Table 1 shows that patients and controls (Section 1) and separately patients-with-CU and controls-without-CU (Section 2) did not significantly differ in terms of age, sex, race, ethnicity or socioeconomic status. The average age for both groups (patients and controls) was about 16 years, about three-quarters were male, two-thirds Caucasian, and about 15% Hispanic.

After Colorado Multiple IRB approval, twenty adolescent patients (15 male) were recruited through a university-based adolescent treatment program. Patients are generally referred to treatment through social services or criminal justice agencies. Previous publications support that most of the patients admitted to this program have both conduct disorder and substance dependence by DSM-IV criteria [25] ; treatment focuses on both these disorders, utilizing behavioral approaches, family-based therapy and medications for comorbid disorders such as depression. Nineteen control adolescents (15 male) were recruited through flyers, advertisements in school newspapers and a market research company. Written informed consent (parents) and assent (adolescents) was obtained from all research subjects. Inclusion criteria for both groups were: 1) age 14–18 years, 2) primary language is English, 3) reading proficiency adequate to understand consent, 4) not mentally retarded, 5) not threatening or dangerous, 6) no physical illness that would exclude participation, 7) adolescent and all first degree relatives have never worked, or volunteered for, or received assistance from the Red Cross, and 8) parent or guardian has adequate English proficiency to provide informed consent and complete the written questionnaires. Recruitment of control subjects was monitored to assure controls had similar age, sex, racial and ethnic distributions to patients and that controls lived in a zip code from which patients are usually referred. Exclusion criteria for controls were: 1) history of court conviction for offenses other than minor traffic or curfew violations, and 2) history on pre-evaluation screening of substance-related treatment or substance-related expulsion from school.

Approximately 3–7 days later, eligible subjects completed Meeting 2 in a quiet private room, chosen by the subject, in the subject's home, a public facility (i.e. library), or our research offices. Subjects again provided urine and breath samples for onsite drug and alcohol testing. Then they viewed an investigator-produced ninety second slide presentation with pre-recorded messages highlighting the positive contributions of the Red Cross. After viewing the slides, subjects indicated how much good they felt the Red Cross does by marking on a line anchored by the terms “No good at all” and “Lots of good” (Red Cross Visual Analogue Scale). Subsequently, subjects viewed the AlAn's game instructions (slides with pre-recorded audio; instructions are included in Material S1 ), and then answered questions about the game to ensure comprehension of the instructions. All participants played a short practice version of the game to familiarize them with the timing and format of the game. We told all participants that we would hold research data in strict confidence. Subjects had the opportunity to ask questions or to replay the practice game. Then subjects played the AlAn's game, which takes approximately 31 minutes. By protocol, seating arrangements ensured that the laptop screen was only visible to the subject. After finishing the game, subjects completed a short debriefing interview and then several self-report measures including: the Inventory of Callous-Unemotional Traits (self-report) [30] ; the Antisocial Process Screening Device (self-report) [33] ; and the Social Desirability Scale [37] . Qualitative data from our debriefing interviews supported that subjects were generally engaged by and understood the AlAn's game. Four research assistants administered the study protocol; each conducted a similar proportion of patient and control interviews.

Description of the AlAn's (Altruism/Antisocial) Game

This game shares some similarities with previous tests of altruism [1]–[3] or behavioral economic games [4], [5]. However, the AlAn's game differs from such games in several fundamental ways. First, in altruistic acts the actor gives up something to benefit others [38]. In contrast, many antisocial acts take something from others to benefit the actor; therefore, in the AlAn's game we offered subjects the opportunity to take some benefit despite expense to another. Second the game was designed to be quantitative, allowing between group comparisons of outcomes. Subjects started with no money; a planned, real donation to the Red Cross started at $16 but could decrease due to choices made by the participant playing the game. Subjects were told that the researchers would (and they did) give the Red Cross the value of the charity donation remaining at the end of the game. Based on previous studies which have demonstrated differences between hypothetical and actual valuations [39]–[41], we designed AlAn's game to use actual money donations to a real charity. Third, we designed the game for future use in the fMRI environment; for example, we included many (72) decision-making trials and also trials which may be used as a contrast in MRI designs (Calculation Trials). Finally, we utilized a single charity, which we hypothesized would generally be considered beneficent; this single “other” standardized the experience across subjects.

We carefully considered whether to utilize a second participant, or a real person who was not a research participant, instead of a charity; empathy and perspective-taking generally are conceptualized as a between-individual, and not person-charity, phenomena. However, we opted to use a charity for these reasons: (1) given that empathy can vary based on how similar the “other” is to one's self (i.e. ingroup vs. outgroup), we worried that failure to appropriately match the “other” to the research participant would introduce error (i.e. subject made more self-benefiting choices because he considered the other player to be very dissimilar to him, rather than because of an inherent tendency toward antisocial behaviors); and, (2) some of our adolescent patients may use manipulation or threats of aggression in interpersonal interactions, or may address grievances through violence. Thus, we worried about pairing adolescent patients together or with controls while playing a game requiring choices that pit self interest against the interests of others. Although paradigms have been constructed where two participants remain anonymous to one another, and even to the researchers [4], we wanted research participants to view the “other” as beneficent and deserving, given that increasing the perceived “deservingness” of the other in economic games increases the quantity of donations from experimental subjects [5]. Thus, we chose to utilize a single charity that we thought would be broadly viewed as beneficent, to standardize the experience across subjects.

Subjects were told directly that the researchers were interested in understanding how people make decisions when they can win but others will lose. No attempts were made to deceive subjects and no cover story was utilized. Instead subjects were informed that there were no “right” or “wrong” answers while playing the game and that all information about how they played the game would be held in strict confidence; we would not inform parents about how subjects played the game, and for patients we would not inform treatment providers.

Subjects made 144 choices (“trials”) divided into two 72-trial sessions. Each trial was approximately 13 seconds in length (see Figure 1): 5 seconds to view and consider the offer, 1 second to press an “accept” or “reject” computer key, 4 seconds to observe results, and 2–4 seconds of a gray fixation screen. The “results” portion of each trial showed the outcomes of the choice; analogue “thermometers” and digital counters showed the subject's accrued money and the current remaining value of the Red Cross donation.

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larger image TIFF original image Download: Figure 1. AlAn's game trials: screen-captures with associated timing (example Active Trial shown). Note: A. An offer in which the subject will receive 32 cents and the Red Cross donation will be reduced by 8 cents (5 seconds); B. When the red ball turns green, subjects have one second to press one computer key to accept the offer, or a different key to reject it; C. The thermometers and counters then show how much money the subject has accrued. Here the subject accepted so the “You” counter now reads 32 cents and the “Red Cross” counter reads $15.92); D. Gray fixation screen. https://doi.org/10.1371/journal.pone.0036158.g001

The AlAn's game contained three trial types (See Figure 2). During Active Trials (“AT” in Figure 2.A) the decision screen showed an amount that the subject would gain (range 2–64 cents) and the Red Cross would lose (same range) if the choice were accepted (“Change both thermometers?”); in most trials the two amounts were unequal. Subjects accepted (“Yes” button) or rejected (“No” button) the choice on the screen; after rejected choices neither counter changed. Attention-Control Trials (“A” in Figure 2.A) have a correct or logical answer. We assumed that most subjects would reject all trials in which both they and the Red Cross would lose money, so these trials forced changes if a subject fell into a repetitive pattern of accepting all offers (to maximize earnings). Conversely, we assumed most subjects would accept all choices in which they would get money and the Red Cross donation would not be reduced, so these trials forced changes if a subject fell into a repetitive pattern of rejecting all offers (to prevent any loss to the Red Cross). These trials also objectively tested subject attention to the task. Calculation Trials (“C” in Figure 2.B) ask, “Is the YOU number bigger?” while presenting one positive number for the subject and another for the Red Cross. Subjects were instructed to press the YES button if the subject's number (the “YOU” number) was bigger than the Red Cross' number, and the NO button if the subject's number (the “YOU” number) was not bigger than the Red Cross' number. These trials assessed whether patients could, in 6 seconds, understand relative numerical values used in the Active Trials. In addition, the AlAn's game was designed for future use in the MRI environment which further motivated inclusion of multiple Calculation Trials. Calculation Trials, compared with Active Trials, present the same visual cues, require the same motor responses, and require subjects to judge the relative numerical values but are devoid of self vs. other consideration. Thus they may serve as a comparison for Active Trials in future MRI studies. Each of the two sessions contained 36 Active Trials, 6 Attention-Control Trials and 30 Calculation Trials (72 per session, 144 total). The order of amounts offered (order of trial presentation) was randomized before the beginning of the study and was the same for all subjects.

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larger image TIFF original image Download: Figure 2. The AlAn's game: description of three trial types. 2.A: Matrix with “You Gain” values (Y axis) and “Red Cross Loses” values (X axis) in cents. Each cell in the matrix represents a single offer. AT = active trials; arrow from AT points to a screencapture showing a sample active trial in which “You” will get 2 cents but the Red Cross donation will decrease by 64 cents. Note that the “You” amount varies independently from the “Red Cross” amount. A = Attention-Control; trials in which either a “Yes” results in a loss for both subject and Red Cross (top row of matrix), or a “Yes” results in gain for the subject and no loss for the Red Cross (first column of matrix); arrow from A points to a screencapture showing a sample Attention-Control trial in which “You” will lose 2 cents and the Red Cross donation will decrease by 32 cents. Note: Thicker borders indicate the quadrants for which percentage of “Yes” responses across those 9 Active Trials were calculated (see Materials and Methods, Data Analyses, Between-Group analyses). 2.B: C = Calculations, in which subjects decide whether the “You” number is bigger than the Red Cross number; arrow points to a screencapture showing a sample calculation trial in which the “You” number (4 cents) is not bigger than the “Red Cross” number (8 cents). https://doi.org/10.1371/journal.pone.0036158.g002

Subjects received $15 for their time and effort. To provide real monetary consequences to their within-game decisions, they were given their earnings from the game (Active and Attention-Control Trials). Money left to the Red Cross in the game actually was donated to the Red Cross. To incentivize attention to Calculation Trials, subjects received $0.25 if they correctly answered at least 75% of the Calculation Trials. Subjects were paid in cash at the end of Meeting 2.