A role for primate subgenual cingulate cortex insustaining autonomic arousalPeter H. Rudebeck1,2,3, Philip T. Putnam1, Teresa E. Daniels, Tianming Yang, Andrew R. Mitz, Sarah E. V. Rhodes,
and Elisabeth A. Murray
Section on the Neurobiology of Learning and Memory, Laboratory of Neuropsychology, National Institute of Mental Health, National Institutes of Health,Bethesda, MD 20892
Edited* by Leslie G. Ungerleider, National Institute of Mental Health, Bethesda, MD, and approved February 14, 2014 (received for review September
18, 2013)

Area 25| infralimbic | pupil size | anticipatory arousal

emotion and arousal, by comparison, remains unclear. We
addressed this imbalance by assessing the effect of subgenual
ACC lesions on autonomic arousal in response to a positive
emotional event: the receipt of a reward.
Results
Task. We recorded pupil size, a measure of autonomic and emo-

tional arousal (18), in six monkeys as they performed a task
in which they could anticipate fluid rewards. Three monkeys
sustained bilateral aspiration lesions of the subgenual ACC.
The remaining three monkeys served as unoperated controls(CON). The task involved Pavlovian conditioning of stimulus–
reward associations superimposed on instrumental conditioning of active visual fixation (see Methods, Fig. 1A, and SI
Methods). We chose to examine pupil size because the relatively fast (<0.5 s) response of the pupil to external stimuli
allows alterations in autonomic arousal to be correlated withbehavioral events on a trial-by-trial basis with good temporal
resolution.
On each trial, monkeys were operantly conditioned to maintain gaze on a central fixation spot for 4 s. If they did so, theyreceived three small drops of water (3 × 0.1 mL) as a reward.
A variable 4- to 6-s intertrial interval (ITI) followed immediately.
On half of the fixation trials, randomly selected, we superimposed a Pavlovian conditioning procedure. One stimulus, thepositive conditioned stimulus (CS+), always predicted a reward;
another stimulus (CS–) never did so. On Pavlovian trials, either
a CS+ or CS– was presented for 1.0 s (CS period) during eitherThe ability to regulate emotion and arousal in response topleasurable and aversive situations is essential for adapting toour environment and, ultimately, for our mental health. The
anterior cingulate cortex (ACC), specifically its subgenual part,has been implicated in a number of psychiatric disorders, including major depressive disorder (1). Dysfunction and degeneration in the subgenual ACC have been reported in patients
suffering from depression (2, 3), and the degree of activation inthis area correlates with anhedonia, the loss of positive emotions(4). Based on these findings, new approaches for treatmentresistant depression target the subgenual ACC with deep brain
stimulation (5). Determining the causal role of subgenual ACC
in the regulation of affect and arousal would advance our understanding of emotional regulation and could provide insight
into the pathophysiology of depression.
A long history of research implicates the ACC as a whole inthe control of autonomic arousal, emotional responses, and behavior (6–8). Much of what is known about the function of the
ACC, however, relates to the more dorsal parts of the ACC and
its role in higher cognition and arousal (9–11). Less is known
about the function of the ventral ACC, especially the subgenual
ACC, in part because lesions of the ventromedial prefrontal
cortex often include the subgenual ACC as well as adjacentportions of orbitofrontal cortex and the dorsal ACC (12–14).
Where research has focused on the primate subgenual ACC, it
has emphasized its role in mediating responses to threateningor fear-inducing situations, as work in rodents has (15–17).
The contribution of the subgenual ACC to regulating positive
www.pnas.org/cgi/doi/10.1073/pnas.1317695111

SignificanceDysregulation of emotion is central to the etiology of mooddisorders, such as depression. A causal understanding of how
neural structures regulate emotion and arousal could help toimprove treatments for these psychiatric disorders. Studies ofpatients with depression indicate that a particular part of thefrontal lobe, the subgenual cingulate cortex, plays an important role in affective processing, though its precise contribution
remains unclear. Here we show that, in macaque monkeys, thissmall part of the frontal cortex is necessary for sustaining elevated arousal in anticipation of positive emotional events.
This finding suggests a mechanism for the contribution of thisarea to affective regulation, including an account for the lackof pleasure and passivity that characterizes mood disorders.
Author contributions: P.H.R., P.T.P., T.E.D., T.Y., A.R.M., S.E.V.R., and E.A.M. designed
research; P.H.R., P.T.P., T.E.D., T.Y., A.R.M., and S.E.V.R. performed research; P.H.R., P.T.P.,
T.E.D., T.Y., A.R.M., and S.E.V.R. analyzed data; and P.H.R., P.T.P., S.E.V.R., and E.A.M.
wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1

P.H.R. and P.T.P. contributed equally to this work.

2

To whom correspondence should be addressed. peter.rudebeck@mssm.edu.

3

Present address: Icahn School of Medicine at Mount Sinai, New York, NY 10029.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1317695111/-/DCSupplemental.

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The subgenual anterior cingulate cortex (subgenual ACC) plays animportant role in regulating emotion, and degeneration in thisarea correlates with depressed mood and anhedonia. Despite thisunderstanding, it remains unknown how this part of the prefrontal cortex causally contributes to emotion, especially positive
emotions. Using Pavlovian conditioning procedures in macaquemonkeys, we examined the contribution of the subgenual ACC toautonomic arousal associated with positive emotional events.
After such conditioning, autonomic arousal increases in response
to cues that predict rewards, and monkeys maintain this heightened state of arousal during an interval before reward delivery.
Here we show that although monkeys with lesions of thesubgenual ACC show the initial, cue-evoked arousal, they fail tosustain a high level of arousal until the anticipated reward isdelivered. Control procedures showed that this impairment did
not result from differences in autonomic responses to reward
delivery alone, an inability to learn the association between cuesand rewards, or to alterations in the light reflex. Our data indicatethat the subgenual ACC may contribute to positive affect bysustaining arousal in anticipation of positive emotional events. A
failure to maintain positive affect for expected pleasurable events
could provide insight into the pathophysiology of psychologicaldisorders in which negative emotions dominate a patient’s affective experience.

correct per testing session: effect of lesion, F(1, 139) = 0.02, P >0.8]. Toward the end of training (final day of acquisition), themonkeys aborted fixation during trials with a CS+ stimulus more
frequently than trials with a CS–, but this difference failed toreach statistical significance [effect of CS, F(1, 35) = 3.35, P =0.07]. Nevertheless, this trend served as one indication that themonkeys had learned the association between the CS+ and reward. Though this might seem counterintuitive, we believe themonkeys were more likely to break fixation during CS+ trials
because they had learned that delivery of the 0.5-mL reward
associated with the Pavlovian stimulus was not contingent upon
continued fixation. Evidently, after CS+ presentations, the costof maintaining effortful fixation was not always worth the additional rewards (3 × 0.1 mL) to be obtained for successfullycompleting the fixation trial. Indeed, one control monkey had to
be excluded from the study because it would immediately breakfixation upon presentation of the CS+ (see SI Methods).
Conditioned Autonomic Responses. Both groups of monkeys showed
differential autonomic responses to the CS+ and CS–; they
exhibited an increased pupil size to the CS+, the stimulus thatpredicted a reward, but not to the CS– (Fig. 2A). These differences appeared after a similar number of testing sessions in
the two groups (mean ± SEM, CON = 18.2 ± 1.9 sessions;subgenual ACC = 14.2 ± 0.9; Wilcoxon test, χ2 = 1.76, P = 0.18).
Thus, monkeys with lesions of the subgenual ACC acquired a
conditioned autonomic response at the same rate as unoperated controls. Pupillary responses to the two sets of conditioned

Fig. 1. Trial sequence, stimuli, and lesions. (A) On each trial, monkeys had
to hold their gaze within 3.0° (visual angle) of the red fixation spot for 4 s toreceive three small drops of water. On 50% of trials, conditioned stimuli,
either CS+ or CS−, were presented for 1 s (CS period) during either the 4-s
fixation period or the ITI. If the CS+ was presented, a large drop of water
was delivered 0.5 s after the offset of the stimulus (trace period). (B)Intended extent of the subgenual ACC lesion (shaded region) shown on a
medial view of a macaque brain (Left) and coronal sections through the frontal
lobe (Center). Nissl-stained sections from corresponding levels show the location and extent of the subgenual ACC lesions in cases 1–3 (Right). Colors represent the degree of overlap of the lesions, as indicated in the legend.
Numerals indicate the distance in millimeters from the interaural plane.

the 4-s fixation period (38% of fixation trials, randomly selected)
or during the 4- to 6-s ITI (12% fixation trials, randomly selected). Conditioned stimuli, either CS+ or CS–, were subtle
alterations in the gray mask stimulus that was present on thescreen throughout testing (Fig. 1A). The CS+ was followed by
the delivery of a large drop of water (0.5 mL), 0.5 s after the
stimulus was turned off (trace period). Monkeys were initiallytested with one pair of conditioned stimuli (set 1) before being
tested on a second pair (set 2) to replicate the observations(Fig. S1).
The experimental design ensured that the instrumental fixation
task and Pavlovian conditioning procedure were independent.
Accordingly, autonomic responses to the Pavlovian conditioned
stimuli were not under instrumental control. For example, if amonkey broke fixation during the 4-s fixation period (while noconditioned stimulus was present), the fixation spot was extinguished, no reward was delivered, and a penalty ITI was enforced. If, however, the monkey broke fixation when a Pavlovianconditioned stimulus was presented during the fixation period,
the fixation spot was extinguished but the Pavlovian trial continued, unaffected by the oculomotor behavior.
The subgenual ACC lesion did not affect the ability of themonkeys to perform the fixation task. Monkeys in both the lesion
and control groups successfully maintained fixation during thetask, completing more than 70% of the fixation trials [proportion
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Fig. 2. Conditioned responses. (A) Pupillary dilation as a function of time
(percent change, mean ± SEM). Control (Upper, n = 3) and lesion group(Lower, n = 3). Data from stimulus sets 1 and 2, combined. Because the pupil
response lags the trial events by ∼250 ms, each analysis period is shifted 250
ms relative to CS onset and offset (dashed vertical lines). Shaded regions
show SEM. (B) Mean (±SEM) difference in pupillary responses during the CS
period, CS+ minus CS–, during the final day of acquisition (ACQ), and two
extinction sessions (EXT 1 and 2). Symbols represent the responses of individual monkeys. Negative values indicate pupillary contraction.

Rudebeck et al.

between the groups could arise if the monkeys with subgenual
ACC lesions showed diminished autonomic arousal to the rewards per se; to explore this, we tested monkeys in the same task
as before, but without conditioned stimuli. In this setting, thedelivery of large drops of water (0.5 mL) on 50 randomly
selected trials was unsignaled (see Methods, Fig. 3A andSI Methods).
Both groups showed increases in pupil size after the delivery ofunsignaled rewards, as contrasted with trials in which no reward
was delivered [reward period: effect of reward F(1, 420) = 127.7,
P < 0.0001; Fig. 3B]. Along with the CS+ response during the CS
period (Fig. 2A), this finding rules out a diminished autonomic
arousal to positive emotional events as an explanation of theimpairment observed in anticipatory autonomic arousal. In fact,monkeys with a lesion of the subgenual ACC exhibited greater
increases in pupil size following unsignaled reward than didmonkeys in the control group [reward × lesion interaction,
F(1, 420) = 12.37, P = 0.0005]. We interpret this group difference with caution, however, because slight differences in the
dynamic range of the pupil among monkeys could have accentuated the response to unsignaled rewards.
Pupil Size Changes in Response to Varying Luminance. Modulation of
pupil size is dominated by the amount of light on the retina,
known as the light reflex (21). We controlled for the light reflex
by using a stimulus mask (vertical gray bar presented before andafter CS+/− onset; Fig. 1A) and conditioned stimuli of equal luminance. Still, it is possible that the subgenual ACC lesions
produced alterations in the basic physiology of the pupil, generally, or the light reflex in particular, changes that led to groupdifferences in the pupillary response. Indeed, one might posit

Extinction of Conditioned Autonomic Responses. To confirm thatthe increase in pupil size was related to the positive emotional
nature of the reward associated with CS+ and not other variables, such as stimulus identity, we tested monkeys under extinction conditions. Monkeys were tested for an additional 2 d using
the same task parameters as before, but now no reward followed
CS+ presentation.
Compared with the final acquisition session, pupillary responses
decreased during extinction sessions, as judged by the difference
between CS+ and CS– trials during the two extinction sessions[CS period: cue × session interaction, F(1, 586) = 26.53, P <0.001; Fig. 2B]. Monkeys in both groups showed this decrease
to the same extent [effect of lesion or lesion × cue interaction,
F(1, 4/586) < 0.5, P > 0.7]. Extinction abolished the differencein pupillary response to the conditioned stimuli by the second
session (Fig. 2B). That both groups extinguished responding to
the CS+ suggests that the previously observed increases in pupil
size were directly related to the positive nature of the reward
associated with the CS+.
Previously, deficits in retention of extinction, characterized byhigher rates of spontaneous recovery of responding during thesecond extinction session, have been reported in rats with lesionsof the area homologous to the macaque subgenual ACC, infralimbic cortex (19, 20). In light of this work, we looked forheightened autonomic arousal at the start of the second compared with the end of the first extinction session. If anything,monkeys in both groups exhibited slightly decreased pupil
responses to the CS+ early in the second compared with theend of the first extinction session [Fig. S2; effect of session,
F(2, 189) = 13.7, P < 0.001]. Monkeys with subgenual ACC
lesions were no different to controls [effect of lesion or interaction
involving lesion, F(1, 4/189) < 0.75, P > 0.4]. At least in thepresent paradigm, subgenual ACC lesions were not associatedwith decreased retention of extinction.
Unconditioned Autonomic Responses. The findings from the ex-tinction sessions strongly suggest that group differences in thesustained autonomic arousal are indeed related to the anticipation of reward. It is possible, however, that the difference
Rudebeck et al.

Fig. 3. Autonomic responses to unsignaled reward. (A) On 25% of trials,
a large drop of water (0.5 mL) was delivered either during the fixation period or the ITI, with no prior indication. (B) Pupillary dilation as a functionof time (percent change, mean ± SEM) for the control (Upper) and lesion(Lower) groups. Format as in Fig. 2A.

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stimuli were highly similar [effect of set, F(1, 2,559) < 0.5,
P > 0.9], and data from the two sets were therefore combinedfor subsequent analyses.
To examine the time course of the conditioned change in pupil
size we divided the period after the presentation of the conditioned stimuli into two parts, taking into account the response
latency for changes in pupil size (see SI Methods, Data Processing
and Analysis): (i) the CS period, the time that conditioned
stimuli were present and (ii) the trace period, the interval between CS offset and reward delivery (Fig. 1A). Our initial analysis of monkeys’ pupil responses during these two periods
revealed that controls and monkeys with subgenual ACC lesions
differed in their responses (cue × lesion × period interaction,
F(1, 4316) = 36.87, P < 0.0001). Subsequent analyses were thenconducted on these two periods of the trial separately. During
the CS period, both groups exhibited increased pupil size to theCS+ relative to the CS– [for sample responses from individualmonkeys, see Fig. S1B; CS period: effect of cue, F(1, 36) =614.78, P < 0.00001; effect of lesion, F(1, 4) = 0.81, P > 0.3].
Although there was a marginally significant difference between
the groups during this period [cue × lesion interaction, F(1, 26) =3.87, P = 0.06], additional tests revealed that both groups
exhibited robust differential responses to the conditioned stimuli(Fs > 200, P < 0.0001). During the trace period, by comparison,
the lesion had a marked effect. Monkeys with lesions of subgenual ACC did not show the same sustained increase in autonomic arousal that the control group exhibited [trace period:
lesion × cue interaction: F(1, 486) = 121.78, P < 0.00001].
These findings indicate that the subgenual ACC may be criticalfor maintaining heightened autonomic arousal in anticipationof rewards.

that monkeys with subgenual ACC lesions are unable to maintain increases in pupil size, even to increases in luminance, andthat this might account for our results.
To rule out effects due to the light reflex, we tested monkeys insettings nearly identical to those of the main task. Instead ofpresenting either the CS+ or CS–, however, we either extinguished or brightened the mask stimulus (see Methods and Fig.
S3A). Changes in the luminance of the mask stimulus were notassociated with the delivery of an additional reward.
Extinguishing or brightening the mask stimulus led to a sustained dilation or contraction of the pupil, respectively, in allmonkeys [CS period: effect of cue, F(1, 221) = 1,186.26, P <0.0001; Fig. S3B]. Comparing the magnitude of pupil size responses during the period where the luminance of the mask wasaltered did not reveal any differences between the two groups[effect of lesion or lesion by cue interaction, F(1, 4/221) < 1.64,
P > 0.25]. Similarly, the latency, defined as the first 20-ms bin inwhich there was a significant difference between extinguishingor brightening the mask, was comparable between controls
and lesioned monkeys [mean ± SEM, CON = 16.67 ± 2.19;
subgenual ACC = 14.67 ± 0.33]. Thus, alterations in the lightreflex cannot account for the effect of subgenual ACC lesions on
autonomic arousal in anticipation of reward.
Acquisition and Extinction of Object–Reward Associations. To confirm that lesions of the subgenual ACC did not affect the abilityof monkeys to learn stimulus–reward associations and then subsequently modify such learned associations and behavior, monkeys were tested on an object–reward association and extinctiontask. This task has been shown to be sensitive to lesions of boththe orbitofrontal cortex (OFC) and amygdala (22). During acquisition sessions consisting of 30 trials each, monkeys wereallowed to displace a single object and to obtain a food reward
hidden underneath. Monkeys were tested until they displaced
the object on nearly every trial. Following acquisition, two
extinction sessions were conducted. Extinction sessions wereidentical to those in acquisition with the exception that no food
reward was available under the object. Omissions, i.e., trials on
which monkeys failed to displace the object, were counted as ameasure of extinction.
All monkeys readily displaced the object for food reward.
During acquisition, controls and monkeys with lesions of thesubgenual ACC learned at a similar rate (mean omissions ±SEM: CON = 1.75 ± 1.75; subgenual ACC = 5 ± 5; Kruskal–
Wallis test χ2 = 0.19, P = 0.66). During extinction, displacementof the unrewarded object decreased markedly across sessions[effect of session, F(2, 10) = 31.15, P < 0.001; Fig. S4A]. Thischange in behavior was similar in the two groups [F(1, 5) = 0.005,
P = 0.95; all interactions involving group, F < 1.74, P > 0.1]. We
also investigated whether monkeys with subgenual ACC lesions
exhibited spontaneous recovery of responding at the start of thesecond extinction session. Though both groups exhibited elevated responding early in the second session by comparison withthe end of the first session [Fig. S4B; effect of session, F(2, 10) =13.08, P < 0.002], monkeys with subgenual ACC lesions performed similarly to controls [effect of lesion, F(1, 5) = 0.03, P >0.8]. Thus, lesions of subgenual ACC failed to disrupt theacquisition, extinction, and retention of extinction of object–reward associations.

Discussion
We found that lesions of the subgenual ACC caused a specific
impairment in autonomic arousal during the anticipation of
rewards, as measured by pupillary dilation. In contrast to thesustained prereward arousal observed in intact monkeys, monkeys with lesions of the subgenual ACC showed only transientarousal at the time of the CS+ (Fig. 2A). The lesions did notaffect the extinction of conditioned autonomic arousal (Fig. 2B),
autonomic arousal to unsignaled rewards (Fig. 3B), pupillary
responses to light (Fig. S3), or the acquisition and extinction of
object–reward associations (Fig. S4). Our findings suggest that5394 | www.pnas.org/cgi/doi/10.1073/pnas.1317695111

the subgenual ACC is necessary for sustaining elevated autonomic arousal in anticipation of rewards, and implicate this areain the regulation of positive affect.
Interpretational Issues. This study sought to identify the previously
uncharacterized effects of lesions specifically targeting the subgenual ACC, and is one of only a handful to assess the effects ofcortical lesions on autonomic arousal in primates (23–26). The
subgenual ACC is one of the least accessible parts of the frontal
cortex. To investigate its function, we chose to make lesions byaspiration because it allowed direct visualization of landmarksin this region and had a better chance of success; it also had thepotential to cause less damage to other parts of the medial
prefrontal cortex relative to other approaches, such as stereotaxically placed injections of excitotoxins or pharmacologicalinactivation, either of which could have caused inadvertent
damage to other parts of the ACC. Our lesions involved thetransection of the genu of the corpus callosum, which was required to gain access to the subgenual ACC. Consequently, we
cannot rule out a contribution by the genu. In addition, it ispossible that inadvertent damage to the white matter subjacent
to subgenual ACC might have contributed to our results. One
possible site of termination for these fibers is the OFC, an adjacent part of the prefrontal cortex that has been associated withautonomic control and emotion regulation (6). In marmosetmonkeys, lesions of the OFC cause alterations in autonomic
arousal that differ from those reported here. Specifically, OFC
lesions render monkeys less able to suppress arousal when thecontingency between a CS+ and reward is abolished. By contrast,
lesions of this area spare acquisition and expression of conditioned autonomic arousal (24). It therefore seems unlikely thatdamaging fibers running to or from the OFC could account forthe present pattern of results. An alternative possibility is thatthe lesions might have damaged fibers coursing to or from themore dorsal parts of the ACC. We are not aware of any lesion
studies that have assessed the role of the dorsal ACC in autonomic arousal in monkeys. Lesion and functional studies inhumans indicate that the dorsal ACC plays a role in the control
of autonomic arousal, but these studies often emphasize a role
for this area in arousal in relation to effortful responding andaction as opposed to subjective emotional experience (9, 27).
Either way, delineating the effects on arousal of damage limited
to cell bodies within the subgenual ACC, potentially by using
excitotoxic lesions, is an important avenue for future research.
Subgenual ACC, Emotion, and Arousal. Fundamentally, the autonomic nervous system is part of the motor system in the broad
sense, in that it is one way that the central nervous system controls the body (28). It has long been recognized that the subgenual ACC contributes to autonomic control (for a review, seeref. 9). Electrical stimulation of this area produces changes inbreathing and heart rate (6, 8, 29), and the subgenual ACC isdensely interconnected with structures that play a central role invisceromotor control, such as the hypothalamus (30).
Despite this understanding, the role of the primate subgenual
ACC in positive affect has remained unclear; in part, this isbecause lesions of the ventromedial frontal cortex often include
parts of the dorsal ACC and/or medial OFC as well as subgenual
ACC (12–14). Likewise, there are few neurophysiology studies ofthe subgenual ACC. Though one study found slight changes
in tonic firing rates in response to emotional events, such as
rewards (31), another suggested that the subgenual ACC mightplay a role in internally driven motivational behavior (32), an
issue we take up later.
Here we provide evidence that the role of the subgenual ACC
in visceromotor behavior is specific to regulating conditioned
autonomic arousal (Fig. 2), as opposed to unconditioned arousal(Fig. 3), in anticipation of positive emotional events—specifically, fluid rewards. We note that the pattern of effects is subtly
different from those reported after lesions of the amygdala inmarmoset monkeys (25). Whereas lesions of the subgenual ACC,
Rudebeck et al.

Subgenual ACC and Mental Health. Patients with psychiatric dis-orders involving mood dysregulation, as a group, display both
functional and architectonic changes within the subgenual ACC(2, 3). Based on these and related findings, clinicians have useddeep brain stimulation techniques targeting this portion of the
ACC as a potential treatment for depression (5). Here we provide evidence that the subgenual ACC contributes to maintaining heightened arousal in anticipation of positive emotional
events, especially in the absence of predictive cues. Our findingspresent a starting point for future studies seeking to determine
the role of the subgenual ACC in affect by providing an initialautonomic fingerprint of the effects of damage within this area.
They might also inform studies of autonomic function in patientswith depression (36) and potentially contribute to efforts tounderstand the neural mechanisms underlying deep brain stimulation techniques for mood disorders such as depression.
Anhedonia, characterized by a loss of pleasure from previously
rewarding events, is associated with depression and dysfunction
within the subgenual ACC (4). From one perspective, the present results would appear inconsistent with a role for the subgenual ACC dysfunction in anhedonia. Monkeys with subgenual
ACC lesions showed normal patterns of arousal following thereceipt of rewards and also in response to stimuli (CS+) thatpredicted rewards. Anhedonia, however, is thought to incorporate a motivational or anticipatory component, related tomaintaining positive affect in advance of appetitive stimuli, as
well as a “consummatory” component related to the experienceof primary rewards (37, 38). Our findings indicate that the subgenual ACC may play a specific role in the anticipatory aspectsof positive affect. Indeed, a recent functional neuroimaging study
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has reported that depressed individuals are unable to sustain
neural activation associated with positive emotional affect in theventral striatum (39), a portion of the basal ganglia directly
connected with the subgenual ACC (40).
Using measures of autonomic arousal in conjunction withmanipulations of subgenual ACC function potentially provides
a means of modulating the motivational and anticipatory aspectsof anhedonia. Our results also demonstrate the value of establishing causal links between specific neural structures and theregulation of emotion and arousal.
Methods
Subjects. Seven adult rhesus monkeys (Macaca mulatta, six male and one
female) served as subjects. Three monkeys sustained bilateral aspiration
lesions of the subgenual ACC, and the remaining four were retained as
unoperated controls. Six monkeys (three controls and three subgenual ACC
lesion) were tested on the tasks assessing autonomic function. The remaining control monkey was not tested on the tasks assessing autonomic function due to an inability to meet behavioral performance requirements (SIMethods). All seven monkeys were tested on the object–reward and extinction task. Monkeys were at least 4.5 y old at the start of testing. Eachanimal was individually or pair housed, and kept on a 12-h light-dark cycle.
Monkeys’ access to food and water was controlled for 6 d a week. All procedures were reviewed and approved by the National Institute of Mental
Health Animal Care and Use Committee.
Surgical Procedures. Surgery was conducted using standard aseptic procedures. For full a description of the surgical methods, see SI Methods. Bilateralremoval of subgenual ACC was carried out in a single operation. A large,
symmetrical bone flap was turned over the dorsal aspect of the cranium andthe dura mater opened over the frontal lobe. With the aid of an operating
microscope, key landmarks on the medial surface of the hemisphere andalong the midline (e.g., the cingulate sulcus, corpus callosum, and anterior
cerebral artery) were identified. The subgenual ACC was then removed bysubpial aspiration, i.e., the cortex ventral to the genu and rostrum of the corpus
callosum was removed. The cortical removal was carried out in the samemanner in the other hemisphere via the same opening. Thus, the intended
lesion includes both areas 25 and anterior area 24 ventral to the genu of
the corpus callosum. Approximately 1 mo before training, all monkeys were
implanted with a titanium head post using asceptic procedures.
Lesion Assessment. Lesions of the subgenual ACC were assessed using T1weighted MRI scans acquired shortly after surgery and at the conclusion of
the experiment by histological examination (Fig. 1B). In all three cases,
lesions encompassed the intended parts of the cortex, with damage centering on areas 25 and area 24 ventral to the genu of the corpus callosum.
There was systematic sparing of the cortex across the two hemispheres in themost caudal parts of area 25. Inadvertent damage was limited to parts ofareas 14, 24a/b and 32.
Pavlovian and Fixation Task: Acquisition. Previously, monkeys had been trained
to fixate the central red spot within 3.0° of visual angle (dva) for 4 s on every
trial (for full details, see SI Methods). A Pavlovian trace-conditioning procedure was then superimposed, and both tasks were conducted at the sametime (Fig. 1A). There was a random delay of between 200 and 600 ms after
fixation or ITI onset before the presentation of either of the conditioned
stimuli. The CS+ was followed by a 0.5-mL fluid reward, delivered 500 ms(trace period) after the stimulus was turned off, whereas the CS– was followed by an unfilled interval and no reward was delivered.
Monkeys were first tested with stimulus set 1, in which the CS+ and CS–
consisted of the gray mask stimulus rotated 45° clockwise and counterclockwise, respectively (Fig. S1). To replicate the effects, we repeated thetask with a second pair of conditioned stimuli (stimuli: set 2; Fig. S1). At least6 d intervened between training with stimulus set 1 and 2. Altering the mask
stimulus to present either the CS+ or CS– meant that the same number ofgray pixels were physically present on the screen throughout Pavlovian
conditioning trials, minimizing differences in luminance; this was done sothat any within-trial alterations in pupil size would most likely reflectchanges in autonomic arousal and not simply changes associated withturning on or off the conditioned stimuli or differences between the CS+
and CS–. The assignment of CS+ and CS– was counterbalanced across lesion
groups. Monkeys received 50 CS+ and 50 CS– presentations per session overthe course of ∼200 fixation trials. One session was conducted per day, untilmonkeys exhibited a stable difference in pupil size during the CS+ and CS–

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like amygdala lesions, diminished arousal in anticipation of
rewards, in the case of the subgenual ACC this effect was limited
to the trace interval (i.e., the period between the offset of the CS
and reward delivery). Furthermore, in the marmoset experiments, the CS+ (food) was visible throughout the anticipatory
period. Thus, there was no possibility to examine sustained autonomic arousal in the absence of the CS+.
An indirect, as opposed to a direct, role for the primate subgenual ACC in the control of autonomic arousal is underscoredby tract-tracing studies that show that the subgenual ACC in
primates, unlike rodents (33), is not directly connected to autonomic effector regions in the brainstem (30). Such a pattern ofconnections in primates could account for the differences between the present findings and those reported in rodents after
lesions of the homologous area, infralimbic cortex (29, 34). In
rodents, lesions of the infralimbic cortex have been reported tolead to heightened respiration (34), but also attenuated heart
rate changes in response to stimuli that predict aversive footshock (35). It is possible, however, that other factors, such as thevalence of the stimuli used, might have contributed, and this is
one clear difference between the present study and those inrodents. Indeed, whether our results would hold for negative
emotional events invites empirical investigation. Based on studies in rats (19, 20), we would predict that the subgenual ACC
would play a similar role in the control of emotion and arousal
in anticipation of aversive events, as it does in anticipation of
positive events.
Lesion studies in rodents and functional MRI investigations inhumans suggest that subgenual ACC is important for retentionof extinction (15–17, 20). Here, we did not see evidence fordecreased extinction retention in the form of increased spontaneous recovery of either autonomic or behavioral responding
during the second extinction session in monkeys with subgenual
ACC lesions (SI Methods and Figs. S2 and S4B). It is possiblethat the task we used to assess conditioned autonomic responding,which used concurrent Pavlovian and fixation trials, might havecontributed to these negative findings. In addition, it is possiblethat the potential for the object–reward association task to besolved using either Pavlovian or instrumental strategies might
have been a factor.

presentation periods for four consecutive sessions, referred to from here
onward as criterion sessions (SI Methods, Data Processing and Analysis).
Pavlovian and Fixation Task: Extinction. At the conclusion of the four criterion
sessions, we conducted an extinction procedure. Monkeys were tested for
two additional sessions, one per day, using the same task as before, but now
neither the CS+ nor CS– led to reward delivery. There were 50 presentationsof each of the conditioned stimuli per session, and monkeys completed ∼200
fixation trials per session. Extinction sessions were conducted after themonkeys reached criteria for both stimulus sets 1 and 2. Data from extinction sessions with stimulus set 1 from one monkey, subgenual ACC case 1,
was not available for analysis due to experimenter error.
Unsignaled Reward Task. The same general task design and trial structure as in
the main task was used to provide unsignaled rewards, but neither the CS+
nor CS– stimuli were presented (SI Methods). Monkeys were required to
fixate a central spot for fluid rewards (3 × 0.1 mL). On 50 randomly chosen
trials, large rewards (1 × 0.5 mL of fluid) were delivered either during the
fixation period or during the ITI. Thus, by design, the frequency and timingof unsignaled reward delivery matched the parameters used in the maintask. Monkeys were tested for four consecutive days, ∼200 fixation trials per
daily session. As in the Pavlovian and fixation task, monkeys had to maintaina criterion of successfully completing >80% of fixation trials during each of
the 4 d of testing.

stimulus (mask turned white, 50 trials) for 1 s. Extinguishing or brightening
the mask stimulus occurred randomly on 50% of trials and was not associated with the delivery of additional fluid reward. Matching the previous
tasks, either extinguishing or brightening of the mask stimulus could occur
either during the fixation period or the ITI. Monkeys were tested for threeconsecutive days, ∼200 fixation trials per daily session.
Object–Reward and Extinction Task. We used highly similar methods to thoseused previously in the laboratory (22). Two separate tests, acquisition followed by extinction, were conducted.
Acquisition phase. On each trial, the monkey was presented with a single
object, novel at the start of acquisition, covering the central well of the test
tray. Monkeys were given 30 s to displace the object and retrieve the food
reward hidden underneath. If the monkey retrieved the food, the trial wasscored as correct; however, the trial ended only after the full 30 s had elapsed.
If the monkey failed to retrieve the food within the limit of 30 s, it was scoredas an omission. At the end of each 30-s trial, the screen was lowered. Trials
were separated by 15 s. Monkeys were tested at the rate of 30 trials persession, one session per day. The criterion for acquisition was set at 93% over
five consecutive days i.e., 140/150.
Extinction phase. At the conclusion of the acquisition phase, monkeys received
two consecutive extinction sessions. Trials were conducted exactly as in acquisition with the exception that no food rewards were provided.

Luminance Test. The effect of varying luminance on extent and latency ofmonkey’s pupil size responses was assessed in a separate task (Fig. S3A). This
procedure differed from the main task in several ways. Monkeys were required to fixate a central spot for fluid rewards (3 × 0.1 mL). Over the courseof a session, instead of introducing a CS+/CS–, we either extinguished themask stimulus (mask stimulus removed, 50 trials) or brightened the mask

ACKNOWLEDGMENTS. We thank Vincent Costa, Bruno Averbeck, andSteven Wise for advice on analyses and comments on an earlier versionof the manuscript. We also thank Yogita Chudasama for assistance withsurgery and John Kakareka, Randal Pursely, and Tom Pohida for theirassistance in processing physiological signals. This work was supported by
the Intramural Research Program of the National Institute of Mental Health.

1. Price JL, Drevets WC (2010) Neurocircuitry of mood disorders. Neuropsychopharmacology
35(1):192–216.
2. Drevets WC, et al. (1997) Subgenual prefrontal cortex abnormalities in mood disorders. Nature 386(6627):824–827.
3. Ongür D, Drevets WC, Price JL (1998) Glial reduction in the subgenual prefrontal
cortex in mood disorders. Proc Natl Acad Sci USA 95(22):13290–13295.
4. Keedwell PA, Andrew C, Williams SC, Brammer MJ, Phillips ML (2005) The neuralcorrelates of anhedonia in major depressive disorder. Biol Psychiatry 58(11):843–853.
5. Mayberg HS, et al. (2005) Deep brain stimulation for treatment-resistant depression.
Neuron 45(5):651–660.
6. Kaada BR, Pribram KH, Epstein JA (1949) Respiratory and vascular responses inmonkeys from temporal pole, insula, orbital surface and cingulate gyrus; a preliminary report. J Neurophysiol 12(5):347–356.
7. Papez J (1937) A proposed mechanism of emotion. Arch Neurol Psychiatry 79:
217–224.
8. Smith WK (1945) The functional significance of the rostral cingular cortex as revealed
by its responses to electrical stimulation. J Neurophysiol 8:241–255.
9. Critchley HD (2005) Neural mechanisms of autonomic, affective, and cognitive integration. J Comp Neurol 493(1):154–166.
10. Rushworth MF, Walton ME, Kennerley SW, Bannerman DM (2004) Action sets anddecisions in the medial frontal cortex. Trends Cogn Sci 8(9):410–417.
11. Botvinick MM, Braver TS, Barch DM, Carter CS, Cohen JD (2001) Conflict monitoring
and cognitive control. Psychol Rev 108(3):624–652.
12. Hadland KA, Rushworth MF, Gaffan D, Passingham RE (2003) The effect of cingulate
lesions on social behaviour and emotion. Neuropsychologia 41(8):919–931.
13. Damasio AR, Tranel D, Damasio H (1990) Individuals with sociopathic behavior causedby frontal damage fail to respond autonomically to social stimuli. Behav Brain Res
41(2):81–94.
14. Bechara A, Damasio H, Damasio AR, Lee GP (1999) Different contributions of thehuman amygdala and ventromedial prefrontal cortex to decision-making. J Neurosci
19(13):5473–5481.
15. Phelps EA, Delgado MR, Nearing KI, LeDoux JE (2004) Extinction learning in humans:role of the amygdala and vmPFC. Neuron 43(6):897–905.
16. Quirk GJ, Garcia R, González-Lima F (2006) Prefrontal mechanisms in extinction of
conditioned fear. Biol Psychiatry 60(4):337–343.
17. Milad MR, et al. (2007) Recall of fear extinction in humans activates the ventromedial
prefrontal cortex and hippocampus in concert. Biol Psychiatry 62(5):446–454.
18. Bradley MM, Miccoli L, Escrig MA, Lang PJ (2008) The pupil as a measure of emotionalarousal and autonomic activation. Psychophysiology 45(4):602–607.
19. Rhodes SE, Killcross S (2004) Lesions of rat infralimbic cortex enhance recovery andreinstatement of an appetitive Pavlovian response. Learn Mem 11(5):611–616.
20. Quirk GJ, Russo GK, Barron JL, Lebron K (2000) The role of ventromedial prefrontal
cortex in the recovery of extinguished fear. J Neurosci 20(16):6225–6231.
21. Lowenstein O, Loewenfeld IE (1969) The pupil. The Eye, ed Davson H (Academic, NewYork), 2nd Ed, Vol 3.

22. Izquierdo A, Murray EA (2005) Opposing effects of amygdala and orbital prefrontal
cortex lesions on the extinction of instrumental responding in macaque monkeys. Eur
J Neurosci 22(9):2341–2346.
23. Marshall LB, Smith OA (1975) Prefrontal control of conditioned suppression and associated cardiovascular variables in the monkey (Macaca mulatta). J Comp Physiol
Psychol 88(1):21–35.
24. Reekie YL, Braesicke K, Man MS, Roberts AC (2008) Uncoupling of behavioral andautonomic responses after lesions of the primate orbitofrontal cortex. Proc Natl Acad
Sci USA 105(28):9787–9792.
25. Braesicke K, et al. (2005) Autonomic arousal in an appetitive context in primates: A
behavioural and neural analysis. Eur J Neurosci 21(6):1733–1740.
26. Kimble D, Bagshaw MH, Pribram KH (1965) The GSR of monkeys during orienting andhabituation after selective partial ablations of the cingulate and frontal cortex.
Neuropsychologia 3(2):121–128.
27. Critchley HD, et al. (2003) Human cingulate cortex and autonomic control: Converging neuroimaging and clinical evidence. Brain 126(Pt 10):2139–2152.
28. Swanson LW (2000) Cerebral hemisphere regulation of motivated behavior. Brain Res
886(1-2):113–164.
29. Buchanan SL, Powell DA (1993) Cingulothalamic and prefrontal control of autonomic
function. Neurobiology of Cingulate Cortex and Limbic Thalamus, eds Vogt BA,
Gabriel M (Birkhauser, Cambridge, MA), pp 1381–1414.
30. Freedman LJ, Insel TR, Smith Y (2000) Subcortical projections of area 25 (subgenual
cortex) of the macaque monkey. J Comp Neurol 421(2):172–188.
31. Monosov IE, Hikosaka O (2012) Regionally distinct processing of rewards andpunishments by the primate ventromedial prefrontal cortex. J Neurosci 32(30):10318–10330.
32. Bouret S, Richmond BJ (2010) Ventromedial and orbital prefrontal neurons differentially encode internally and externally driven motivational values in monkeys.
J Neurosci 30(25):8591–8601.
33. Hurley KM, Herbert H, Moga MM, Saper CB (1991) Efferent projections of the infralimbic cortex of the rat. J Comp Neurol 308(2):249–276.
34. Frysztak RJ, Neafsey EJ (1991) The effect of medial frontal cortex lesions on respiration, “freezing,” and ultrasonic vocalizations during conditioned emotional responses in rats. Cereb Cortex 1(5):418–425.
35. Frysztak RJ, Neafsey EJ (1994) The effect of medial frontal cortex lesions on cardiovascular conditioned emotional responses in the rat. Brain Res 643(1-2):181–193.
36. Lane RD, et al. (2013) Subgenual anterior cingulate cortex activity covariation withcardiac vagal control is altered in depression. J Affect Disord 150(2):565–570.
37. Treadway MT, Zald DH (2011) Reconsidering anhedonia in depression: Lessons fromtranslational neuroscience. Neurosci Biobehav Rev 35(3):537–555.
38. Tomarkenand A, Keener A (1998) Frontal brain asymmetry and depression: A selfregulatory perspective. Cogn Emotion 12(3):387–420.
39. Heller AS, et al. (2009) Reduced capacity to sustain positive emotion in major depression reflects diminished maintenance of fronto-striatal brain activation. Proc Natl
Acad Sci USA 106(52):22445–22450.
40. Haber SN, Kunishio K, Mizobuchi M, Lynd-Balta E (1995) The orbital and medial
prefrontal circuit through the primate basal ganglia. J Neurosci 15(7 Pt 1):4851–4867.

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Rudebeck et al.

Supporting Information
Rudebeck et al. 10.1073/pnas.1317695111
SI Methods
Apparatus and Materials. All training and testing involving as-

sessment of pupil size took place within a custom-made soundattenuating chamber (Crist Instrument Co.). Monkeys wereseated in a primate chair 57 cm from a computer monitor (640 ×
480 pixels). The behavioral task was controlled by a computerrunning National Institutes of Mental Health (NIMH) CORTEX software (http://dally.nimh.nih.gov/). Stimuli, presented on
the screen, were 48 × 48-pixel 256-color bitmaps [1.4° of visual
angle (dva)]. Fluid rewards were delivered through a spout positioned in front of monkeys using a pressurized delivery system (1).
During training and testing, eye position and pupil size weremonitored with an infrared camera (ISCAN). Signals were filtered at 200 Hz and sampled at a rate of 1 kHz. Trial events, eyeposition, and pupil size signals were recorded using a Plexon
multiacquisition system (Plexon).
For the object–reward task, all testing was conducted withina modified Wisconsin general testing apparatus (WGTA). The
animal compartment of the WGTA held a wheeled transport
cage in which monkeys were situated during training and testing.
The test compartment of the WGTA contained a test tray (19.2 ×
72.7 × 1.9 cm) with three food wells, 6 mm deep and 38 mm indiameter, positioned in the center of the tray. An opaque screen
separated the two compartments of the WGTA and could beraised and lowered to control monkeys’ access to the test tray.
An additional screen, one that allowed one-way viewing of thetest compartment, separated the experimenter from the testcompartment. Two novel junk objects were used, one for eachtest. A half peanut served as the food reward for all monkeys,
apart from subgenual anterior cingulate cortex (ACC) case-3,
which received single M&M’s (Mars Food, Inc.).
Surgical Procedures. Anesthesia was induced with ketamine hydrochloride (10 mg/kg, i.m.) and maintained with isoflurane (1.0–
3.0%, to effect). The monkeys received isotonic fluids via an i.v.
drip. Aseptic procedures were used, and heart rate, respiration
rate, blood pressure, body temperature, and expired CO2 levels
were monitored throughout the procedure. After the lesion wascompleted, the wound was closed in anatomical layers. Dexamethasone sodium phosphate (0.4 mg/kg) and Cefazolin antibiotic (15 mg/kg) were administered for 1 d before surgery and
1 wk after surgery to reduce swelling and prevent infection, respectively. At the end of surgery, and for two additional days, themonkeys received the analgesic ketoprofen (10–15 mg); ibuprofen (100 mg) was provided for five additional days. During
surgery, monkeys were given 30 mL of mannitol (20%, 1 mL/min,i.v.) to increase access to midline structures and control edema.
Bilateral removal of subgenual ACC was carried out in a single
operation. A large, symmetrical bone flap, roughly 4 cm on a side,
was turned over the dorsal aspect of the cranium. The dura mater
was cut near the lateral edge of the bone opening and reflected
toward the midline. The cerebral hemisphere was gently retracted
and, with the aid of an operating microscope, key landmarks on
the medial surface of the hemisphere and along the midline (e.g.,
the cingulate sulcus, corpus callosum) were identified. Bloodvessels that interfered with access to the midline structures werecauterized, as necessary. The anterior portion of the corpus
callosum was visualized and sectioned using gentle suction
through a fine glass sucker. The anterior cerebral artery, whichsurrounds the genu, served as a guide. Using a combination ofsuction and electrocautery, the subgenual cortex was removed bydirect, subpial aspiration through a fine-gauge metal sucker inRudebeck et al. www.pnas.org/cgi/content/short/1317695111

sulated except at the tip. The removal extended from just below
the genu and rostrum of the corpus callosum to the base of the
cranium. The ablation was then extended a few millimeters
rostrally and caudally, or as far as possible within the limits of the
opening. The cortical removal was carried out in the same mannerin the other hemisphere via the same opening. Thus, the intended
lesion includes both areas 25 and anterior 24 below the genu of
the corpus callosum (2, 3).
Approximately 1 mo before training, all monkeys were implantedwith a titanium head post using the same anesthetic and standard
surgical procedures described above. Head posts were implanted
between 4–5 y after lesions of subgenual ACC were made.
Behavioral Training and Testing. For all training and testing involving
autonomic arousal measurements, monkeys were seated in a primate
chair facing a computer monitor. Over a period of approximately 1
wk, monkeys were habituated to the test chamber and to having theirhead position fixed via the head post. Before testing, eye position wascalibrated using a nine-point array (3 × 3 grid, 5-dva spacing).
Fixation Task Training. Through successive approximation, monkeys were trained to fixate a central red spot (0.2 dva) to earn fluid
reward. Fixation was initially registered when monkeys moved
their eyes to within 1.5 dva of the central spot; they then had tohold their gaze within 3.0 dva of the spot for the remainder of thetrial before 3 × 0.1-mL rewards were delivered and the fixation
spot extinguished. Over the course of successive training sessions
the fixation requirement was lengthened from 1 to 4 s. The intertrial interval (ITI) was 4–6 s. If monkeys broke fixation during
a trial, the trial was aborted, the red fixation spot extinguished,
and a penalty ITI of 8–10 s ensued. Monkeys completed ∼400
trials per session and were trained to a criterion of >80% or 320/400
trials per session for four consecutive sessions. At the completionof fixation training, monkeys were habituated to the gray “mask”
stimulus (vertical gray rectangle, 1.37 × 0.85 dva or 48/20 pixels).
The stimulus was presented centrally behind the red fixation spotin the center of the screen. Monkeys were tested for a further 3 d
to ensure stable and accurate performance (>80%).
Dual Fixation–Pavlovian Trace-Conditioning Task: Acquisition. Oncemonkeys were reliably completing >80% of trials in the fixation
task they were tested on a dual fixation–Pavlovian trace-conditioning task. As before, monkeys were required to fixate thecentral red spot for 4 s on every trial, but now a Pavlovian traceconditioning procedure was superimposed and both tasks wereconducted at the same time (Fig. 1A). The Pavlovian conditioning procedure was conducted on 50% of fixation trials selected at random and could occur in either the fixation period(38% of trials) or ITI (12% of trials). On these trials, either a
CS+ or CS- was presented for 1,000 ms during either the 4-s
fixation period or during the 4- to 6-s ITI. There was a random
delay of between 200–600 ms after fixation or ITI onset before
the presentation of either of the conditioned stimuli. The CS+
was followed by a 0.5-mL fluid reward, delivered 500 ms (trace
period) after the stimulus was turned off, whereas the CS− wasfollowed by an unfilled interval and no reward was delivered.
Monkeys were first trained with stimulus set 1. In stimulus set 1,
the CS+ and CS− were the gray mask stimulus rotated 45° either
clockwise or counterclockwise, respectively. To replicate the effects
and confirm that any differences in pupil diameter were due tothe association between stimuli and rewards and not simply tophysical features of the conditioned stimuli, we repeated the task
1 of 4

with a second set of conditioned stimuli (stimulus set 2). At least6 d intervened between training with stimulus sets 1 and 2. In
stimulus set 2, the CS+ and CS− were the gray mask stimuluswith the middle block of 16 × 20 pixels shifted horizontally either
10 pixels to the left or the right (Fig. S1A). Altering the mask
stimulus to present either the CS+ or CS− meant that the same
number of gray pixels were physically present on the screen
throughout Pavlovian conditioning trials, minimizing changes
in luminance. This procedure was used so that any within-trialchanges in pupil size would most likely be related to changes inautonomic arousal and not simply changes in luminance associated with turning on or off the conditioned stimuli, or differences
between the CS+ and CS−. Monkeys received 50 CS+ and 50
CS− presentations per session over the course of ∼200 fixation
trials. The assignment of CS+ and CS− was counterbalanced
across lesion groups.
Importantly, we devised the dual-task procedure so that the twotasks (fixation and Pavlovian conditioning) were not contingent
upon each other and were run completely independently. Performance on the fixation task did not affect the Pavlovian conditioning task and vice versa; this was to ensure that behavioral
and autonomic responses to the Pavlovian conditioned stimuli
were not under instrumental control. For example, if a monkey
broke fixation during the 4-s fixation period (while no conditioned
stimulus was present), the fixation spot was extinguished, the 8to 10-s penalty was imposed, and no reward was delivered. If,
however, the monkey broke fixation when a Pavlovian-conditioned stimulus was presented during the fixation period, thefixation spot was extinguished but the Pavlovian trial continued;
presentation of the CS+/− for 1,000 ms was followed by eithera reward or no reward, as appropriate for that trial.
Monkeys were tested on the dual fixation–Pavlovian task, one
session per day, until they exhibited a stable difference in pupil
size during the CS+ and CS− presentation periods for four
consecutive sessions (see next section). During this 4-d criterion
period, monkeys also had to maintain a high level of performance on the fixation task, completing at least 80% of total
trials. If performance fell below 70% during a session, the session was excluded from analysis. If poor performance continued(more than three sessions at <80%), monkeys were stepped backto the fixation task alone until they were performing at >80% on
fixation trials; this was done to ensure that (i) monkeys were notassociating performance on one task with the other; and (ii)monkeys were not extinguishing performance on fixation trials
due to contingency degradation effects, due to receiving “free”
rewards during Pavlovian conditioning. Indeed, one control
monkey had to be excluded from the study because its fixation
performance degraded markedly with the introduction of the
Pavlovian CS+ and CS− stimuli and associated reward. On
presentation of the CS+/CS−, this monkey immediately broke
fixation. Additional training was unable to remedy this behavior,
which meant that only three unoperated control monkeys weretrained on the autonomics task.
Data Processing and Analysis. To assess overall fixation performance on the conditioned autonomic task, the proportion ofcompleted trials per session was compared between the groups.
For the analysis of conditioned autonomic responses, pupil sizeand eye position signals were down-sampled to 50 Hz, and all data
were processed using MatOFF software (4) in MatLab (MathWorks). Only trials on which (i) monkeys maintained their gazewithin the required window for the duration of the fixation period and (ii) events of interest (i.e., CS+/CS–, unsignaled rewards, etc.) fell within the 4-s fixation period were included in
the analysis of conditioned autonomic responses. This procedure
served to remove trials where monkeys either made eye movements or blinks that would have otherwise added additional
noise to the pupil size signals.
Rudebeck et al. www.pnas.org/cgi/content/short/1317695111

All pupil size data were baseline corrected to a 500-ms period
extending from 250 before to 250 ms after CS+/− onset. This
period was chosen because it allowed monkeys’ pupil size themaximum amount of time to stabilize following fixation andbefore CS+/− presentation (earliest response to change in luminance ∼280 ms; Fig. S3). To control for the differences in the
dynamic range of the pupil, the data for each animal were normalized using a z-score before analyses (z = x − μ/σ).
For initial inspection of the data from each session of the maintask, we compared responses across a 2,500-ms time window(125 samples) starting from the onset of the CS+/CS–. Data
were analyzed using a one-way ANOVA conducted at eachtime point, Bonferroni-corrected for multiple comparisons (P <0.0004). Learning was reflected by the emergence of differential
pupil responses to the conditioned stimuli. Criterion was set atsignificantly different pupil response to the CS+ and CS– at
more than three sample points during the CS period. This
screening procedure was conducted for each session untilmonkeys achieved four consecutive sessions with pupil responses at or above criterion.
Pupil size data from the four consecutive days and two replications (stimulus pairs 1 and 2) were analyzed together usinga nested mixed-model ANOVA. To account for the slow change
in pupil size following an event, analysis periods were shifted 250
ms forward in time. Pupil size responses to conditioned stimuli
were analyzed separately within two periods: the CS period, 250–
1,250 ms after conditioned stimulus onset and the trace period,
1,250–1,750 ms after conditioned stimulus onset (Fig. 2A). The
analysis included within-subjects factors of conditioned stimulus,
period (either CS or trace period) and session. A variable foreach individual monkey was included as a random factor in theanalysis, nested below the between-subjects factor, lesion group.
Session was nested below the variable for each monkey.
A mixed-model ANOVA was fitted to the difference in pupil
size between the CS+ and CS–, during the CS period in the lastsession of Pavlovian conditioning and the two sessions of extinction. Within, between, and random effects were modeled in
the same way as in the analysis of the data from the Pavlovian
conditioning task with the addition of session (ACQ, EXT-1,
or EXT-2) as a factor. For the analysis of extinction retention,a mixed-model ANOVA was fitted to the data with factors oflesion, monkey (random effect), cue, set (random effect), andsession (either final five trials of extinction session 1 or first five
trials of extinction session 2). Monkey was nested below lesion.
Highly similar statistical procedures were applied to theunsignaled reward and luminance tasks. A mixed-model ANOVA
was conducted on the period after the unsignaled reward wasdelivered (reward period, 250–1,000 ms after reward delivery) or
luminance analysis period (250–1,250 ms after change in luminance). We also assessed when the pupil first responded tochanges in luminance. To determine if there were any groupdifferences in the latency of responses, time points were compared using a one-way ANOVA.
For the instrumental object–reward task, we analyzed the total
number of omissions scored during the acquisition phase as wellas the number of sessions that it took monkeys to reach criterionusing separate Wilcoxon rank-sum tests for the two replications.
For the extinction phase, the two 30-trial sessions were separated
into six 5-trial blocks, and the data were analyzed using repeatedmeasures ANOVA that included within-subjects factors of session and block and a between-subjects factor of group. For the
analysis of extinction retention, we used methods identical tothose applied to the conditioned autonomic responses. A mixedmodel ANOVA was fitted to the data with factors of lesion(between-subjects effect), monkey (random effect), set (random
effect), and session (either final five trials of extinction session 1
or first five trials of extinction session 2). Monkey was nested
below lesion.
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1. Mitz AR (2005) A liquid-delivery device that provides precise reward control forneurophysiological and behavioral experiments. J Neurosci Methods 148(1):19–25.
2. Barbas H, Pandya DN (1989) Architecture and intrinsic connections of the prefrontal
cortex in the rhesus monkey. J Comp Neurol 286(3):353–375.

Stimuli
Set 1

CS+

CS-

Set 2

B Control - case 3 (Set 1)% Change in pupil sizeA

3. Vogt BA, Pandya DN, Rosene DL (1987) Cingulate cortex of the rhesus monkey:
I. Cytoarchitecture and thalamic afferents. J Comp Neurol 262(2):256–270.
4. Genovesio A, Mitz AR (2007) MatOFF: A tool for analyzing behaviorally complex
neurophysiological experiments. J Neurosci Methods 165(1):38–48.

CS off

6

Rewarddelivery

4
2
0
-2
-4
CS+
CS-

-6
-8

% Change in pupil size

Subgenual ACC - case 3 (Set 1)
6
CS analysis period

4

Trace analysis
period

2
0
-2
-4
CS+
CS-

-6
-8
0

500

1000

1500

2000

Time from CS Presentation (ms)Fig. S1. Pavlovian-task stimuli and individual case responses. (A) Conditioned stimuli sets used in main Pavlovian and fixation task. (B) Pupillary dilation as
a function of time (percent change, mean ± SEM) from individual monkeys. (Upper) Selected unoperated control monkey. (Lower) Selected monkey witha lesion of subgenual ACC. Data from stimulus set 1. Because the pupil response lags the trial events by ∼250 ms, each analysis period is shifted 250 ms relative
to CS onset and offset (dashed vertical lines).

1
Control
Subgenual ACC% Difference in pupil size during CS period(CS+ minus CS-)

0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
Ext. session 1
final 5 trials

Ext. session 2
first 5 trials

SessionFig. S2. Extinction retention of conditioned autonomic responses. Mean percent difference in pupil size responses during the CS period to the CS+ and CS−
stimuli during the final five trials of extinction session 1 and the first five trials of the extinction session 2. White bars represent the responses of the control
group, and gray bars represent responses of the group with lesions of the subgenual ACC. Symbols represent individual subjects. Error bars show SEM.

Rudebeck et al. www.pnas.org/cgi/content/short/1317695111

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A

Fixation rewards
delivered

Stimulus period (1 s)
Fixation &brighten

Fixation spot on

ITI (4-6 s)

Acquire fix
Fixation &extinguish

Luminance change within fixation
Fixation
only trial

Maintain fixation
Fixation period(4 s)Fixation trial

BC% Change in pupil size

Control case 1

Control group

Return to baseline
luminance
1050-5Brighten
Extinguish

-10
0

500

1000

1500

2000

2500

% Change in pupil size

500

1000

1500

2000

2500

500
1000
1500
2000
Time from change in luminance (ms)2500

Subgenual ACC group

Subgenual ACC case 1
Luminance analysis period
10
5
0
-5
Brighten
Extinguish-100Return to baseline
luminance

10
8
6
4
2
0
-2
-4
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-8
-10
0

2500

500
1000
1500
2000
Time from change in luminance (ms)Luminance analysis period

10
8
6
4
2
0
-2
-4
-6
-8
-10
0

Fig. S3. Pupil size responses to changes in luminance. (A) Trials started with the presentation of a red fixation spot. Monkeys had to hold their gaze within
3.0 dva of the spot for 4 s to receive three drops of water (3 × 0.1 mL). On 50% of trials, the gray vertical mask stimulus was either brightened or extinguishedfor 1 s (luminance period) during either the 4-s fixation period or the ITI. (B) Mean ± SEM time course of percent change in pupil size from an unoperated
control (Upper, blue) and a monkey with a lesion of subgenual ACC (Lower, green) following the brightening (darker colors) or removal (lighter colors) of themask. (C) Mean ± SEM time course of percent change in pupil size for the unoperated control group (Upper, n = 3) and the group of monkeys with lesions ofsubgenual ACC (Lower, n = 3). Group data show combined responses on the luminance task. Shaded regions show SEM.

Control
Subgenual ACC

B 1.0
Probability of response

Probability of response

A 1.0
0.8

0.6

0.4

Control
Subgenual ACC

0.8

0.6

0.4

0.2

0.2

0.0

0.0
Acquisition
final session

Extinction
session 1

SessionExtinction
session 2

Ext. session 1
final 5 trials

Ext. session 2
first 5 trials

SessionFig. S4. Acquisition, extinction, and retention of extinction of object–reward associations. (A) Mean probability of object displacement during the final acquisition and two extinction sessions. During extinction sessions, the reduced probability of a response indicates an increased number of omissions. (B) Mean
probability of object displacement during the final five trials of extinction session 1 and the first five trials of the extinction session 2. In A and B, white bars
represent the performance of the control group and gray bars represent performance of monkeys with lesions of the subgenual ACC. Symbols represent
individual subjects. Error bars show SEM.

Rudebeck et al. www.pnas.org/cgi/content/short/1317695111

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