ORIGINAL INVESTIGATION

Response requirement and increases in accuracyproduced by stimulant drugs in a 5-choice serialreaction-time task in rats

Mikhail N. Koffarnus & Jonathan L. Katz

Received: 7 May 2010 /Accepted: 16 September 2010 /Published online: 6 October 2010# Springer-Verlag 2010

AbstractRationale Increased signal-detection accuracy on the 5-choice serial reaction time (5-CSRT) task has been shownwith drugs that are useful clinically in treating attentiondeficit hyperactivity disorder (ADHD), but these increasesare often small and/or unreliable. By reducing the reinforcerfrequency, it may be possible to increase the sensitivity of thistask to pharmacologically induced improvements in accuracy.Methods Rats were trained to respond on the 5-CSRT taskon a fixed ratio (FR) 1, FR 3, or FR 10 schedule ofreinforcement. Drugs that were and were not expected toenhance performance were then administered before exper-imental sessions.Results Significant increases in accuracy of signal detectionwere not typically obtained under the FR 1 schedule withany drug. However, d-amphetamine, methylphenidate, andnicotine typically increased accuracy under the FR 3 andFR 10 schedules.

Conclusions Increasing the FR requirement in the 5-CSRTtask increases the likelihood of a positive result withclinically effective drugs, and may more closely resembleconditions in children with attention deficits.

Keywords 5-Choice serial reaction time task .

Sustained attention . Attention deficit hyperactivitydisorder . Fixed ratio . Psychostimulants . Rats

A number of animal models of attention deficit hyperac-tivity disorder (ADHD) have been proposed with varyingdegrees of success in predicting clinical outcome (for recentreviews, see Russell 2007; Sagvolden et al. 2005; van derKooij and Glennon 2007). One animal model of attentionwhich has been used extensively is the 5-choice serialreaction-time (5-CSRT) task (see Robbins 2002). In thistask, the subject (typically a rodent) is faced with an arrayof holes which can be briefly illuminated from behind. Theresponse of poking its nose in the hole most recentlyilluminated is reinforced. Drugs that are used clinically totreat ADHD have been shown to increase the percentage ofcorrect responses (nose poking in the hole that was mostrecently illuminated), and often to decrease responselatency and the number of illuminations that were notfollowed by a response (omissions). As with manycognitive tasks in animals, however, pharmacologicallyinduced increases in performance are typically small inmagnitude and often not reliably obtained at a particulardose. For example, d-amphetamine has been shown tosignificantly increase signal-detection accuracy on this task(Bizarro et al. 2004; Grottick and Higgins 2002), but otherreports show small increases that are not statisticallysignificant (Bizarro and Stolerman 2003; Cole and Robbins

Electronic supplementary material The online version of this article(doi:10.1007/s00213-010-2027-0) contains supplementary material,which is available to authorized users.

M. N. Koffarnus (*)Department of Psychiatry,Johns Hopkins University School of Medicine,5200 Eastern Ave., Suite 142W,Baltimore, MD 21224, USAe-mail: mkoffar1@jhmi.edu

J. L. KatzPsychobiology Section, National Institute on DrugAbuse–Intramural Research Program,National Institutes of Health,251 Bayview Blvd., Rm. 06A711A,Baltimore, MD 21224, USA

Psychopharmacology (2011) 213:723–733DOI 10.1007/s00213-010-2027-0

1987). Methylphenidate has also been reported to increaseaccuracy on this task (Bizarro et al. 2004; Navarra et al. 2007;Paine et al. 2007), with one report only showing an effect inlow-performing rats (Puumala et al. 1996). Atomoxetine hasbeen shown to both increase the percentage of correctresponses (Navarra et al. 2007), or have no effect (Robinsonet al. 2008). Nicotine, which is not used clinically to treatADHD, also increases accuracy on this task, although theseincreases are sometimes only seen under specific experimen-tal conditions (Bizarro et al. 2004; Bizarro and Stolerman2003; Grottick and Higgins, 2002; Hahn and Stolerman2002; Mizra and Stolerman 1998; Young et al. 2004).

Previous attempts to increase the sensitivity of the 5-CSRT task to pharmacologically induced increases inaccuracy usually involved some manipulation to decreasebaseline levels of performance accuracy, thereby makinglarger increases possible. Prefeeding rats before experimen-tal sessions did not significantly affect accuracy, andchanged the effective dose of nicotine without changingthe magnitude of that effect (Bizarro and Stolerman 2003;Grottick and Higgins 2002). Increasing the level of fooddeprivation also did not significantly affect accuracy or theeffects of nicotine or amphetamine on accuracy (Bizarroand Stolerman 2003). Alterations to the duration of theintertrial interval have reliably resulted in decreasedbaseline accuracy (Bizarro et al. 2004; Mizra and Stolerman1998; Navarra et al. 2007). However, these decreases inbaseline accuracy were not associated with further increasesin accuracy produced by d-amphetamine, nicotine, methyl-phenidate, or atomoxetine (Bizarro et al. 2004; Navarra etal. 2007). Mizra and Stolerman (1998) also tested theeffects of nicotine with very short brief stimulus presenta-tions (0.25 s), but found no increases in accuracy afternicotine administration.

All of these previously studied manipulations arrange areinforcer to be delivered contingent upon each correctresponse recorded during the response period in theilluminated nose-poke hole, a fixed-ratio one (FR 1)response schedule. These conditions typically support highlevels of baseline accuracy. However, it has been arguedthat children with ADHD primarily exhibit sustainedattention deficits in environments in which infrequent orintermittent reinforcement is arranged (Aase and Sagvolden2006; Douglas 1985; Douglas and Parry 1994; van derMeere 1996). In the present study, the sensitivity of the 5-CSRT task to behaviorally active drugs was examinedunder conditions in which the response requirement wasaltered. In three separate groups of rats, performance wasassessed with the schedule of required accurate responsesset to an FR 1, FR 3, or FR 10. It was hypothesized thatincreases in response requirement would be associated withdecreases in baseline performance and would more closely

resemble conditions where ADHD symptoms are observedclinically. The effects of drugs used clinically to treatADHD (d-amphetamine, methylphenidate, and atomoxe-tine) were compared to nicotine and several other com-pounds that are not used clinically.

Methods

Subjects

Twenty-three male Sprague-Dawley rats (Taconic Farms,Hudson, NY) served as subjects. Diet was controlled tomaintain the subjects at approximately 85% of their adultfree-feeding weights, which resulted in weights rangingfrom 325 to 360 g. When not in session, subjects wereindividually housed with fresh water continuously avail-able. Husbandry and other care were in accordance withNIDA institutional animal care and use guidelines andthe Guide for the Care and Use of Laboratory Animals(1996).

Apparatus

Sessions were conducted in rodent operant-conditioningchambers designed for the 5-CSRT task (MED-NP5Lpackage; Med-Associates Inc., St. Albans, VT, USA). Onone wall of the chamber were five holes capable of beingilluminated from behind. The opposite wall held a food trayinto which 45-mg food pellets could be delivered. Thechambers were contained within light-proof, ventilatedenclosures that provided sound attenuation. White noisewas delivered to the chamber at all times to maskextraneous noise.

Procedure

Sessions were conducted during the light cycle (7:00A.M. to7:00P.M.) with sessions for each subject conducted atapproximately the same time each day. All subjects wereinitially trained to poke their noses into a hole under an FR1 schedule of reinforcement. An 8-s stimulus light wasrandomly illuminated behind one of the five holes of theoperant conditioning chamber, and responses to the lit hole(correct responses) produced a 45-mg food pellet to ahopper on the opposite wall, followed by a 5-s intertrialinterval (ITI). Responses to any other hole (incorrectresponses) were followed by the ITI only, and responsesduring the ITI had no scheduled consequence. After the ITIa randomly selected hole was illuminated, which markedthe beginning of a new trial. This 8-s stimulus duration wasgradually decreased to 1 s over successive sessions, but the

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response period remained 5.5 s from the onset of thestimulus (i.e., responses were still correct for a period afterthe stimulus was turned off). Any response during theresponse period immediately ended the trial, so only oneresponse per trial was possible. Sessions ended after 50food presentations or 40 min (FR 3 and FR 10 groups) or15 min (FR 1 group), whichever occurred first. This trainingprocedure was in place for a median duration of 30 sessions(interquartile range = 24 to 38) before testing (FR 1 group) orFR escalation (FR 3 and FR 10 groups; see below).

After responding stabilized with a stimulus duration of1 s, subjects were split into groups. Seven subjectscontinued on the schedule described above and comprisedthe FR 1 group. For the remaining 16 subjects, the FR(number of correct responses required to produce a foodpellet) was gradually increased with a target of FR 10.When the FR was greater than one, correct responses thatdid not fulfill the response requirement and incorrectresponses both led to a 5-s ITI. Only correct responsescounted toward the FR requirement and incorrect responsesdid not reset the response requirement. Correct responsesthat fulfilled the response requirement were reinforced asdescribed above. The FR was gradually increased untileither an FR 10 was reached or responding was notmaintained at or above approximately 50% correctresponses. Ten subjects reached an FR 10, and thesesubjects comprised the FR 10 group. Of the remaining sixsubjects, five reached an FR≥3 before performancedeteriorated, and these rats were placed on an FR 3schedule and comprised the FR 3 group. One subject didnot respond at any response requirement greater than FR 1,and was subsequently studied under a variety of conditionsthat were not successful in developing responding underintermittent reinforcement. Data for this subject are notpresented in this report.

Once responding for any individual subject was deemedstable (at least 50% accuracy with no apparent increasing ordecreasing trend for at least five sessions), drug testingbegan. Injections were administered intraperitoneally im-mediately before subjects were placed in the operantchamber, and a 5-min blackout period elapsed before thesession start. Sessions were conducted 5 days per week,with drug tests occurring the second and fifth days. Thefirst and fourth days of each week were control sessions,with vehicle administrations on the fourth day and noinjections administered on the first and third days. Not allsubjects received all drug treatments. The order that drugswere allocated to individual subjects is detailed in OnlineResource 1 and resulted in five subjects per drug conditionper FR group. The median duration of the drug testingperiod was 237.5 days (interquartile range = 175–311 days).

Drugs

Methylphenidate HCl, d-amphetamine SO4, atomoxetine(R-tomoxetine) HCl, morphine SO4, nicotine hydrogentartrate, pentobarbital Na, desipramine HCl, nortriptyline,HCl, clonidine HCl, and selegiline (R(−) deprenyl) HClwere obtained from Sigma-Aldrich (St. Louis, MO, USA).All drugs were dissolved in sterile water and doses in mgrefer to the salt forms.

Data analysis

Four dependent variables were of interest in the current study.First, percent correct was defined as the percent of trials inwhich a response was emitted to the correct hole within theresponse period (correct responses divided by the sum ofcorrect and incorrect responses). Latency was defined as thetime to any response during the response period after a briefstimulus presentation was initiated. Trials in which responseswere not emitted were not included in latency calculations.Omissions were defined as the proportion of trials in which noresponse was emitted during the response period, expressed asa percent of total trials. ITI responses were defined as thoseresponses that were emitted in any hole during the ITIimmediately preceding a stimulus presentation.

The dependent measures from control sessions werecompared among groups using a two-way analysis ofvariance (ANOVA) with Bonferroni-corrected post-hoctests (FR value and the drug for which the sessions servedas control were the two factors), while FR 1 performancewas compared with a one-way ANOVA. Dependentmeasures were compared after drug pretreatments using aone-way repeated-measures ANOVA with Bonferroni-corrected post-hoc tests comparing each drug dose to thecontrol value. Percent correct and response latency datawere calculated for any session if the number of trialsomitted by the subject was less than 90% (OnlineResource 2 shows the number of trials with suppressedresponding for each drug dose). At least 25 responses wereemitted in all sessions that remained after this exclusioncriterion was applied. Data for any dose of any drug wereincluded in statistical analyses and graphical presentationsif at least two subjects omitted less than 90% of trials. ITIresponse data and percent omissions were included for allsubjects at all doses. Statistical tests were conducted usingSystat SigmaStat 3.5 (San Jose, CA, USA). In the case ofmissing data with repeated-measures ANOVAs, SigmaStatconducts a mixed-models ANOVA which allows for theinclusion of all available data while still accounting forbetween-subjects variance in the model. In four instances,percent correct data were not normally distributed and arepeated-measures ANOVA on ranks (Friedman test) was

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conducted. Both the parametric and nonparametric statisticsin each of these cases produced the same result (notstatistically significant).

Results

Control performances differed among groups maintained onthe FR 1, FR 3, or FR 10 schedules (Fig. 1, left column). Inparticular, accuracy (the percentage of correct responses)significantly differed among groups when maintained at thefinal FR schedule value [F(2,120)=48.4, p<0.001]. Com-pared to the FR 1 group (mean [M] = 79.2%), accuracy washigher in the FR 3 group (M=85.6%) and lower in the FR10 group (M=71.9%), and each group differed significantlyfrom the other two (all p<0.001). Though reduced,accuracy under the FR 10 schedule was much higher thanthe 20% chance level. Latency to respond also differedamong the groups after FR escalation [F(2,120)=48.9, p<0.001], with latency marginally lower in the FR 3 group(M=0.85s, p=0.073) compared to the FR 1 group (M=0.93s), and highest in the FR 10 group (M=1.2s, p<0.001vs. FR 1 and FR 3). Percent trials omitted increased withresponse requirement [F(2,120)=47.0, p<0.001]; the FR 10group (M=55.1%) omitted more trials than the FR 3 (M=43.3%, p=0.006) and FR 1 (M=20.0%, p<0.001) groups,and the FR 3 group omitted more trials than the FR 1 group(p<0.001). The frequency of ITI responses increased withFR value [F(2,120)=17.3, p<0.001], with the FR 10 group(M=0.89) emitting significantly more responses per trialthan the FR 3 (M=0.53, p<0.001) or FR 1 (M=0.42, p<0.001) groups. For each of these measures, there was nomain effect of drug for which the control session preceded,indicating that no baseline shift was apparent.

Differences among groups were typically absent instable performances under the FR 1 schedule before thechanges in schedule requirement (Fig. 1, right column).Percent correct in the three groups when maintained underthe FR 1 schedule did not differ by group [F(2,19)=0.3, p=0.719]; however, differences in latency were evident[F(2,19)=5.0, p=0.017], with the FR 1 group (M=0.79s)exhibiting a significantly lower latency than did the groupthat was eventually maintained under the FR 10 schedule(M=1.07 s, p=0.021). Neither the percentage of trialsomitted [F(2,19)=1.0, p=0.390] nor ITI responses per trialdiffered among groups before FR escalation [F(2,19)=1.8,p=0.194].

Within session response patterning differed considerablyunder the three FR schedules (Fig. 2). Responding underthe FR 1 schedule was characterized by relatively fewomitted trials with responding at a consistent rate through-out the session. The infrequent pauses in correct responseswere often though not universally correlated with periods of

incorrect responding (gray line), that in this exampleincreased toward the end of the session. Responding onthe FR 3 schedule was characterized by more omitted trials,which were more likely to occur immediately after a pellet

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Fig. 1 Performance in the absence of any drug treatments in the FR 1(black bars), FR 3 (gray bars), and FR 10 (white bars) groups. Controldata in the left panels are the mean of the sessions immediately prior todrug test sessions, the same data that comprise the control data forstatistical analyses of drug effects. Data in the right panels represent theperformance of each group responding on an FR 1 schedule ofreinforcement, taken from the mean of the last five sessions of respondingon an FR 1 schedule either before drug testing was initiated (FR 1 group)or before FR escalation (FR 3 and FR 10 groups). Baseline percentcorrect, latency, omissions, and ITI responses differed among the three FRgroups, and asterisks depict significant Bonferroni-corrected post-hoc testsbetween specific group pairings (*p<0.05, **p<0.01, ***p<0.001)

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delivery. Therefore the pattern of correct responses oftenapproached a typical pattern of “break and run” seen withless complex responding under FR schedules. Overallaccuracy was significantly higher in this group, thoughthere were consistent incorrect responses throughout thesession (gray line). These incorrect responses often oc-curred after a short pause, and then decreased before thecorrect responses fulfilled the FR requirement. Subjectsresponding under the FR 10 schedule omitted the largestpercentage of trials, which were more likely to occurimmediately after a pellet delivery. The pattern of correctresponses often approached a “break and run” pattern;however there were also instances in which the accelerationof correct responses between reinforcers was more gradual.As under the FR 3 schedule, incorrect responding under theFR 10 schedule was often characterized by a pausefollowed by a transition to a higher rate, which subsequent-ly decreased before reinforcement.

Dose of d-amphetamine significantly altered accuracy inthe FR 1 [F(6,24)=3.10, p=0.022] and FR 3 [F(5,16)=5.89,p=0.003] groups, but not in the FR 10 group (Fig. 3, leftcolumn). Accuracy was significantly increased after 0.56,1.0, and 1.7 mg/kg D-amphetamine in the FR 3 group (all pvalues <0.05), but no individual dose reached statisticalsignificance compared to control in the FR 1 or FR 10groups. Latency to respond was altered in the FR 1 [F(5,16)=6.58, p<0.001], but not FR 3 or FR 10 groups. Thepercentage of trials omitted was also altered by d-amphetamine in the FR 1 [F(6,24)=7.94, p<0.001] and FR3 [F(6,24)=9.18, p<0.001] groups, with the highest doses

tested in each group significantly increasing omissions(3.0 mg/kg in the FR 1 group and 5.6 mg/kg in the FR 3group, both p<0.001). The frequency of ITI responses wasnot significantly altered by any of the doses tested underany of the FR values examined.

Methylphenidate altered accuracy in the FR 10 group[F(4,16)=8.13, p<0.001], with a significant increase (p=0.005) at 3.0 mg/kg (Fig. 3, center column). Latency wasgenerally decreased with increasing dose, and this effectwas significant in the FR 3 [F(4,16)=13.3, p<0.001] and FR10 [F(4,16)=3.52, p=0.030] groups, with 3.0 mg/kg signif-icantly decreasing latency in the FR 3 group (p<0.001).Omissions also tended to decrease with increasing dose ofmethylphenidate. This trend was significant in the FR 3group [F(4,16)=5.86, p=0.004], specifically at 3.0 mg/kg(p=0.005). ITI responses per trial were not altered bymethylphenidate in any group.

Neither accuracy nor latency was altered with atom-oxetine up to 10.0 mg/kg in any of the groups (Fig. 3, rightcolumn). At the 10.0 mg/kg dose, omissions were increasedin each group (all p values <0.05) [FR 1, F(4,16)=4.68, p=0.011; FR 3, F(4,16)=12.3, p<0.001; FR 10, F(4,16)=9.22,p<0.001], and ITI responses were decreased in the FR 3group [F(4,16)=13.3, p<0.001] and FR 10 [F(4,16)=13.3, p<0.001] groups.

Nicotine significantly increased accuracy in the FR 3[F(5,20)=4.81, p=0.005] and FR 10 [F(5,16)=10.7, p<0.001]groups at 0.56 and 1.0 mg/kg (all p values <0.05), but hadno effect in the FR 1 group (Fig. 4, left column). Latency wasonly decreased at 1.0 mg/kg (p=0.018) in the FR 10 group

Fig. 2 Cumulative records fromthree representative sessions onthe FR 1, FR 3, and FR 10schedules with no drug pretreat-ment. Black lines depictresponses to the illuminatednose-poke hole during the re-sponse period and downwarddeflections of these lines depictpellet deliveries. Gray linesdepict incorrect responses dur-ing the response period. Lines inthe upper-right portion of thefigure indicate scaling of timeand responses, respectively

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[F(5,16)=3.95, p=0.016]. Omissions were not significantlyaltered in any FR group, and while ITI responses tended todecrease with increasing nicotine dose, this effect was onlysignificant in the FR 3 group [F(5,20)=3.12, p=0.030].

Morphine only affected the measures of performance atthe highest doses that disrupted behavior in general (Fig. 4,center column). Accuracy [F(5,16)=7.59, p<0.001] andlatency [F(5,16)=9.65, p<0.001] was altered at 10.0 mg/kg

(both p<0.001) in the FR 3 group. Morphine dose-dependently increased omissions in each group [FR 1,F(5,20)=5.20, p=0.003; FR 3, F(5,20)=8.63, p<0.001; FR10, F(5,20)=10.4, p<0.001] and ITI responses were dose-dependently decreased with morphine in the FR 3 [F(5,20)=4.49, p=0.007] and FR 10 groups [F(5,20)=6.74, p<0.001].

Pentobarbital, like morphine, had little effect on anymeasure up to doses that disrupted behavior in general

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Fig. 3 Effects of drug pretreat-ments in the FR 1 (blackcircles), FR 3 (gray triangles),and FR 10 (white squares)groups on percent correct, la-tency, omissions, and ITIresponses. The effects of d-amphetamine, methylphenidate,and atomoxetine pretreatmentsare displayed in the left, center,and right columns, respectively.Control points above C repre-sent performance in sessionsimmediately preceding each ofthe drug sessions for that group,consisting of both sessions inwhich saline was administeredor no injection was given. Sig-nificant main effects of dose foreach group are represented byasterisks (FR 1), crosses (FR 3),or double crosses (FR 10) nextto the panel title. Symbols nextto a specific point indicate sig-nificant Bonferroni-correctedpost-hoc tests for that dose ascompared to the control point(one symbol, p<0.05; two sym-bols, p<0.01; three symbols,p<0.001)

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(Fig. 4, right column). This was true for percent correct inthe FR 1 group [F(3,11)=6.73, p=0.008] and latency in theFR 3 group [F(3,11)=5.90, p=0.012]. Omissions weredramatically and dose-dependently increased in all groups[FR 1, F(4,15)=12.9, p<0.001; FR 3, F(4,16)=15.8, p<0.001;FR 10, F(4,16)=29.6, p<0.001] at 17.0 mg/kg and in the FR10 group at 10 mg/kg (all p values <0.001). ITI responseswere dose-dependently decreased with pentobarbital, which

was significant in the FR 1 [F(4,15)=4.29, p=0.016] and FR10 groups [F(4,16)=8.57, p<0.001].

Desipramine did not have a selective effect onaccuracy or latency, only disrupting behavior at highdoses (Fig. 5, first column). Latency was only altered inthe FR 10 group [F(3,10)=3.89, p=0.045] with no signifi-cance of any doses in post-hoc tests. However, percenttrials omitted was dose-dependently increased in each

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Fig. 4 Effects of nicotine, mor-phine, and pentobarbital pre-treatments are shown in the left,center, and right columns, re-spectively. All other details areas in Fig. 3

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group [FR 1, F(4,16)=31.5, p<0.001; FR 3, F(4,16)=5.93, p=0.004; FR 10, F(4,16)=9.85, p<0.001], and ITI responseswere dose-dependently decreased in all groups [FR 1,F(4,16)=6.43, p=0.003; FR 3, F(4,16)=6.69, p=0.002; FR10, F(4,16)=6.62, p=0.002].

The pattern of behavior with nortriptyline was similar tothat observed with desipramine (Fig. 5, second column).Accuracy and latency were not affected at any desipraminedose tested in any group. Omissions were dose-dependentlyincreased in each group [FR 1, F(4,16)=10.5, p<0.001; FR

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Fig. 5 Effects of desipramine, nortriptyline, clonidine, and selegiline pretreatments are shown in the first, second, third, and fourth columns,respectively. All other details are as in Fig. 3

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3, F(4,16)=3.06, p=0.047; FR 10, F(4,16)=5.67, p=0.005],and ITI responses were also dose-dependently decreased ineach group, although this effect was only significant in theFR 1 [F(4,16)=38.5, p<0.001] and FR 3 groups [F(4,16)=4.14, p=0.017].

Clonidine had little effect on behavior except at 0.03 mg/kg, the highest dose tested (Fig. 5, third column). Accuracywas only decreased in the FR 1 group [F(4,16)=5.76, p=0.005] at 0.03 mg/kg (p=0.037), which corresponded withan increase in latency [F(4,16)=14.0, p<0.001] at 0.03 mg/kg (p<0.001). Doses of 0.01 and 0.03 mg/kg (both p<0.05)also slightly increased latency in the FR 3 group [F(4,16)=5.64, p=0.005]. Omissions were dose-dependently in-creased in each group [FR 1, F(4,16)=4.10, p=0.018; FR3, F(4,16)=12.0, p<0.001; FR 10, F(4,16)=3.12, p=0.045],and ITI responses were dose-dependently decreased in theFR 3 [F(4,16)=3.16, p=0.043] and FR 10 [F(4,16)=3.75, p=0.024] groups.

Selegiline had a significant effect on percent correct inthe FR 10 group [F(5,15)=3.00, p=0.045], but not at theother FR values (Fig. 5, fourth column). None of theindividual doses in the FR 10 group had a significant effectin post-hoc tests, though the effects at 30 mg/kg approachedsignificance (p=0.054). This dose also increased trialsomitted, such that only two subjects were included in theaverage for this dose. Latency was not altered in any group,but omissions were dose-dependently increased in the FR10 group [F(5,20)=4.79, p=0.005]. ITI responses weresignificantly decreased in the FR 1 [F(5,20)=4.32, p=0.008] and FR 3 [F(5,20)=3.24, p=0.027] groups at30 mg/kg (both p values <0.05).

Discussion

Increasing the FR value on the 5-CSRT task increasedsensitivity to d-amphetamine, methylphenidate, and nico-tine on accuracy without altering the profile of other drugsnot expected to enhance response accuracy. While therewere trends, neither d-amphetamine nor methylphenidatesignificantly increased accuracy at any of the doses testedin the FR 1 group. Further, nicotine in the FR 1 group didnot affect percent correct at doses up to 1.0 mg/kg.However, in at least one of the groups studied at higherFR schedule values, these drugs did significantly increasepercent correct. Nicotine significantly increased accuracy at0.56 and 1.0 mg/kg in both the FR 3 and FR 10 groups, d-amphetamine significantly increased accuracy in the FR 3group, and methylphenidate increased accuracy in the FR10 group. Atomoxetine, morphine, pentobarbital, desipra-mine, nortriptyline, clonidine, and selegiline did not signif-icantly increase accuracy at any dose tested at any of the FRvalues studied. Taken together, these results indicate that

increasing the response requirement in the 5-CSRT taskincreases the sensitivity to accuracy-increasing effects ofdrug administration without altering the profile of drugs thathave not been shown to enhance accuracy on this task.

In the absence of any drug administration, baselineperformance differed among the three FR groups. Percentcorrect was lowest in the FR 10 group, and was actuallyhighest in the FR 3 group. This effect may have been due tothe nonrandom way in which subjects were assigned to theFR 3 and FR 10 groups. For all the subjects in these twogroups, the FR value was increased until either an FR 10was reached or response accuracy or rate was substantiallylowered. Thus, the FR 3 group consisted of those subjectsthat did not respond reliably and at 50% accuracy or greaterat FR 10. The highest baseline accuracy rates in the FR 3group may have resulted from the history of training at thehigher FR values, but this notion remains untested. Baselinelatency to respond, trials omitted, and ITI responses alsodiffered among the three FR groups. Latency was highest inthe FR 10 group, but the FR 3 group actually had thelowest average latencies. Latencies in this group tended tobe lower than even the FR 1 group, although this effect wasnot statistically significant. Response latency and percentcorrect on the 5-CSRT task are known to be inverselycorrelated (Puumala et al. 1996), and it is possible that thenonrandom group selection procedure also resulted in thisordering of latencies. In addition, some differences inlatency were evident before the increases in FR value,suggesting that the differences in this measure were not dueentirely to schedule parameter. Trials omitted and ITIresponses were both positively correlated with responserequirement, with the FR 10 group both omitting the mosttrials and emitting the most ITI responses per trial. Theeffect on trials omitted was not surprising, as increases inFR response requirement increases post-reinforcementpause duration (Ferster and Skinner 1957). In the currentprocedure, pausing after reinforcement manifests as one ormore omitted trials. Further, omitted trials tended to occurimmediately after reinforcement (Fig. 2), creating a tempo-ral pattern of responding typical of FR schedules.

Statistically significant increases in percent correct weremore likely to be observed with nicotine and the psychos-timulants commonly used to treat ADHD at the higher FRvalues than at FR 1. Although nicotine is not currently anapproved drug for ADHD treatment, a number of clinicaltrials show beneficial effects on attention with transdermalnicotine in adults with or without ADHD, includingalleviation of ADHD symptoms in those with an ADHDdiagnosis (Conners et al. 1996; Levin and Rezvani 2000;Levin et al. 1996, 1998, 2001). These findings suggest thatintermittent reinforcement of performance in the 5-CSRTtask increases its predictive power for compounds that maybe clinically useful.

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Percent correct was not altered in any group withatomoxetine pretreatments up to doses that significantlyincreased trials omitted. Atomoxetine has previously beenshown to either not affect accuracy on the 5-CSRT task(Robinson et al. 2008), or do so inconsistently and non-dose-dependently (Navarra et al. 2007). Thus, a limitationof the predictive validity of the 5-CSRT seems to be thelack of concordance between the clinical utility of atom-oxetine and increases in performance on this task. Atom-oxetine has also been shown to decrease ITI responding, apurported measure of impulsivity (Robinson et al. 2008;Navarra et al. 2007). This was also observed in the currentstudy in the FR 3 and FR 10 groups, but only at a dose of10 mg/kg which also increased omissions. ITI responses inthe current study are probably not equivalent to the“premature response” measure often discussed as a measureof impulsivity (for recent review, see Dalley et al. 2008)because the consequences of each are different. In thecurrent study, there were no programmed consequencescontingent upon ITI responses, whereas previous studies inwhich ITI responses are presented as a behavioral measureof impulsivity punish those responses with a timeout. Sincemost definitions of impulsivity require that the behavior inquestion persists despite being disadvantageous (Ainslie1975; Logue 1995), it is probably not appropriate toconsider ITI responses in the current experiment asindicative of impulsivity.

The other drugs administered had roughly similarprofiles: no significant effect on any measure up to dosesthat increased trials omitted. Neither morphine, pentobarbi-tal, desipramine, nortiptyline, clonidine, nor selegelineincreased percent correct or decreased response latency,while all typically increased trials omitted and decreasedITI responses at the highest dose or doses administered.None of these drugs have demonstrated clinical utility intreating ADHD, further supporting the predictive validity ofthe 5-CSRT procedure, though the results with atomoxetinewarn that the predictive validity of this procedure may berestricted to compounds sharing a particular mechanism.

Longer sessions under the FR 3 and FR 10 schedulescompared to the FR 1 schedule may have contributed to thedifferences observed under the different schedules. Toaddress this, an additional analysis was conducted (notshown) that compared only responding in the first 5 min ofall sessions This analysis indicated that data from these 5-min windows were largely confirmative of data from theentire session. No increases or insignificant increases inpercent correct were obtained with all drugs in the first5 min of sessions under the FR 1 schedule, whereasincreases in percent correct under the FR 3 and FR 10schedules during the first 5 min of the sessions were similarto those described above for entire sessions. Thus, durationsof sessions contributed minimally if at all to the enhance-

ment of the effects obtained with d-amphetamine, methyl-phenidate and nicotine under conditions of intermittentreinforcement.

The current results not only provide further evidence thatthe 5-CSRT task has predictive validity, but it may point toimportant factors involved in ADHD that should beincorporated in studies of drugs used to treat the disorder.It has been argued that children with ADHD primarilyexhibit sustained attention deficits in environments inwhich infrequent or intermittent reinforcement is arranged(Aase and Sagvolden 2006; Douglas 1985; Douglas andParry 1994; van der Meere 1996). This considerationcoincides with the results in the current experiments inwhich significant increases in accuracy of detecting anoccasional but brief signal were obtained when reinforce-ment was intermittent. This was even true for the FR 3group which had higher baseline accuracy than the FR 1group, but less frequent reinforcer deliveries. Perhaps theintermittency of reinforcement on this task is a more relevantfactor than level of baseline performance in contributing toits validity as a model of disorders of attention.

Acknowledgements The authors would like to express theirgratitude to Bettye Campbell and Dawn French for their experttechnical assistance. This research was supported by the NationalInstitute on Drug Abuse Intramural Research Program. The content issolely the responsibility of the authors and does not necessarilyrepresent the official views of the National Institute on Drug Abuse orthe National Institutes of Health.

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