ( p values>0.25), so, as in all other analyses, the order term was removed from the analytic model. Adjusting for the difference between conditions in change in exhaled CO from baseline to 300 min, the TYR/PHE-free challenge was associated with a significant increase in intensity of demand for cigarettes (F [1, 10]=8.96, p= 0.01). There were no differences in breakpoint (F [1, 10]= 0.29, p=0.60), Omax (F [1, 10]=0.48, p=0.50), Pmax (F [1,

Fig. 3 Mean performance on the CPT by challenge condition. a Demand for cigarettes (self-reported consumption) over the session and in proportionate log–log coordinates. Demand curves after both challenges exhibit the prototypical deceleration in response to escalating cost, initially exhibiting inelastic demand and then becoming elastic and decreasing to zero consumption. Zero values are recoded as trivial nonzero values to permit logarithmic transformation, but values are only shown to two decimal points. b Output for cigarettes (associated monetary expenditure) over the experimental period. The prototypical inverted U-shaped curve is evident, reflecting escalating expenditure to a peak followed by subsequent decreases, eventually to zero, is evident, but no effect on maximum expenditure (i.e., Omax). Zero values are recoded as trivial nonzero values to permit logarithmic transformation, but values are only shown to two decimal points. c The significant effect of the TYR/PHE-free challenge on intensity of demand for cigarettes (i.e., initial demand at zero cost), which is obscured by the logarithmic coordinates in a; **p0.18), with the exception of a high-magnitude negative correlation between Pmax and percent change in tyrosine, r=−0.69, p< 0.05. This relationship indicated an inverse relationship such that the greater the reduction in plasma tyrosine, the higher the level of Pmax (i.e., price at which demand becomes elastic), reflecting greater desire for cigarettes. Subjective cigarette craving The repeated-measures MANCOVA of QSU-Brief scores revealed that mixture × time interaction was statistically nonsignificant (F [1, 11]=0.01, p=0.92). A significant main effect of time (F [1, 11]=30.7, p0.08). For the VAS ratings of psychological and physical symptoms, there were significant main effects of time for difficulty concentrating (F [1,12]=11.0, p=0.006), restlessness (F [1,12]=19.0, p=0.001), and headache (F [1,12]=7.1, p=0.02), indicating in all cases an increase from baseline to 300 min in both conditions. For irritability, there was a significant main effect of time (F [1,12]=5.3, p=0.04), which was qualified by a time × mixture interaction (F [1,12]=10.72, p=0.007), indicating an increase from baseline to 300 min in the TYR/PHE-free condition only. For hunger, there was a significant main effect of time (F [1,11]=5.9, p=0.03), which was qualified by time × mixture (F [1,11]=12.1, p=0.005) and time × mixture × order (F [1,11]=11.4, p=0.006) interactions, indicating an

increase from baseline to 300 min, with this effect greater among participants who received the TYR/PHE-free mixture second. No other symptoms showed changes, including those for depression and anxiety ( p values>0.06).

Discussion We found that ATPD among nonabstaining male and female smokers was associated with increased intensity of demand for cigarettes relative to money and reduced attentional bias to smoking-related cues, although the latter effect depended upon the order in which the challenge conditions were administered. Despite standardizing the number of cigarettes smoked and achieving comparably very low levels of nicotine withdrawal between conditions, we also found that ATPD was associated with increased intensity of smoking behavior as measured by exhaled CO, although this effect only constituted a trend when all intermediate timepoints between baseline and approximate time of peak depletion were included in the analysis. The effects on smoking behavior, attentional bias to smoking-related cues, and cigarette reward value appeared in the absence of subjective craving. They also occurred independently of any changes in anxious or depressed mood or symptoms, as all were unaffected by ATPD. Our results are consistent with preclinical and smoking treatment studies evaluating acute and longer-duration manipulations of dopamine. Dopaminergic agonists have been found to reduce smoking behavior, whereas antagonists have the opposite effect (Caskey et al. 1999, 2002; Dawe et al. 1995; George et al. 2003; Houtsmuller et al. 2002; Jarvik et al. 2000). Caskey and colleagues (2002) found that the dopaminergic agonist bromocriptine reduced smoking behavior, as indicated by alterations in smoking topography, whereas the dopaminergic antagonist haloperidol increased smoking. Selegiline, which enhances dopaminergic functioning through inhibition of monoamine oxidase B, has been shown to exert a modest effect on short-term smoking abstinence (George et al. 2003; Houtsmuller et al. 2002). Finally, treatment involving bupropion, which acts as both a dopaminergic and noradrenergic reuptake inhibitor, doubles long-term quit rates as compared with placebo (Mooney and Sofuoglu 2006). Unlike in the current study, the aforementioned pharmacological studies typically have observed effects on subjective craving (Brody et al. 2004; Caskey et al. 2002; Houtsmuller et al. 2002). As in Casey and colleagues’ (2006) study of male smokers after overnight abstinence, ATPD did not influence craving in our sample of nonabstaining smokers. This discrepancy with the literature may reflect differing mechanisms of action between ATPD and agents used in the general pharmacological challenge

Psychopharmacology

and treatment studies. Whereas ATPD influences dopaminergic function by decreasing the amount of presynaptic dopamine available to postsynaptic receptors, most pharmacological challenges studied either stimulate or block postsynaptic receptor sites leading to more extreme changes in dopaminergic function. They also have substantial secondary influences on other neurochemical systems. The dissociation of smoking-related reward and motivational processes from conscious awareness is consistent with formulations of incentive motivation (Berridge and Winkielman 2003; Uslaner et al. 2006) and compulsive drug use (Tiffany and Carter 1998; Tiffany and Conklin 2000). That ATPD-reduced attentional bias was also unexpected given the theory that attentional bias is a cognitive correlate of craving (Sayette et al. 2000). If accepted, a reduction in central dopamine levels, which may serve to model some of the effects of acute nicotine abstinence, should increase craving and, in parallel, increase attentional bias. Our results, in contrast, are consistent with a study by Munafò and colleagues (Munafò et al. 2007), which reported a reduction in attentional bias following ATPD among female smokers in acute withdrawal. Dopamine blockade using haloperidol has been found to attenuate attentional bias to heroin-related cues but not subjective craving (Franken et al. 2004). In another study, combined dopaminergic and serotonergic blockade using olanzapine reduced smoking cue-elicited craving for cigarettes (Hutchison et al. 2004). These findings are consistent with the hypothesis that dopaminergic neurotransmission may serve to signal the expectation of reward (de la Fuente-Fernandez et al. 2002), with conditioned cues stimulating dopaminergic release as a result of their association with addictive substances such as nicotine (Di Chiara et al. 2004). Thus, attentional bias for smoking-related cues may reflect expectation of reward and reduced dopaminergic neurotransmission via ATPD may attenuate this effect. Moreover, attentional bias may not be a simple cognitive correlate of subjective craving. It may be necessary to distinguish between phasic and tonic dopamine neurotransmission, with the former mediating cue reactivity and attentional bias to drug cues (Zhang and Sulzer 2004), and the latter mediating withdrawal symptoms associated with acute abstinence (Mansvelder et al. 2003). A reduction in central dopamine levels may therefore serve to reduce tonic dopamine levels (thereby elevating withdrawal symptoms such as craving) while attenuating phasic dopamine release (thereby reducing cue reactivity and attentional bias). One qualification of the modified Stroop effect is that it was observed only among the subset of participants who completed the BAL challenge first. The differential effect as a function of condition order (BAL first but not TYR/ PHE-free first) likely reflects a combination of an attenuation of the modified Stroop effect with practice (i.e.,

repeated administration from session 1 to session 2), which has been observed in a prior study (Munafò et al. 2003), plus attenuation caused by ATPD. This would serve to maximize the difference between challenge conditions in the BAL-first group and decrease it in the TYR/PHE-free group. This interpretation is supported by our observation that, at the first test session, there was a marginal effect of ATPD (reflecting a depletion effect), while at the second test session, there was no evidence of a modified Stroop effect in either group (reflecting a practice effect). Despite observing no effects of ATPD on subjective craving, it was notable that ATPD also increased the relative value of cigarettes. Five hours after ATPD, participants reported wanting to smoke on average two more cigarettes over the next 3 h than at the same time-point during the balance amino acid challenge. The pattern of expenditure reflected more persistent motivational drive, although this was based on observation, not on a significant effect of ATPD on the expenditure-related index of Omax. The CPT results are consistent with studies reporting state-dependent effects on other behavioral economic indices of drug use motivation (Field et al. 2006; Giordano et al. 2002; Mitchell 2004) and research indicating a dissociation between subjective craving and objective indices of motivation (e.g., Perkins et al. 1997). It is important to highlight two issues. First, the amino acid mixture used in this study (90-g mixture developed by McTavish et al. 1999b) lacked histidine, which is the essential amino acid precursor of histamine in the brain. Therefore, it is possible that the effects observed in our study are due to reduced synthesis of histamine in addition to dopamine. Second, participants in this study, although selected on the basis of cigarettes smoked per day (10 or more), were relatively young smokers. There is debate regarding the processes that lead to attentional bias for drug-related cues. Incentive salience theories regard such bias as a consequence of the ascription of incentive salience to initially neutral cues through repeated pairing with drug reinforcement (Franken et al. 2005) so that attentional bias should increase with exposure. Reward probability theories (Fiorillo et al. 2003), in contrast, argue that dopamine encodes prediction error (i.e., the discrepancy between predicted and actual reward), so that attentional bias should be greater when prediction error is greater (i.e., in inexperienced drug users) compared with cues that have been learned to robustly predict drug reinforcement (i.e., in experienced users). Although our data do not allow us to evaluate these two possibilities directly, use of ATPD in inexperienced vs experienced smokers might offer a means to test these competing theories on the basis that it should attenuate dopamine-mediated effects. The issue is complex, however—the difference may instead reflect attention to conditioned cues vs the reward itself, while the difference

Psychopharmacology

between phasic and tonic dopamine release may be relevant. It has also been suggested that dopamine may mediate both the rewarding and aversive effects of nicotine, such that blockade of mesolimbic dopamine signaling may selectively reduce the aversive properties of nicotine (Laviolette and van der Kooy 2003). These possibilities will require detailed investigation to elucidate the exact relationship between dopaminergic neurotransmission and cigarette-smoking behaviors. In conclusion, this study provides preliminary evidence that acute dopaminergic depletion (and possibly histidine depletion) via ATPD may influence smoking behavior, as well as cognitive and behavioral factors reflecting motivational drive to smoke in nonabstaining daily smokers. These effects were not captured by self-reported craving as measured using the QSU-Brief, and they occurred in the absence of acute changes in negative mood. Acknowledgements This study was funded by a National Institute on Drug Abuse Mentored Clinical Scientist Development Award (K08 DA017145) to Brian Hitsman. We are grateful to Karin Mogg and Brendan Bradley for supplying the code for the original modified Stroop task used in this study.

References Benwell ME, Balfour DJ (1992) The effects of acute and repeated nicotine treatment on nucleus accumbens dopamine and locomotor activity. Br J Pharmacol 105:849–856 Berridge KC, Winkielman P (2003) What is an unconscious emotion: the case for unconscious ‘liking’. Cogn Emot 17:181–211 Brody AL, Mandelkern MA, Lee G, Smith E, Sadeghi M, Saxena S, Jarvik ME, London ED (2004) Attenuation of cue-induced cigarette craving and anterior cingulate cortex activation in bupropion-treated smokers: a preliminary study. Psychiatry Res 130:269–281 Caroll JB, Davies P, Richman B (1971) The American heritage word frequency book. American Heritage, New York Casey KF, Benkelfat C, Young SN, Leyton M (2006) Lack of effect of acute dopamine precursor depletion in nicotine-dependent smokers. Eur Neuropsychopharmacol 16:512–520 Caskey NH, Jarvik ME, Wirshing WC (1999) The effects of dopaminergic D2 stimulation and blockade on smoking behavior. Exp Clin Psychopharmacol 7:72–78 Caskey NH, Jarvik ME, Wirshing WC, Madsen DC, Iwamoto-Schaap PN, Eisenberger NI, Huerta L, Terrace SM, Olmstead RE (2002) Modulating tobacco smoking rates by dopaminergic stimulation and blockade. Nicotine Tob Res 4:259–266 Corrigall WA, Coen KM, Adamson KL (1994) Self-administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area. Brain Res 653:278–284 Cox LS, Tiffany ST, Christen AG (2001) Evaluation of the brief questionnaire of smoking urges (QSU-brief) in laboratory and clinical settings. Nicotine Tob Res 3:7–16 Dawe S, Gerada C, Russell MA, Gray JA (1995) Nicotine intake in smokers increases following a single dose of haloperidol. Psychopharmacology 117:110–115 de la Fuente-Fernandez R, Phillips AG, Zamburlini M, Sossi V, Calne DB, Ruth TJ, Stoessl AJ (2002) Dopamine release in human

ventral striatum and expectation of reward. Behav Brain Res 136:359–363 Di Chiara G (2000) Role of dopamine in the behavioural actions of nicotine related to addiction. Eur J Pharmacol 393:295–314 Di Chiara G, Bassareo V, Fenu S, De Luca MA, Spina L, Cadoni C, Acquas E, Carboni E, Valentini V, Lecca D (2004) Dopamine and drug addiction: the nucleus accumbens shell connection. Neuropharmacology 47(Suppl 1):227–241 Epping-Jordan MP, Watkins SS, Koob GF, Markou A (1998) Dramatic decreases in brain reward function during nicotine withdrawal. Nature 393:76–79 Field M, Santarcangelo M, Sumnall H, Goudie A, Cole J (2006) Delay discounting and the behavioural economics of cigarette purchases in smokers: the effects of nicotine deprivation. Psychopharmacology 186:255–263 Fiorillo CD, Tobler PN, Schultz W (2003) Discrete coding of reward probability and uncertainty by dopamine neurons. Science 299:1898–1902 Franken IH, Hendriks VM, Stam CJ, Van den Brink W (2004) A role for dopamine in the processing of drug cues in heroin dependent patients. Eur Neuropsychopharmacol 14:503–508 Franken IH, Booij J, van den Brink W (2005) The role of dopamine in human addiction: from reward to motivated attention. Eur J Pharmacol 526:199–206 Furst P, Pollack L, Graser TA, Godel H, Stehle P (1990) Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials. J Chromatogr 499:557–569 George TP, Vessicchio JC, Termine A, Jatlow PI, Kosten TR, O’Malley SS (2003) A preliminary placebo-controlled trial of selegiline hydrochloride for smoking cessation. Biol Psychiatry 53:136–143 Giordano LA, Bickel WK, Loewenstein G, Jacobs EA, Marsch L, Badger GJ (2002) Mild opioid deprivation increases the degree that opioid-dependent outpatients discount delayed heroin and money. Psychopharmacology 163:174–182 Hamilton M (1967) Development of a rating scale for primary depressive illness. Br J Soc Clin Psychol 6:278–296 Harmer CJ, McTavish SF, Clark L, Goodwin GM, Cowen PJ (2001) Tyrosine depletion attenuates dopamine function in healthy volunteers. Psychopharmacology 154:105–111 Heatherton TF, Kozlowski LT, Frecker RC, Fagerstrom KO (1991) The Fagerstrom test for nicotine dependence: a revision of the Fagerstrom Tolerance Questionnaire. Br J Addict 86:1119–1127 Hodos W (1961) Progressive ratio as a measure of reward strength. Science 134:943–944 Houtsmuller EJ, Thornton JA, Stitzer ML (2002) Effects of selegiline (L-deprenyl) during smoking and short-term abstinence. Psychopharmacology 163:213–220 Hughes JR, Hatsukami D (1986) Signs and symptoms of tobacco withdrawal. Arch Gen Psychiatry 43:289–294 Hursh SR, Raslear TG, Shurtleff D, Bauman R, Simmons L (1988) A cost–benefit analysis of demand for food. J Exp Anal Behav 50:419–440 Hutchison KE, Rutter MC, Niaura R, Swift RM, Pickworth WB, Sobik L (2004) Olanzapine attenuates cue-elicited craving for tobacco. Psychopharmacology 175:407–413 Jacobs EA, Bickel WK (1999) Modeling drug consumption in the clinic using simulation procedures: demand for heroin and cigarettes in opioid-dependent outpatients. Exp Clin Psychopharmacol 7:412–426 Jarvik ME, Caskey NH, Wirshing WC, Madsen DC, Iwamoto-Schaap PN, Elins JL, Eisenberger NI, Olmstead RE (2000) Bromocriptine reduces cigarette smoking. Addiction 95:1173–1183 Koob GF, Le Moal M (1997) Drug abuse: hedonic homeostatic dysregulation. Science 278:52–58

Psychopharmacology Laviolette SR, van der Kooy D (2003) Blockade of mesolimbic dopamine transmission dramatically increases sensitivity to the rewarding effects of nicotine in the ventral tegmental area. Mol Psychiatry 8:50–59 Leyton M, Young SN, Pihl RO, Etezadi S, Lauze C, Blier P, Baker GB, Benkelfat C (2000) Effects on mood of acute phenylalanine/ tyrosine depletion in health women. Neuropsychopharmacology 22:52–63 Leyton M, Dagher A, Boileau I, Casey K, Baker GB, Diksic M, Gunn R, Young SN, Benkelfat C (2004) Decreasing amphetamineinduced dopamine release by acute phenylalanine/tyrosine depletion: a PET/[11C]raclopride study in healthy men. Neuropsychopharmacology 29:427–432 MacKillop J, Murphy JG (2007) A behavioral economic measure of demand for alcohol predicts brief intervention outcomes. Drug Alcohol Depend 89:223–233 Mackillop J, Menges DP, McGeary JE, Lisman SA (2007) Effects of craving and DRD4 VNTR genotype on the relative value of alcohol: an initial human laboratory study. Behav Brain Funct 3:11 Mansvelder HD, De Rover M, McGehee DS, Brussaard AB (2003) Cholinergic modulation of dopaminergic reward areas: upstream and downstream targets of nicotine addiction. Eur J Pharmacol 480:117–123 McNair D (1971) Profile of mood states manual. Educational and Industrial Testing Service, San Diego McTavish SF, Callado L, Cowen PJ, Sharp T (1999a) Comparison of the effects of alpha-methyl-p-tyrosine and a tyrosine-free amino acid load on extracellular noradrenaline in the rat hippocampus in vivo. J Psychopharmacol 13:379–384 McTavish SF, Cowen PJ, Sharp T (1999b) Effect of a tyrosine-free amino acid mixture on regional brain catecholamine synthesis and release. Psychopharmacology 141:182–188 Mitchell SH (2004) Effects of short-term nicotine deprivation on decision-making: delay, uncertainty and effort discounting. Nicotine Tob Res 6:819–828 Mogg K, Bradley BP (2002) Selective processing of smoking-related cues in smokers: manipulation of deprivation level and comparison of three measures of processing bias. J Psychopharmacol 16:385–392 Montgomery AJ, McTavish SF, Cowen PJ, Grasby PM (2003) Reduction of brain dopamine concentration with dietary tyrosine plus phenylalanine depletion: an [11C]raclopride PET study. Am J Psychiatry 160:1887–1889 Mooney ME, Sofuoglu M (2006) Bupropion for the treatment of nicotine withdrawal and craving. Expert Rev Neurother 6:965– 981

Munafò MR, Hitsman B (2006) Neurocircuitry of attentional processes in addictive behaviours. In: Munafò MR, Albery I (eds) Cognition and addiction. Oxford University Press, Oxford Munafò MR, Mogg K, Roberts S, Bradley B, Murphy M (2003) Selective processing of smoking-related cues in current smokers, ex-smokers and never-smokers on the modified Stroop task. J Psychopharmacol 17:310–316 Munafò MR, Mannie ZN, Cowen PJ, Harmer CJ, McTavish SF (2007) Effects of acute tyrosine depletion on subjective craving and selective processing of smoking-related cues in abstinent cigarette smokers. J Psychopharmacol (in press) Murphy JG, MacKillop J (2006) Relative reinforcing efficacy of alcohol among college student drinkers. Exp Clin Psychopharmacol 14:219–227 Oldendorf WH, Szabo J (1976) Amino acid assignment to one of three blood–brain barrier amino acid carriers. Am J Physiol 230:94–98 Pardridge WM (1977) Kinetics of competitive inhibition of neutral amino acid transport across the blood–brain barrier. J Neurochem 28:103–108 Perkins KA, Grobe J, Fonte C (1997) Influence of acute smoking exposure on the subsequent reinforcing value of smoking. Exp Clin Psychopharmacol 5:277–285 Ratcliff R (1993) Methods for dealing with reaction time outliers. Psychol Bull 114:510–532 Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive-sensitization theory of addiction. Brains Res Rev 18:247–291 Robinson TE, Berridge KC (2000) The psychology and neurobiology of addiction: an incentive-sensitization view. Addiction 95 (Suppl 2):S91–S117 Sayette MA, Shiffman S, Tiffany ST, Niaura RS, Martin CS, Shadel WG (2000) The measurement of drug craving. Addiction 95 (Suppl 2):S189–S210 Spielberger CD, Gorusch RL, Lushene R, Vagg PR, Jacobs GA (1983) Manual for the state trait anxiety inventory. Consulting Psychologists Press, Mountain View Tiffany ST, Carter BL (1998) Is craving the source of compulsive drug use? J Psychopharmacol 12:23–30 Tiffany ST, Conklin CA (2000) A cognitive processing model of alcohol craving and compulsive alcohol use. Addiction 95 (Suppl 2):S145–S153 Uslaner JM, Acerbo MJ, Jones SA, Robinson TE (2006) The attribution of incentive salience to a stimulus that signals an intravenous injection of cocaine. Behav Brain Res 169:320–324 Zhang H, Sulzer D (2004) Frequency-dependent modulation of dopamine release by nicotine. Nat Neurosci 7:581–582