Neuronal activity in the primate dorsomedialprefrontal cortex contributes to strategicselection of response tacticsYoshiya Matsuzakaa, Tetsuya Akiyamaa, Jun Tanjib, and Hajime Mushiakea,c,1

aDepartment of Physiology, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan; bBrain Science Center, Tohoku University, Sendai980-8577, Japan; and cCore Research for Evolutional Science and Technology (CREST), Tokyo 102-0075, Japan

Edited by Ranulfo Romo, Universidad Nacional Autónoma de México, Mexico City, D.F., Mexico, and approved February 1, 2012 (received for reviewDecember 6, 2011)

The functional roles of the primate posterior medial prefrontalcortex have remained largely unknown. Here, we show that thisregion participates in the regulation of actions in the presence ofmultiple response tactics. Monkeys performed a forelimb task inwhich a visual cue required prompt decision of reaching to a leftor a right target. The location of the cue was either ipsilateral(concordant) or contralateral (discordant) to the target. As a resultof extensive training, the reaction times for the concordant anddiscordant trials were indistinguishable, indicating that the mon-keys developed tactics to overcome the cue-response conflict.Prefrontal neurons exhibited prominent activity when the concor-dant and discordant trials were randomly presented, requiringrapid selection of a response tactic (reach toward or away fromthe cue). The following findings indicate that these neurons areinvolved in the selection of tactics, rather than the selection ofaction or monitoring of response conflict: (i) The response periodactivity of neurons in this region disappeared when the monkeysperformed the task under the behavioral condition that requireda single tactic alone, whereas the action varied across trials. (ii) Theneuronal activity was found in the dorsomedial prefrontal cortexbut not in the anterior cingulate cortex that has been implicatedfor the response conflict monitoring. These results suggest thatthe medial prefrontal cortex participates in the selection of a re-sponse tactic that determines an appropriate action. Furthermore,the observation of dynamic, task-dependent neuronal activitynecessitates reconsideration of the conventional concept of corti-cal motor representation.

cortical plasticity | frontal executive function

The primate dorsomedial prefrontal cortex (PFC), and partic-ularly its posterior region (pmPFC), is intimately connected

with higher-order cortical motor areas in the frontal lobe, in-cluding the presupplementary motor area (pre-SMA), the rostralcingulate motor area, and the dorsal premotor area (1–4). Thisrelationship with cortical motor areas resembles connectionsfrom the dorsolateral PFC (dlPFC), suggesting that both areasinfluence downstream motor areas during the selection, prepa-ration, and execution of actions (5–8). Despite numerous studiesdemonstrating the involvement of the dlPFC in various aspects ofbehavioral control (9–12), however, little is known about the roleof the pmPFC in the performance of motor behaviors.A few studies have attempted to explore this area. For example,

Petrides proposed that the dorsomedial PFC plays importantroles in monitoring multiple events and self-ordering actions (13),although his later study suggested that the dlPFC is also involvedin this process (14). Bon and Lucchetti reported that electricalstimulation in area 8b evoked ear or eye movements, and neuronsin this region showed activity modulation related to these types ofmovements (15, 16). Human brain-imaging studies identifiedactive areas including the mPFC that were related to the selectionof actions and conflict monitoring (17) or to the generation andadjustment of action plans (18). These reports represent initial

attempts to elucidate the functions of the mPFC, providing afoundation for more exact identification of medial prefrontalareas participating in specific aspects of motor behavior.Here, we describe regulatory roles of the pmPFC for voluntary

actions in the presence of multiple response tactics. When per-forming highly practiced or routine motor behaviors, we oftenattempt to enhance efficiency by using a variety of tactics to selectan action (19). The selection of an action and the selection oftactics differ in that the former is the process of determining whatto do, whereas the latter reflects the internal protocol of how todecide what to do. The selection of appropriate tactics involvestheir strategic application and, therefore, is thought to constitutean aspect of supervisory control over motor behavior in whichparticipation of the PFC in general has been inferred (20, 21).

ResultsTwomonkeys performed a visually cued, two-choice arm reachingtask in which red and green cues signaled the monkey to reach forthe right and left targets, respectively (Fig. 1A andMethods). Thecue appeared either ipsilaterally (concordant condition) or con-tralaterally (discordant condition) to the target. Therefore, con-cordant cues required the monkey to reach toward the visualstimulus, whereas discordant cues required the monkey to reachaway from the visual stimulus. Cues were randomly presented.

Behavior of the Monkeys. Both monkeys quickly learned to per-form the behavioral task. After <3 wk of training, the rate ofcorrect responses in both concordant and discordant trials wasalmost 100% for each monkey. On the other hand, as has beenreported previously for human and other animal studies, thespatial discrepancy between the target and cue adversely affectedthe monkeys’ performance during early training (Fig. S1). Dur-ing the training sessions, the reaction time (interval from the“go” signal until the monkey released the hold button) was sig-nificantly longer for discordant trials than for concordant trials(difference of 42.4 ms for monkey F and 42.8 ms for monkey H).This effect, however, diminished with extensive training over thecourse of 3 mo. By the time neuronal activity was recorded, thedifference between the reaction times for concordant and dis-cordant trials had disappeared (Fig. S1). The multiple regressionanalysis of monkeys’ reaction times (Methods) revealed no sig-nificant effect of target–cue concordance. The absence of dis-cordance effects on the animals’ performance indicated that the

Author contributions: Y.M., J.T., and H.M. designed research; Y.M. and T.A. performedresearch; Y.M. and T.A. analyzed data; and Y.M., J.T., and H.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: hmushiak@med.tohoku.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1119971109/-/DCSupplemental.

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behavior was highly practiced and behavioral conflict no longeraffected motor behavior. To confirm this, we also examinedpotential Gratton effects—modulation of behavior owing to aconflict experienced in the preceding trials (22). We comparedthe reaction time during discordant trials subsequent to a con-cordant trial with the reaction time during discordant trialspreceded by a discordant trial. The differences were not signifi-cant and close to nil (Fig. S2).

Neuronal Activity.We examined neuronal activity in broad regionsof the medial frontal lobe (Fig. 1B). Within the surveyed area, weidentified the supplementary motor area (SMA) and pre-SMAon the basis of criteria established previously (sensory responseprofiles and microstimulation effects) (23, 24). Rostral to thepre-SMA, we found an additional focus of task-related neurons(Fig. 1C). This region could be distinguished from the adjacentpre-SMA on the basis of a number of properties. First, unlikepre-SMA neurons, which showed clear responses to visualstimuli, neurons in this region did not respond to visual or tactilestimuli not associated with the task (Fig. 1D). Second, monkeys’eye or arm movements were not evoked by electrical stimulation.These findings suggest that this area, which we refer to as the

pmPFC in this report, is distinct from the previously identifiedpre-SMA and the supplementary eye field (SEF).In the pmPFC, we recorded 412 neurons that exhibited changes

in activity during the behavioral task.We also recorded 308 neuronsin the pre-SMA and 288 in the SMA, which were distributed incortical locations that matched previous studies (25–29). In thisstudy, we concentrated on analyzing pmPFC activity during theresponse period after cue presentation. For some neurons, firingactivity depended on whether the target was concordant with theilluminated cue position, whereas other neurons did not show thiseffect (Fig. 2).In the first example of pmPFC neurons (Fig. 2A), firing was

preferential during discordant trials; activity was observed well be-fore the monkey began to reach for the target and was more pro-nouncedwhen the target was on the left. In a secondpmPFCneuron(Fig. 2B), higher levels of activity were observed during concordanttrials, particularly preceding a movement to the right target. In thethird example, a prominent increase in neuronal activity was ob-served in response to all four conditions, without a preference foreither concordance or discordance (Fig. 2C). We analyzed 330pmPFC neurons that exhibited activity changes during the responseperiod after the visual cue appeared. Among them, 167 neurons(51%) showed different activity levels during concordant or

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B C D

Fig. 1. Behavioral task and relevant cortical areas. (A) The monkeys wererequired to fixate on a central spot (cross), hold a lever, and wait until a vi-sual cue appeared. A red cue signaled the monkey to reach for the righttarget, whereas a green cue signaled the monkey to reach for the left target.After the cue, the monkeys were required to hit the target button within 1 s(reaction time plus movement time). The cue appeared either ipsilaterally(concordant condition, Bottom Left) or contralaterally (discordant condition,Bottom Right) to the correct target. Concordant trials required reachingtoward the visual stimulus, whereas discordant trials required reaching awayfrom the visual stimulus. (Top) Time intervals between the behavioral eventsare included. (B) A top view of a cortical surface map indicates the threeregions (colored rectangles) identified with neuronal recording. pmPFC,posterior medial prefrontal cortex; pre-SMA, presupplementary motor area;SMA, supplementary motor area. (C) Spatial distribution of task-relatedneurons on an enlarged cortical map. Points of microelectrode entry intoboth hemispheres are also indicated (monkey F). The sizes of circles denotethe number of neurons; minus signs (−) indicates no task-related neurons.(D) Spatial distribution of sensory responses tested outside of the task per-formance. V and S represent neuronal responses to visual and somatosen-sory stimuli. The somatosensory receptive fields were topographicallyorganized in accordance with previous studies. Numbers to the right are theanterior–posterior levels of Horsley-Clarke’s coordinates.

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Fig. 2. Responses of three pmPFC neurons exhibiting distinct activitychanges before the monkey began to reach for the target in response tovisual cues. Rasters and spike density functions are aligned on the basisof the onset of a reaching movement. Tick marks indicate neural spikes,whereas small squares, circles, and triangles represent onset of the visualcue, release of the hold key, and reaching the target, respectively. (A) Anexample of a neuron showing preferential activity for the discordant con-dition. In this example, neural activity was also greater for the Left target.(B) A neuron showing preferential activity for the concordant condition. (C)A neuron that did not show preferential activity for either concordant ordiscordant conditions. Neuronal activity was assessed on the basis of two-way ANOVA, with the target direction (Left vs. Right) and behavioral con-dition (concordant vs. discordant) as independent variables and spike num-bers as dependent variables.

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discordant trials (Table 1), whereas the others did not exhibit re-sponse period selectivity for concordant or discordant conditions.The concordance-based selectivity could have resulted from

the discordant cue requiring target selection based on a colorconditional rule (right, red; and green, left) and the concordantcue necessitating target selection based on a spatial rule. To ex-amine this possibility, we performed a control experiment in whicha cyan cue signaled the monkey to reach toward the illuminatedcue, whereas a blue cue signaled the monkey to reach away fromthe illuminated cue. We counted the number of spikes during theresponse period of each trial. Multivariate regression analysis wasthen performed using the target location (left or right), concor-dance (concordant or discordant), and rule (color conditional orspatial) as factors that could potentially affect spiking activity. Wecounted neurons displaying activity that was significantly affectedby the rule (P < 0.01). Most pmPFC neurons exhibited similaractivity regardless of which rule was tested (Table 2 and Fig. S3).These control experiments suggested that the monkeys did notrespond to the cue as either a color-conditioned cue or a spatialcue, depending on whether the red cue appeared on the right andthe green cue appeared on the left or vice versa.One question that remained was why we observed abundant

response period activity in the pmPFC, whereas previous studiesfailed to detect this activity (25–29). One plausible explanation liesin the nature of the behavioral task used in the present study. Afterintensive behavioral training, reaction times in the behavioral taskwere similar under concordant and discordant conditions. Thus,the monkeys were proficient at immediately responding to the cueand reaching toward or away from the illuminated target. Mon-keys were prepared to select either of these two tactics after thecue appeared, suggesting that the participation of pmPFC neuronswas required when the behavioral strategy involved multiplepotential tactics.To test this hypothesis, we retrained the monkeys for 2 wk using

only concordant or discordant conditions; (i.e., only a single tacticwas required). We then examined neuronal activity in the samemonkeys using conditions in which the animals did not need toprepare the two different tactics. The behavioral training andsubsequent neuronal recording were planned to examine neuro-nal activity under each condition incorporating only concordantor discordant cues, interposed between sessions of recording inwhich both types of cues were tested (Fig. 3A). The error rate wasalways low (99–100% correct) in the mixed, concordant-only, anddiscordant-only conditions in both monkeys, indicating that themonkeys’ performance was comparable across these conditions.In both monkeys, the number of pmPFC neurons that were

active in the response period decreased considerably under theconcordant-only condition (compare Fig. 3B and 3C), whereassimilar results were not observed in the pre-SMA or the SMA.The lower number of task-related neurons in the pmPFC was notbecause of damage in the cortex following repeated penetrationwith the electrode: The number of task-related neurons did notdecrease in the similarly examined pre-SMA or SMA, and the

population of task-related pmPFC neurons returned to its orig-inal size when discordant trials were reintroduced to the testingprocedure (Fig. 3D). The number of task-related pmPFC neuronsagain diminished when the monkeys were tested in discordanttrials only (Fig. 3E).We calculated the mean number of task-related neurons in

each electrode penetration in the pmPFC, pre-SMA, and SMAunder the four behavioral paradigms described above (Fig. 4).The number of task-related neurons in the pmPFC decreasedsignificantly under the concordant-only and discordant-onlyconditions, compared with the condition in which both types ofcues were presented (P < 0.01, χ2 test). In contrast, no suchdecrease was found in the pre-SMA or the SMA.In subsequent analyses, we attempted to evaluate how well the

activity of the concordance-selective neurons reflected whetherthe monkeys would reach toward or away from the target. Forthis purpose, we used an entropy estimation technique to cal-culate the strength of predictive information encoding the re-sponse tactics from the instantaneous discharge rates of thepmPFC neurons (SI Methods). Activity in the neuronal ensemblefrom the pmPFC started to distinguish the two tactics at a la-tency of 145 ms for monkey F and 125 ms for monkey H, usinga confidence level of P < 0.05. The onset of neuronal discrimi-nation was faster than the monkeys’ reaction times (313 ms formonkey F and 247 ms for monkey H).Finally, we explored the cingulate cortex, including the rostral

and caudal cingulate motor areas, and the adjacent cingulatecortex further rostral to the cingulate motor areas (Fig. S4) inmonkey F. Among the 260 task-related neurons recorded in thecingulate cortex, very few (5 neurons) exhibited differential ac-tivity selective for concordant or discordant trials, in accordancewith a previous report (30).

DiscussionIn the present study, we found a previously unknown focal areain the pmPFC that encodes part of the behavioral operationsrequired to complete a forelimb motor task. The pmPFC andadjacent pre-SMA differed in a number of physiologic and an-atomic properties. First, the focal area that contained the task-related pmPFC neurons was rostral to the pre-SMA. Second,unlike pre-SMA neurons, the pmPFC neurons responded poorlyto visual and somatosensory stimuli. Third, electrical micro-stimulation of the pmPFC did not elicit forelimb or eye move-ments, which distinguishes this region from the pre-SMA andSEF. And finally and most importantly, the representation ofa forelimb task by pmPFC neurons was dynamic; (i.e., the rep-resentation appeared selectively when the monkeys were testedwith the mixed behavioral condition in which concordant anddiscordant trials were randomly intermixed).Interestingly, previous studies have failed to identify neurons

representing forelimb tasks in this region (24–29). The discrep-ancy between the present and previous findings may have resul-ted from the response conflict in our study. One may argue thatthe spatial discordance between the visual stimuli and target inthe discordant trials required the monkeys to override the pre-potent response to reach for the visually most prominent stimuli.

Table 1. Distribution of neurons exhibiting concordanceselectivity

pmPFC pre-SMA SMA

Differential 167 (51%) 79 (35%) 48 (26%)Concordant preferred 77 40 21Discordant preferred 90 39 27Nondifferential 163 (49) 146 (65) 137 (74)Total 330 225 185

Neurons are classified according to the selectivity to the concordant/dis-cordant cues. For differential neurons, the numbers of concordant-preferredand discordant-preferred neurons are shown. Data from the two monkeysare combined.

Table 2. Number of rule-selective and nonselective neurons inthe three areas

pmPFC pre-SMA SMA

Rule selective 13 (4%) 5 (2%) 2 (1%)Nonselective 317 (96) 220 (98) 183 (99)Total 330 225 185

All neurons summarized in Table 1 were analyzed for rule selectivity, asexplained in the text and SI Methods.

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This condition likely caused conflict between stimulus-boundand task-required responses. A number of findings in the presentstudy, however, are not compatible with this interpretation. First,extensive training ensured that the speed at which the monkeyscompleted the arm-reach task (reaction time) did not differ whenthe behavioral condition was concordant or discordant (Fig. S1).Thus, training allowed the animals to obtain a state in whichresponse conflict no longer affected their behavior. Second, themajority of pmPFC neurons were active during the concordanttrials, which lacked response conflict. And finally, task-relatedactivity in the pmPFC largely diminished when monkeys wereperforming only discordant trials (Fig. 3), a condition likely tocause response conflict.There is an alternative hypothesis that explains the represen-

tation of the behavioral task in the pmPFC based on the selec-tion of behavioral tactics. When concordant and discordant trialswere randomly mixed, the response cue required the monkeys toselect a tactic immediately about how to respond. For the con-cordant cue, they were required to respond rapidly with a tacticthat called for reaching toward the illuminated target, whereasthe discordant cue required selection of a tactic that resulted in

reaching away from the cued target. In contrast, under theconcordant-only or discordant-only condition, response tacticsdid not vary across trials, and the monkeys had to reach onlytoward or away from the illuminated target. Thus, there was noneed to select a tactic each time a cue was presented.The decrease in active task-related pmPFC neurons with only

concordant or discordant trials supports the tactic-selection hy-pothesis. Furthermore, behavioral analysis demonstrated similarlyreduced reaction times in the concordant-only and discordant-only conditions (Fig. S5); this again suggested competition be-tween the two response tactics when the monkey had to choose,which could lengthen the reaction time.If the pmPFC is involved in the selection of response tactics,

how do the concordance-selective and -nonselective pmPFCneurons contribute to this process? We have developed a con-ceptual model for strategic tactic selection that incorporatespmPFC neurons (Fig. S6). In the model, the pmPFC processes aconcordant visual cue leading to tactic 1 (representing reachingtoward the cue) or a discordant cue to facilitate tactic 2 (repre-senting reaching away from the cue). The nonselective neuronsmay provide excitatory drive for both protactic 1 and protactic 2,

A

B C D EFig. 3. Changes in response period activity in the pmPFC,based on the requirements of the behavioral task. (A) Schedulefor recording neuronal activity under three different behav-ioral conditions. The first recordings were made with mixedconcordant and discordant cues. Then, only the concordant cuewas presented. After a 2-wk training period (hatched bar), weagain recorded neuronal activity. The recording under con-cordant-only condition was followed by another mixed be-havioral condition. Finally, only the discordant cue waspresented. Neuronal recording was resumed after a 2-wktraining period (hatched bar). (B–E) Distributions of neuronsexhibiting response period activity in the pmPFC, pre-SMA, andSMA during the four behavioral paradigms. Neurons fromboth hemispheres of each monkey were recorded. Displayformats are the same as in Fig. 1 B and C. For monkey F, wediscontinued recording from SMA neurons under the discor-dant-only condition (E).

Fig. 4. The distribution of neurons with response period activity in the three areas during the four behavioral paradigms (Fig. 3A). Data represent the meannumber of neurons per electrode penetration ± SE. Red (blue) asterisks indicate significant decreases compared with values obtained during the initial(retested) behavioral condition (P < 0.01, χ2 test). The ordinate shows the number of responsive neurons in each area, normalized according to the sample size.

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enabling them to readily respond to the selective inputs. The se-lectivity that invokes either of the tactics is likely to be transmittedto motor centers (secondary motor areas) situated downstreamof the pmPFC.On the other hand, when the valid tactic is invariant across

trials, the supervisory control to select the tactics is no longernecessary. This would relieve the pmPFC of the necessity to en-gage in the control of action, leading to the disappearance ofactive neurons (Fig. 3 B–E). Further, under the single-tacticcondition, the spatial relationship between the cue and the targetwas constant across trials; (i.e., the target always appeared ipsi-lateral to the cue under the concordant-only condition whereas itwas always contralateral under the discordant-only condition).The monkeys could then have taken advantage of this stimulus-response relationship and developed a habit of automaticallydetermining the reaching direction from the location of the cues.Such a change in the monkeys’ tactics conforms to switching fromcontrolled to automatic action (19, 31). This interpretation issupported by the decreased reaction time under the single-tacticcondition (Fig. S5). The development of habituated behavior isviewed as central to behavioral changes that induce shifts in ac-tivity in cortico-basal ganglia loops (31), which may underlie thedynamic reorganization of the action representation observed inour study. Although our present study dealt with a rather simpleform of tactics selection, both humans and animals could applya more complex form of tactics in their daily lives.Apart from the tactics selectivity, the present results also

showed the abundance of target selectivity among pmPFC neu-rons. Among the 272 neurons that exhibited behavioral selectivity,167 neurons were selective for both target and tactics or target ×tactics interaction, and 105 neurons were selective for the targetonly (see the Venn diagram in Fig. S7). We found that the en-semble activity of the 167 neurons reliably predicted the target ×tactics combination of the impending response (>95% correctrate; SI Methods and Fig. S8). On the other hand, neurons withtarget selectivity outnumber those exhibiting tactics selectivityonly. We interpreted that the present task design requiring boththe tactics and the target decision immediately after the targetappearance strongly emphasized the influence of target selectionon neuronal selectivity. Then a question arises as to whether thetactics selectivity could be observed under the condition whenonly the tactics but not the target should be determined. To an-swer this question, we conducted a separate experiment using thesamemonkeys.We introduced a delay between the determinationof tactics and the determination of the target with a cue (Fig.S9A). Under this condition, we found that 16 of 41 pmPFCneurons with postcue period activity exhibited the tactics selec-tivity during the delay period, as exemplified in Fig. S9B. Thisobservation indicated that the tactics-selective activity appears inthe pmPFC independent of target selectivity.Previous studies reported that the dorsolateral PFC partic-

ipates in a range of processes related to executive behavioralcontrol (32–35). Among them, the role of the lateral PFC in re-sponse-guiding rules and strategies is of particular relevance tothe present study. The involvement of dlPFC neurons in rule-based guidance of behavior was examined with respect to condi-tional vs. spatial rule-based behavioral control (36). Other studieshave examined task-dependent or rule-selective activity in dlPFCneurons during behavioral tasks that conform to object-matchingor location-matching rules (37, 38) or to delayed-matching orunmatched sample rules (39). In the present study, we found thatthe concordance-dependent selectivity of pmPFC neurons couldnot be ascribable to the rule selectivity discriminating color-conditional vs. spatial rules (Fig. S3). This finding, however, doesnot rule out a possibility that pmPFC neurons may participate indiscriminating behavioral rules in general. The contributions ofthe pmPFC and dlPFC, which have different afferent inputs (1),to rule-based behavioral control may differ considerably (36).

A more recent report described dlPFC neural activity repre-senting abstract response strategies (9); the activity differeddepending on whether monkeys adopted a strategy of “repeat,stay” or “change, shift.” In contrast to the behavioral demand forselection and implementation of a behavioral strategy in that study,our present task did not require abstract strategies. Instead, in-tensive training helped the monkeys to develop response tacticsand respond efficiently to randomly presented concordant or dis-cordant cues. By preparing tactics reflecting either reaching towardor reaching away from the illuminated target and selecting one orthe other after the cue was presented, the monkeys were able torespond quickly and efficiently to either cue. We propose that theneed to prepare neuronal processes to enable immediate andcorrect selection of an action—referred to as strategic assignmentof tactics—required the participation of pmPFC neurons. Of note,the medial frontal cortex is thought to take part in selecting “actionsets” (40), which may be related to the selection of response tacticsreflecting reaching toward or away from a cue. Strategic assign-ment of tactics can be categorized as supervisory control of in-formation flow to achieve specific behavioral goals (7, 41), forwhich the PFC is thought to be vital. Finally, several recent studiesdemonstrated that the neuronal selectivity to task-relevant stimulior monkeys’ decision is flexibly modulated by behavioral demands(42–45). In line with these studies, the present results also indicatedthat cortical representations of behavioral action control dependcritically on behavioral demands such that the representation isdetected only when the behavioral task is adequately selected.

MethodsAnimals and Behavioral Tasks.We used two monkeys (Macaca fuscata; a maleweighing 9.5 kg and a female weighing 5.5 kg). The animals were cared forin accordance with the National Institutes of Health guidelines and theguidelines of Tohoku University. During experimental sessions, the monkeyswere seated in a primate chair, facing a panel. The panel was equippedwith a central fixation target—represented by a white-light–emitting diode(LED)—and two buttons, one on the right and one on the left, each back-illuminated by a colored LED. Throughout each trial, the monkeys wererequired to gaze at the center fixation point until a reward was delivered(0.5 s after the correct button was pushed).

A trial began when the monkeys pressed the hold button in front of theprimate chair with their right hand. After a variable waiting period of 1–1.5 s,the LED behind one of target buttons was turned on. The monkeys receiveda liquid reward if they hit either the right or the left button within 1 s on thebasis of the color of the LED: green for left and red for right (Fig. 1A). The cueappeared either ipsilaterally (concordant condition) or contralaterally to thetarget (discordant condition) (Fig. 1A). Concordant trials required reachingtoward the visual stimulus, whereas discordant trials required reaching awayfrom the visual stimulus. The trials were aborted if the monkey released thehold button before the cue was presented, failed to fixate on the centerfixation point until the reward was delivered, or pressed the wrong button.After an error, the same cue was presented again in the next trial. Concor-dant and discordant trials were randomly intermixed during the initial re-cording of the neurons (Fig. 3A, mixed condition). After completing the initialmapping of the cortical areas, we remapped the same cortical areas whilepresenting either concordant or discordant trials separately (single condition)or intermixing them randomly again (retest of mixed condition).

Neuron Recording.While themonkeyswereperformingthebehavioral task,werecorded neuronal activity from the medial frontal cortex, including the SMA,pre-SMA, and pmPFC. Spike activity was recorded with glass-coated elgiloyelectrodes (impedance, 0.8–1.2 MΩ at 1 kHz), amplified, band-pass filtered at300–3.3 kHz, and sorted using a multispike detector (Alpha-Omega). Eyepositions were continuously monitored with an infrared corneal reflectionsystem. The behavioral task and data sampling were controlled using VisualTEMPO (Reflective Computing). Before examining neuronal activity duringperformance of the task, sensory responses of the neurons were tested at eachelectrode site by presenting objects in the monkey’s visual field, touching themonkey’s body surface, and manipulating the monkey’s joints. To examineevoked movements, we also used intracortical microstimulation (cathodalcurrent, 10–80 μA; pulse width, 300 μs; interval, 3 ms; 12–80 pulses).

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Data Analysis. Behavioral data. We used only correctly performed trials in ourdata analysis. Among these trials, corrective trials (i.e., correct trials after anerror) were discarded because the monkeys may have predicted the trial typeand tactics for the required response. For behavioral analysis, we used themonkeys’ reaction times (interval between the LED turning on and release ofthe hold button). Behavioral data from the conditional and spatial trialswere analyzed separately using multiple regression analysis and the re-gression model

Reaction time ¼ a1 × Targetþ a2 × Trial typeþ bþ ε;

where Target is the target’s location (either left or right), Trial type is thespatial concordance (concordant or discordant), a1 and a2 are regressioncoefficients, b is the intercept, and ε is the residual error.Spiking activity. To analyze neuronal activity, we chose correctly performedtrials and excluded trials in which an error was made and those that werecorrective. In this study, we concentrated on neuronal activity during theperiod from cue onset to target button press (response period). We definedthe “response period activity” as activity during the period starting from theonset of the response cue and ending at the button press. To characterizeand look into the behavioral selectivity of neuronal activity, we performedstatistical analysis on neurons that exhibited significant activity modulationduring the response period, using the following procedure. First, we defined

the baseline activity as the firing rate during the precue period of 500 mspreceding the cue onset. Next, we measured the firing rate during the re-sponse period. Because each neuron’s activity may be time locked to the cueonset, hold release, or target hit, we defined three epochs of 300 ms each inthe response period: epoch 1 following the cue onset, epoch 2 beginning150 ms before and ending 150 ms after the hold release, and epoch 3 pre-ceding the target hit. If the firing rate during any of the above three epochssignificantly increased compared with that in the precue period (P < 0.01 byMann–Whitney test), we defined such neurons as response-period related.

To determine whether the spatial concordance affected neuronal activity,we applied two-way ANOVA on neuronal data (spike rate) during the re-sponse period with the trial type (concordant or discordant) and reachingdirection (left or right) as factors. If ANOVA revealed a significant effect (P <0.01) from either the trial type or the target × trial-type interaction on theneuronal activity in any of the above three analysis periods, the neuron wasdefined as concordance specific.

ACKNOWLEDGMENTS. We thank M. Kurama and Y. Takahashi for theirtechnical assistance. This work was supported by Grant-in-Aid 19500262 forScientific Research on Priority Areas (to Y.M.) from the Japanese Ministry ofEducation, Culture, Sports, Science, and Technology of Japan and CREST.

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