nogo task in the monkey posterior insular cortex

Neuroscience 143 (2006) 627– 639

NEURONAL RESPONSES TO A DELAYED-RESPONSE DELAYED-REWARD GO/NOGO TASK IN THE MONKEY POSTERIOR INSULAR CORTEX T. ASAHI,a,d T. UWANO,b,e S. EIFUKU,b R. TAMURA,b,e S. ENDO,d T. ONOa,e AND H. NISHIJOc,e*

regions during cognition and behavioral manifestation. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Molecular and Integrative Emotional Neuroscience, Graduate School of Medicine, University of Toyama, Toyama 930-0194, Japan

Key words: insular cortex, neural responses, go/nogo, delayed task, reward, monkey.

b

Integrative Neuroscience, Graduate School of Medicine, University of Toyama, Toyama 930-0194, Japan

c

System Emotional Science, Graduate School of Medicine, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan

A previous neuroanatomical study has suggested that the orbital-insular-temporopolar region is an integrated cortical region that is part of a paralimbic circuit (Mesulam and Mufson, 1982). The insular cortex, especially its posterior part, is in a unique position to receive information of all sensory modalities: gustation, olfaction, audition, somesthesis and vision (Mesulam and Mufson, 1982). The posterior insula has intimate connections to somatosensory (Friedman et al., 1986) and auditory association areas (Hurst, 1959). The gustatory (Ogawa, 1994) and olfactory (Pribriam et al., 1950; Augustine, 1985) inputs terminate in the anterior division of the insula. Intra-insular connections occur predominantly from the anterior to the posterior sectors. The insular cortex also receives visual inputs from the superior temporal sulcus (Mesulam and Mufson, 1982; Seltzer and Pandya, 1991). Consistent with the above anatomical studies, several neurophysiological studies have reported various sensory and complex responses in the primate insular cortex in association with gustatory (Yaxley et al., 1990; Ogawa, 1994), somatosensory (Shneider et al., 1993), cardiovascular (Zhang et al., 1999), and vestibular (Grusser et al., 1990) functions. Recent noninvasive imaging studies in humans have demonstrated a role of the insular cortex in complex sensory-motor functions; the insular cortex was implicated in visceral sensory (gustatory, esophageal movement), somatosensory (tactile, pain), autonomic (vomiting, cardiovascular function), motor association, and vestibular functions (Augustine, 1996). The insular cortex also has intimate anatomical connections with the brain regions implicated in higher brain functions (emotion, cognition, memory, attention, etc.), such as the amygdala (Mufson et al., 1981; Amaral and Price, 1984; Friedman et al., 1986), entorhinal cortex (Insausti et al., 1987), striatum (Chikama et al., 1997), temporopolar (Mesulam and Mufson, 1982; Markowitsch et al., 1985), orbitofrontal (Mesulam and Mufson, 1982; Morecraft et al., 1992), cingulate (Mesulam and Mufson, 1982), and perirhinal and parahippocampal cortices (Suzuki and Amaral, 1994). In addition, a posterior part of the insular cortex receives afferent inputs from the cortices related to reward expectancy, e.g. the orbitofrontal cortex (Tremblay

d

Department of Neurosurgery, Graduate School of Medicine, University of Toyama, Toyama 930-0194, Japan

e

Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan

Abstract—Anatomical connections of the insular cortex suggest its involvement in cognition, emotion, memory, and behavioral manifestation. However, there have been few neurophysiological studies on the insular cortex in primates, in relation to such higher cognitive functions. In the present study, neural activity was recorded from the monkey insular cortex during performance of a delayedresponse delayed-reward go/nogo task. In this task, visual stimuli indicating go or nogo responses associated with reward (reward trials) and with no reward (no-reward trials) were presented after eye fixation. In the reward trials, the monkey was required to release a button during presentation of the 2nd visual stimuli after a delay period (delay 1). Then, a juice reward was delivered after another delay (delay 2). The results indicated that the neurons responding in each epoch of the task were topographically localized within the insular cortex, consistent with the previous anatomical studies indicating topographical distributions of afferent inputs from other subcortical and cortical sensory areas. Furthermore, some insular neurons 1) nonspecifically responded to the visual cues and during fixation; 2) responded to the visual cues predicting reward and during the delay period before reward delivery; 3) responded differentially in go/nogo trials during the delay 2; and 4) responded around button manipulation. The observed patterns of insular-neuron responses and the correspondence of their topographical localization to those in previous anatomical studies suggest that the insular cortex is involved in attention- and reward-related functions and might monitor and integrate activities of other brain *Correspondence to: H. Nishijo, System Emotional Science, Graduate School of Medicine, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan. Tel: ⫹81-76-434-7215; fax: ⫹81-76-434-5012. E-mail address: [email protected] (H. Nishijo). Abbreviations: BP, button press; BR, button release; CCD, chargecoupled device; DR-DRW, delayed-response delayed-reward go/ nogo; D1, first delay; D2, second delay; EMGs, electromyograms; FIX, fixation period; GNG-D, go/nogo differential; Ia, agranular insula; Id, dysgranular insula; Ig, granular insula; ITI, inter-trial interval; N-D, non-differential; RNR-D, reward/no-reward differential; RW, reward period; SB, start beep; S1, first visual stimulation; S2, second visual stimulation; S2E, end of the second visual stimulation.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.08.008

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Fig. 1. Time sequences of the three behavioral trials (A, go-reward; B, nogo-reward; C, no-reward) in the DR-DRW task. After the FIX period, one of the three visual stimuli indicating a go-reward, nogo-reward or no-reward appeared at the center of the monitor. (A) A green cue indicated a go-reward trial. The monkey could acquire a reward of fruit juice by releasing a button while the S2 (a blue cue) was on. The S2 cue disappeared when the monkey released the button. (B) A gray cue indicated a nogo-reward trial. The monkey could acquire a reward of fruit juice by holding the button down until the end of the S2. (C) A yellow cue indicated a no-reward trial. The monkey could not acquire a juice reward in these trials. S, visual stimulation; D, delay.

and Shultz, 1999), prefrontal cortex (Ono et al., 1984; Watanabe, 1996), cingulate (Shidara and Richmond, 2002), caudate (Lauwereyns et al., 2002), and amygdala (Nishijo et al., 1987, 1988a,b). Noninvasive human studies have demonstrated the relation of the insular cortex to visual attention (Corbetta et al., 1991) and working memory (Paulesu et al., 1993), and reported that activity of the insula increased in response to both reward and penalty during the performance of a gambling task with financial rewards and penalties (Elliott et al., 2000). Taken together, these anatomical, neurophysiological, and noninvasive human studies suggest that the insula is a limbic integration cortex (Augustine, 1996) for complex and preprocessed sensory information. However, there have been few unit recording studies in the insular cortex of primates, particularly in relation to higher cognitive functions. Furthermore, functional heterogeneity in the insular cortex is not well known. In the present study, single-unit activity was recorded from a posterior part of the insula of monkeys during a delayedresponse delayed-reward go/nogo (DR-DRW) task in order to analyze the insular functions.

EXPERIMENTAL PROCEDURES Animals and experimental apparatus Two adult monkeys (Macaca fuscata), weighing 4.6 and 5.9 kg, were used. During the training and recording sessions, the monkey sat in a primate chair facing an apparatus for behavioral tasks. The primate chair was equipped with a responding button, which was positioned so that the monkey could easily manipulate it. An infrared charge-coupled device (CCD) camera for eye-movement

monitoring was firmly attached to the chair by a steel rod. The monkey’s eye position was calculated with 33-ms time resolution by an eye-monitor system (EM100; Toyo Sangyo, Toyama, Japan). Within the behavioral-task apparatus, a 19-inch CRT monitor was set so that its center was on the same horizontal plane as the monkey’s eyes. Liquid was accessible to the monkey through a small spout controlled by an electromagnetic valve. A Psyscope system (Carnegie Mellon University, Pittsburgh, PA, USA) received the eye position data from the eye-monitor system and the state signal (on/off) of the responding button, and controlled the timing for outputs to the CRT monitor, the electromagnetic valve and sound signal.

Behavioral paradigms (Fig. 1) A trial was initiated by a start beep (SB) for 500 ms. When the monkey pushed a button (BP) and held it pushed, a fixation point (0.13°⫻0.13°) appeared at the center of the monitor. The monkey was required to watch the fixation point for 1.5 s (fixation period, FIX) while the button was held down. Then, a first visual stimulus (S1) appeared for 1.0 s. Go trial with reward. A green cue (1.5°⫻1.5°) appeared in the S1 phase, followed by a first delay (D1) of 1.0 –1.5 s. Then, a blue cue (second visual stimulus, S2) was presented in the S2 phase for at most 1.0 s. When the monkey released the button (BR) during the S2, the blue cue disappeared and juice was given as a reward (reward period, RW) for 1.0 s after a second delay (D2) of 1.5 s. Nogo trial with reward. A gray cue appeared in the S1 phase. When the monkey held the button down during the S2, juice was given in the same manner as in the go trials after a delay (D2) of 1.5 s. No-reward trial. A yellow cue appeared in the S1 phase. Regardless of the monkey’s behavioral responses (go or nogo), no reward was given.

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Fig. 2. Recording area in the insular cortex shown in the 3-D MRI of the monkey brain. Top (A) and lateral (B) views of the monkey brain. The blue-colored areas in A and red-colored area in B indicate the right insular cortex. (C) The area within the white square in A is shown in an expanded scale. Stereotaxic coordinates of the insula were determined in reference to a marker (a tungsten marker was inserted 18 mm anterior from the interaural line, and 20 mm from the midline in this case) in the insular cortex in the 3-D MRI. Pixel size, 1⫻1 mm. Red filled star, position of the tungsten marker.

When the monkey failed to choose correct behavioral responses (either holding down or releasing the button) during the S2 phase in reward trial, or released the button before the S2 phase in both reward and no-reward trials, the trials were aborted. In these cases, the same trial was repeated until it was completed successfully. Inter-trial intervals (ITI) were 5–10 s.

Training and surgery The monkey was trained with the DR-DRW task for 3 h/day, 5 days/week. The monkey required about 6 months of training to reach a 90% correct-response rate. After completion of this training period, a head-restraining device (a U-shaped plate made of epoxy resin) was attached to the skull under aseptic conditions and sodium pentobarbital anesthesia (35 mg/kg, i.m.). The plate was anchored with dental acrylic to titanium bolts inserted in keyhole slots in the skull. During the surgery, heart and respiratory functions and rectal temperature were monitored on a polygraph system (Nihon Kohden, Tokyo, Japan). The rectal temperature was controlled at 37⫾0.5 °C by a blanket heater. Antibiotics were administrated topically and systemically for 1 week to protect against infection. Two weeks after surgery, the monkey was retrained. The performance criterion was again attained within 10 days. All experimental protocols were approved by the Animal Care and Use Committee of University of Toyama and conformed to NIH guidelines on the humane care and use of laboratory animals. Every effort was made to minimize the number of animals used and their suffering.

Stereotaxic localization of the insular cortex for recording Before recording, a marker consisting of a tungsten wire (diam., 500 ␮m) was inserted nearby the target area (the right insular cortex) under anesthesia, and the 3-D MRI scans of the monkey head were performed. The 3-D pictures of the monkey brain with the marker were reconstructed by computer rendering using software for image guided neurosurgical navigation system (Evans; Tomiki Medical Instruments, Kanazawa, Japan) (Fig. 2). The 3-D stereotaxic coordinates of the target area were determined in reference to the marker in the 3-D reconstructed brain (Asahi et al., 2003). After the last recording session, several small marking lesions were made in the right insular cortex by passing 20 –30 ␮A of anodal current for 30 s through an electrode placed stereotaxically and monitored by X-ray. Subsequently the monkey was deeply anesthetized with an overdose of sodium pentobarbital (50 mg/kg, i.m.) and perfused transcardially with 0.9% saline followed by 10% buffered formalin. The brains were removed from the skulls and cut into 50 ␮m sections through the right insular cortex. Sections were stained with Cresyl Violet, and three cytoarchitectonic areas of the insula were identified (Mesulam and Mufson, 1982; Chikama et al., 1997); granular insula (Ig), dysgranular insula (Id), and agranular insula (Ia). The sites of electrical lesions were determined microscopically. The location of each recording site was then calculated by comparing the stereotaxic coordinates of recording sites with those of lesions.

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Recording procedures and data acquisition A glass-insulated tungsten microelectrode (0.8 –1.2 M⍀ at 1 kHz) was stereotaxically inserted into the recording area on a plane vertical to the orbito-meatal plane in a stepwise fashion by a pulse motor-driven manipulator (SM-21; Narishige, Tokyo, Japan). Extracellular signals were passed through a high-input impedance preamplifier, amplified by a main amplifier (DPA-2016; DiaMedical System, Tokyo, Japan), monitored on an oscilloscope, and recorded on videotape by a data recorder (RX-8000; TEAC, Tokyo, Japan). The outputs from the amplifier were digitized and sent to a microcomputer. The software (WorkBench; DataWave Technologies, Longmont, CO, USA) collected an epoch of the digitized analog signals for every event that exceeded a user-set threshold. During recording, eye movements were also monitored by the eye monitor system using an infrared CCD camera. When the monkey failed to maintain eye fixation within ⫾2° from the center of the monitor during the FIX, S1, and S2 periods, the trial was aborted. Electromyograms (EMGs) were recorded from the masseter muscle during the task. If the monkey exhibited signs of fatigue, such as closing its eyes for several seconds or moving its eyes or hand slowly, the experimental session was stopped immediately. In most cases, the unit recording experiment was terminated within 3– 4 h.

Data analysis For purposes of statistical analysis, task phases were re-defined and the tasks were divided into the following nine phases: control (for 1.0 s before the SB), SB (for 0.5 s during presentation of the SB), BP (for 0.4 s from 0.2 s before to 0.2 s after the button press, BP), FIX (for 1.5 s from eye fixation to the onset of the S1), S1 (for 1.0 s during presentation of the S1), D1 (for 1.0 –1.5 s between the first and S2), S2 (for 0.8 s utmost from the onset of the S2, i.e. the S2 period minus the last 0.2 s), end of the second visual stimulation (S2E) (for 0.4 s from 0.2 s before to 0.2 s after the end of the S2), D2 (for 1.3 s from 0.2–1.5 s after the end of the S2, i.e. the D2 period minus the last 0.2 s), and RW (for 1.0 s from 1.5–2.5 s after the end of the S2). The RW in the no-reward trials was defined in the same way as in the reward trials, no reward was delivered in no-reward trials tough. In the following description, definition of the task phases refers to these re-defined phases noted above for statistical analysis. The three types of trials (go-reward, nogoreward, and no-reward) were randomly performed, and data in each phase were accumulated separately for each type of trial for statistical analyses. First, significant excitatory or inhibitory responses in each phase were determined for each kind of trial; the mean firing rate in each phase for a particular trial type was compared with the mean firing rate in the control phase for that trial type by Dunnett’s test (P⬍0.05) after one-way ANOVA across the nine phases for that trial type (P⬍0.05) (ANOVA-I). Second, the insular neurons with significant excitatory or inhibitory responses were functionally categorized into three neuronal types based on the responses from the S1 to the RW in which behavior-relevant stimuli (i.e.

go-reward, nogo-reward, no-reward, and juice reward) were presented (see the Results section for details). For this categorization, neuronal responses in each phase were compared among the three kinds of trials (go-reward, nogo-reward, and no-reward) by a post hoc test (Neuman-Keuls test, P⬍0.05) after one-way ANOVA across the three kinds of trials (P⬍0.05) (ANOVA-II). Third, the insular neurons were also categorized based on responsiveness in the BP and S2E before, during and after button manipulation (i.e. ‘around’ button manipulation). These neuronal types (BP- and BR-responsive neurons) were defined by both ANOVA-I and ANOVA-II (see Results for details). Most neurons were recorded from the Ig and Id, and only one neuron was recorded from the Ia (see Results). Therefore, ratios for each type of neurons noted above were compared between the Ig and Id by Fisher’s exact probability test (P⬍0.05).

RESULTS General In almost all recording sessions, the monkeys performed the operant task more than 90% correctly. The activity of 519 neurons was recorded from the middle to posterior part of the right insular cortex in two monkeys while they performed the DR-DRW task. Of these 519 neurons recorded from the insular cortex, 117 (22.5%) responded in one or some phases of the task. The number of neurons showing a significant response in each period is shown in Table 1. Most neurons responded in the BP, FIX, S2E, and D2 phases. Excitatory responses were especially dominant in the SB, BP, FIX, D1, S2E and D2 phases. Responses in the SB phase were all excitatory. Fig. 3 shows representative records of masseter muscle EMGs and eye movements during the task. The EMG results indicated slight mouth movements during the D2 period followed by stronger mouth movements in the RW (Fig. 3A). The task required the monkeys to fixate the center of the monitor from the beginning of the FIX period to the end of the S2 period except the D1 period (Fig. 3B). Although eye movements were allowed in the D1, only slight eye movements were observed in this phase. In the D2 and RW periods, larger eye movements were observed in some trials. Activity of two neurons was correlated to saccades, and these neurons were excluded from the responsive neurons since they did not display significant responses during the task (data not shown). Activity of the remaining neurons was not correlated to eye movements.

Table 1. Number of responsive neurons in each phase of the DR-DRW task among the 117 responsive neurons Response

Excitation Inhibition Subtotal No response Total

Phases of the DR-DRW task SB

BP

FIX

S1

D1

S2

S2E

D2

RW

9 0 9 108 117

24 5 29 88 117

20 12 32 85 117

12 11 23 94 117

12 5 17 100 117

8 6 14 103 117

34 2 36 81 117

20 8 28 89 117

11 6 17 100 117

Note that the 117 neurons responded during some of the task phases but not all the phases.

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Fig. 3. Representative records of EMGs recorded from the masseter muscle and eye movements during the task. (A) EMGs recorded from the masseter muscle. (B) Traces of eye movements in the horizontal (a) and vertical (b) directions. Note that the eye position was fixed at the center of the monitor in the FIX, S1 and S2, and that EMG activity occurred in the D2 and RW. D, dorsal; L, left; R, right; V, ventral. Other acronyms are as defined in Fig. 1.

Neuronal classification Insular neurons were classified into the following three types based on the responses occurring between the S1 and the RW: non-differential (N-D), reward/no-reward differential (RNR-D), and go/nogo differential (GNG-D) neurons (Table 2) (see below for details). The insular neurons were also classified into the following two groups based on their responsiveness around button manipulation: BP- and BR-responsive neurons (Table 3) (see below for details). N-D neurons. The N-D neurons were defined as those that significantly responded in a given phase of the task in all three kinds of trials (go-reward, nogo-reward, and no-reward) compared with the mean firing rates in the control phase (Dunnett’s test after ANOVA-I, P⬍0.05), but displayed no significant differences in neuronal responses during that task-phase among the three kinds of trials (ANOVA-II, P⬎0.05). Of 117 responsive neurons, 23 (19.7%) were N-D neurons (Table 2). Most responses of Table 2. Number of N-D, RNR-D, and GNG-D neurons in each phase Response

N-D RNR-D only GNG-D only RNR-D and GNG-D Subtotal Other responses Total

Phases of the DR-DRW task S1

D1

S2

D2

RW

Total

17 4 2 0 23 94 117

7 9 1 0 17 100 117

5 7 2 0 14 103 117

2 14 10 2 28 89 117

0 13 4 0 17 100 117

23 38 17 2 80 37 117

RNR-D only, the neurons satisfying the criteria for the RNR-D neurons, but not those for the GNG-D neurons; GNG-D only, the neurons satisfying the criteria for the GNG-D neurons, but not those for the RNR-D neurons; RNR-D and GNG-D, the neurons satisfying both the RNR-D and GNG-D criteria. Note that simple addition of the number of the neurons responding in each phase (S1⫹D1⫹S2⫹D2⫹RW) makes the number exceeding a real total number of the neurons since some neurons responded in multiple phases.

this type of neuron were found in the S1 period. Fig. 4 shows an example of an N-D neuron. This neuron responded during the FIX, S1, and S2 phases irrespective of the type of trial [i.e. go (Aa), nogo (Ab), reward (A), and no-reward (B) trials]. RNR-D neurons. The RNR-D neurons were defined as those that showed significant responses in a given phase of go-reward and nogo-reward trials compared with the mean firing rate in the control phase (Dunnett’s test after ANOVA-I, P⬍0.05), and that showed significant differences in neuronal responses in that phase among the three kinds of trials (ANOVA-II, P⬍0.05). Furthermore, these neurons displayed significant differences in neuronal responses in that phase between go-reward and no-reward trials (Newman-Keuls test after ANOVA-II, P⬍0.05), and significant differences in neuronal responses in that phase between nogo-reward and no-reward trials (Newman-Keuls test after ANOVA-II, P⬍0.05). Of the 117 responsive neurons, 40 (34.2%) were RNR-D (RNR-D only plus RNR-D and GNG-D) neurons (Table 2). Sixty percent (24/40) of the RNR-D neurons displayed significant responses in the D1 and/or D2 phases. Fig. 5 depicts a typical response of an RNR-D neuron. The neuron responded in reward trials (go-reward and nogo-reward trials) (A). The activity gradually inTable 3. Number of each type neurons related to button manipulation Response

Excitation Inhibition Total

Neuron type BP only

BR only

BP and BR

Total

9 3 12

13 1 14

11 1 12

33 5 38

BP only, the neurons satisfying the criteria for the BP-responsive neurons, but not those for the BR-responsive neurons; BR only, the neurons satisfying the criteria for the BR-responsive neurons, but not those for the BP-responsive neurons; BP and BR, the neurons satisfying the both criteria for the BP- and BR-responsive neurons.

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Fig. 4. Responses of an N-D neuron. (A) Raster displays of neuronal activity and summed peri-event histograms in the reward trials with go (a) and no-go (b) responses. (B) Raster displays of neuronal activity and summed peri-event histograms in the no-reward trials. Note that neuronal activity increased in the FIX, S1 and S2 phases in all three types of trials. Bars under the raster displays indicate the duration of the S1, S2, and RW phases. Zero in the time scale indicates the moment when the monkey fixed its eyes at the center of the monitor. Bin width, 100 ms. Other acronyms are as described in Fig. 1.

creased toward the RW phase, and decreased after acquisition of reward. However, there were no changes in activity during the no-reward trials (B). Of the 16 RNR-D neurons that responded in the D2 (D2-responsive RNR-D neurons), 13 did not respond in the RW, in which larger mouth movement occurred due to ingestion of juice. The remaining three neurons responded during both D2 and RW. This suggests that the responses of these 13 D2responsive RNR-D neurons were not related to mouth movements. Furthermore, there were no correlations between neuronal and EMG activity in these D2-responsive RNR-D neurons. These results indicate that the activity of at least 27 RNR-D neurons—i.e. all RNR-D neurons except the 13 that responded during the RW (three showed responses in the D2 and RW, and 10 responded only during the RW)— changed in relation to reward contingency irrespective of either the go/nogo responses or mouth movements. GNG-D neurons. The GNG-D neurons were defined as those that 1) showed significant responses in a given phase of go-reward or nogo-reward trials compared with the mean firing rate in the control phase (Dunnett’s test after ANOVA-I, P⬍0.05); 2) showed significant differences in neuronal response during that task-phase among the three kinds of trials (ANOVA-II, P⬍0.05); and 3) showed significant differences in neuronal responses during that

task-phase between go-reward and nogo-reward trials (Newman-Keuls test, P⬍0.05). Of 117 responsive neurons, 19 (16.2%) were GNG-D (GNG-D only and RNR-D and GNG-D) neurons (Table 2). Most GNG-D neurons (12/19, 63.2%) responded in the D2 phase. Fig. 6 shows an example of a GNG-D neuron. The neuron responded in the D2 phase in go-reward trials (Aa), but not in nogo-reward trials (Ab). However, although the monkey displayed both go and nogo behavioral responses in the no-reward trials, there were no significant differences in neuronal responses in the D2 phase between the go and nogo trials (B). The remaining GNG-D neurons also displayed no significant differences in neuronal responses between the go and nogo trials with no reward. Furthermore, the activity of these neurons did not change when the monkey touched the button during the ITI phase. These results indicated that there were no direct relations between the activity of GNG-D neurons and motor movements, and that the activity of the neurons reflected go/ nogo behaviors in a specific situation (i.e. reward trials). BP- and BR-responsive neurons. The neurons whose activities changed around button manipulation (BP and/or BR) were identified. The BP-responsive neurons were defined as those that showed significant responses in the BP compared with the control phase in each type of trial (Dunnett’s test after ANOVA-I, P⬍0.05), and that dis-

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Fig. 5. Responses of an RNR-D neuron. (A) Raster displays of neuronal activity and summed peri-event histograms in go-reward (a) and nogo-reward (b) trials. (B) Raster displays of neuronal activity and summed peri-event histograms in no-reward trials. Note that neuronal activity gradually increased before the RW only in the reward trials (A), but not in the no-reward trials (B). Zero in the time scale indicates the offset of the S2. Other acronyms are as defined in Fig. 1.

played significant differences between neuronal responses in the SB and BP phases in each type of trial (NeumanKeuls test after ANOVA-I, P⬍0.05). The BR-responsive neurons were defined as those that showed significant responses in the S2E phase compared with the control phase in go-reward and/or nogo-reward trials (Dunnett’s test after ANOVA-I, P⬍0.05), and significant differences in neuronal responses in the S2E phase between go-reward and nogo-reward trials (Neuman-Keuls test after one-way ANOVA-II, P⬍0.05). The responses of BP- and BR-responsive neurons are summarized in Table 3. Twelve and 14 neurons were specifically identified as BP- (BP only in Table 3) and BR-responsive (BR only in Table 3), respectively. Twelve neurons were identified as both BP- and BR-responsive (BP and BR in Table 3). A typical neuron identified as both BP- and BR-responsive is shown in Fig. 7. The activity of the neuron increased around the BP in both go-reward (Aa) and nogo-reward (Ab) trials, and also increased around the end of the S2 phase in go-reward trials (i.e. BR) (Ba), but not in nogo-reward trials (Bb). It is worth noting that, in 47.4% (18/38) of the neurons in this category, neuronal responses in the latter half of the BP and/or BR phase after button manipulation were significantly larger than those in the former half before button manipulation (paired t-test, P⬍0.05), and only in 10.5% (4/38) of the neurons, neuronal responses in the former half of the phase before button manipulation were significantly larger

than those in the latter half (paired t-test, P⬍0.05). Furthermore, in 76.3% (29/38) of the neurons in this category, the activity peaked after button manipulation. Location of neuronal types Most neurons except one were found in the Ig and Id; of the 519 insular neurons, 88, 430, and one were recorded from the Ig, Id, and Ia, respectively. Of these 519 insular neurons, 117 (22.5%) responded in one or some phases of the task. Of these responsive neurons, 14, 102, and one were located in the Ig, Id, and Ia, respectively. Fig. 8A and B shows the distributions of neurons that responded during the SB and FIX phases, respectively. The FIX-responsive neurons were significantly more clustered in an anterior part of the recorded area (Id) (Fisher’s exact probability test, P⬍0.05); of the 430 neurons recorded from the Id, 32 responded to FIX, whereas, of 88 neurons recorded from the Ig, no neurons responded to FIX (B). The SB-responsive neurons were located in a ventral part of the Ig and Id (A). Fig. 9 shows the distributions of the three neuronal types (N-D, RNR-D, and GNG-D neurons) according to their response during the S1 (A), D1 (B), S2 (C), and D2 (D) phases, respectively. In the individual phases from the S1 to D2, there were no significant differences between the ratios of the RNR-D neurons in the Ig and in the Id (Fisher’s exact probability test, P⬎0.05). When the data of the all

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Fig. 6. Responses of a GNG-D neuron. (A) Raster displays of neuronal activity and summed peri-event histograms for go-reward (a) and nogo-reward (b) trials. Note that neuronal activity increased in the D2 only in the go-reward trials (a). (B) Raster displays of neuronal activity and summed peri-event histograms in the no-reward trials. In B, trials with go and nogo responses were re-arranged and shown in order. Zero in the time scale indicates the offset of the S2. Other acronyms are as defined in Fig. 1.

phases were pooled together, ratios of the RNR-D neurons out of the total recorded neurons were larger in the Id than the Ig (Fisher’s exact probability test, P⬍0.05). Similarly, ratios of the N-D neurons were larger in the Id than the Ig (Fisher’s exact probability test, P⬍0.05). However, there

were no differences in ratios of the GNG-D neurons between the Ig and Id (Fisher’s exact probability test, P⬎0.05). The RNR-D neurons that responded in the RW were only found in the Id (Fig. 10A). The GNG-D neurons that

Fig. 7. Responses of a BP- and BR-responsive neuron. (A) Raster displays of neuronal activity and summed peri-event histograms in go-reward (a) and nogo-reward (b) trials aligned with the BP. Note that neuronal activity increased around the BP in both go and nogo trials. Zero in the time scale indicates the moment when the monkey pressed the button. (B) Raster displays of neuronal activity and summed peri-event histograms in go-reward (a) and nogo-reward (b) trials aligned with the end of the S2. Note that neuronal activity also increased around the BR (the end of the S2 in go-reward trials) (a). Zero in the time scale indicates the moment when the S2 disappeared (BR in go-reward trials). Other acronyms are as defined in Fig. 1.

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Fig. 8. Distributions of the neurons that responded in the SB (A) and FIX (B) phases. (A) Neurons that responded in the SB phase (open diamonds) were sparsely located in a ventral posterior part of the recorded area. (B) Neurons that responded in the FIX phase (upright triangles) were located in an anterior part of the recorded area. Dots, non-responsive (NR) neurons in the SB (A) and FIX (B) phases. The white square shown in the inset of the 3-D MRI indicates a posterior part of the insula (red-colored area) corresponding to those in A and B.

responded in the RW were located in both the Id and Ig with no regional difference (Fisher’s exact probability test,

P⬎0.05) (Fig. 10A). Neurons that responded around button manipulation were widely distributed over both re-

Fig. 9. Distributions of the N-D, RNR-D, and GNG-D neurons that responded in the S1 (A), D1 (B), S2 (C), and D2 (D). (A) Both N-D (upright triangles) and RNR-D (open circles) neurons were located in an anterior part of the recorded area. (B), (C) and (D) N-D and RNR-D neurons that responded in the D1 and S2 phases were located in an anterior part of the recorded area (B, C), while distributions of the RNR-D neurons that responded in the D2 phase included a more posterior part of the recorded area (D). Location of the recorded area is shown in the inset of the 3D-MRI in the Fig. 8. Other acronyms are as defined in Fig. 8.

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Fig. 10. Distributions of the RNR-D, and GNG-D neurons that responded in the RW phase (A), and those of the BP- and/or BR-responsive neurons (B). (A) RNR-D (open circles) and GNG-D (open squares) neurons that responded in the RW period were located in an anterior part of the recorded area. (B) BP and/or BR neurons (inverted triangles) were widely distributed throughout the recorded area. Location of the recorded area is shown in the inset of the 3D-MRI in the Fig. 8. Other acronyms are as defined in Fig. 8.

corded areas (Ig, Id) with no regional difference (Fisher’s exact probability test, P⬎0.05) (Fig. 10B).

DISCUSSION Neurons that responded in the FIX and N-D neurons Many neurons [27.4% (32/117) of the responsive neurons] responded during the FIX phase, including 65.2% (15/23) of the N-D neurons. The neurons shown in Fig. 4 responded in the FIX phase, and also responded nonspecifically to all visual stimuli in the S1 and S2 phases. This type of neuron might be related to attention to the visual stimuli presented in the task. The responses of the neurons were suppressed by saccade or when the monkey looked outside the monitor (data not shown). These characteristics of the N-D neurons were comparable to those of the visual fixation neurons in the inferior parietal lobule (Lynch et al., 1977; Duhamel et al., 1992) that had reciprocal connections to the insula (Mesulam and Mufson, 1982; Cavada and Goldman-Rakic, 1989). Furthermore, the Ig and Id receives strong afferent inputs from the pulvinar nucleus of the thalamus (Mufson and Mesulam, 1984; Friedman and Murray, 1986), which plays an important role in attention (Robinson and Petersen, 1992). Human PET studies have reported insular activation during selective visual attention (Corbetta et al., 1991) and spatial attention (Perry and Zeki, 2000). Neglect, a symptom for which attentional deficit is the underlying cause (Mesulam, 1981), has previously been reported in patients with right insular cortex damage (Berthier et al., 1987; Maeshima et al., 1997; Manes et al., 1999). Further studies will be needed to elucidate the role of the N-D neurons in attention. RNR-D neurons The RNR-D neurons differentially responded in the reward or no-reward trials, irrespective of arm movements (i.e.

go/nogo responses). The RNR-D responses in the D2 period were not ascribed to preparatory mouth movements, since all but three of these neurons failed to respond in the RW phase, in which large mouth movements were observed due to ingestion of a juice reward (Fig. 6). The insula has intimate anatomical connections with the limbic cortices, and especially with the following brain regions implicated in reward expectation. The orbitofrontal cortical neurons have been reported to respond before reward delivery and to change their responses depending on reward value (Tremblay and Schultz, 1999). The area 11 in the orbitofrontal cortex, where these neurons were most frequently found, has connections to the posterior insula (Morecraft et al., 1992). Nishijo et al. (1987) found neurons that responded during a delay period in the reward task, and showed that neuronal activity gradually increased before reward delivery in the amygdala. These reward-related neurons were located in the basolateral amygdala, which receives inputs from the Id and Ig (Friedman et al., 1986). Shidara and Richmond (2002) reported neurons related to reward expectancy in the anterior cingulate cortex, where the Id and Ig project (Vogt and Pandya, 1987; Chikama et al., 1997). The anterior cingulate cortex and insula project to the caudate nucleus (Baleydier and Mauguiere, 1980), which has also been implicated in reward expectation (Lauwereyns et al., 2002; Shidara and Richmond, 2002). Furthermore, the insula was implicated in cocaine-induced euphoria (Breiter et al., 1997) and cocaine craving (Wang et al., 1999). Recent human fMRI studies also indicated activation of the insular cortex with anticipation of primary reward (O’Doherty et al., 2002), and a heterogeneous organization within the insula regarding reward anticipation (Tanaka et al., 2004); ventroanterior and dorsoposterior regions were involved in predicting immediate and predicting future rewards, respectively. Thus, the insular cortex and these brain regions form neural circuits for reward value or reward expectancy. The

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present results, therefore, provide neurophysiological evidence that the insular cortex, which is one of the nodal areas in this reward circuit, is involved in reward-related functions. Wilson et al. (1993) suggested the presence of two visual pathways to the prefrontal cortex, i.e. a spatial (dorsolateral) and an object (ventrolateral) vision pathway from the parietal and inferotemporal cortices, respectively. Schultz et al. (2000) suggested a third pathway from the medial temporal lobe, which transmits motivational information to the orbitofrontal cortex. Mesulam (2000) suggested that the insula is one of the major components of the limbic system in the insula–amygdala– orbitofrontal and insula–amygdala– cingulate triangle circuits. The present results expand the idea by Schultz et al. (2000); these triangular circuits including the insular cortex relay motivational information to the prefrontal cortex. We found 13 (11.1%, 13/117) neurons which differentially responded in the RW period. Of these, 10 neurons responded only during ingestion of the juice reward. These neurons were located in a dorsal part of the Id. These results are consistent with previous studies in which gustatory responses were reported in the dorsal Id which receives gustatory inputs from the thalamus (Pritchard et al., 1986; Yaxley et al., 1990).

extremities. The neurons that responded differentially around the BP or BR (BP only and BR only in Table 3) might be related to arm movements. However, it is noted that the activity of these neurons peaked after the actual movements in the present study. Similar responses have been reported in the posterior cingulate motor area (Shima et al., 1991) that projects to the insula (Mesulam and Mufson, 1982) and has been suggested to be involved in encoding of motor efferent copies. The D2-responsive GNG-D neurons responded differentially in go/nogo-reward trials. However, there were no differential responses in no-reward trials, although the monkey displayed both go and nogo responses. Furthermore, it is noted that the activity of the neurons reflected the behavioral responses (go/nogo responses) that had occurred in the preceding phase, but not in the following phase. This suggests that activity of the neurons was not related to execution of actual behavioral responses. It has been reported that the prefrontal cortex, which projects to the insular cortex (Augustine, 1996), was essential in executive functions (Yamatani et al., 1990; Goldman-Rakic, 1995; Fuster, 2000a,b; Lauwereyns et al., 2001). The D2responsive GNG-D neurons might be involved in encoding of efferent copies of go/nogo commands to acquire the juice reward.

Behavior-related neurons (BP-, BR-responsive, GNG-D)

Neurons that responded in the SB

Of the 117 responsive neurons, 12 (10.3%, 12/117) were identified as both BP- and BR-responsive (BP and BR in Table 3). These neurons were distributed widely in the recorded area. The Ig and Id are suggested to be a part of cortico-limbic pathways that subserve tactile learning and memory (Friedman et al., 1986). A neurophysiological study has indicated that a major portion of the Ig is exclusively involved in the somatic processing area, where neurons show large and often bilateral receptive fields (Robinson and Burton, 1980; Schneider et al., 1993). These neurons (BP and BR in Table 3) might respond to somatosensory inputs since they responded nondifferentially during different motor movements (i.e. button pressing and releasing). On the other hand, electric stimulation of the insula induces motor responses, including those of the upper extremities (Showers and Lauer, 1961). Neuroanatomical studies in monkeys have indicated that the insular cortex has connections to the motor association areas (Emmans et al., 1988; Luppino et al., 1993). Frontal medial area 6 consists of two distinct cytoarchitectonic areas: area F3, the supplementary motor area, and area F6, the presupplementary motor area. Area F3 receives afferent inputs from the Ig, whereas the area F6 receives them from the Id (Luppino et al., 1993). PET brain imaging studies have reported that finger opposition and shoulder flexion activated the ipsilateral insula (Colebatch et al., 1991), and that, when patients with stroke moved the fingers of the recovered hand, activity increased in the bilateral insula. These anatomical and imaging studies suggest that the insula is strongly related to motor functions of the upper

The 7.7% (9/117) neurons responded in the SB phase in the present study. All these neurons also responded to various environmental sounds. They were located in a ventral part of the insular cortex that was adjacent to the auditory cortices and that received auditory inputs (Burton and Jones, 1976; Mesulam and Mufson, 1982). The percentage of the SB-responsive neurons was relatively small, consistent with a previous report in which burst noise, clicks, whistles, etc. were presented in monkeys (Schneider et al., 1993). However, Bieser (1998) reported that 22.6% of insular neurons responded to a squirrel monkey’s twitter-call. These results indicated that the insular neurons preferentially responded to complex sounds such as species-specific calls, and suggest that the insula is involved in species-specific rather than simple auditory processing. Functional consideration of the insula cortex There have been few previous reports of functional heterogeneity in the insular cortex. In the present study, the neurons responding in the SB phase were located in a ventral part of the recorded areas (Ig, Id). The neurons that responded in the FIX were clustered in an anterior part of the recorded area (Id). The RNR-D neurons that responded in the RW were located in a dorsal part of the Id. The neurons associated with BP and/or BR were widely distributed across the recorded areas. The distributions of these neurons were largely consistent with those of afferent inputs from other cortical and subcortical areas (see discussion above). Furthermore, the activity of the GNG-D neurons might reflect go/nogo commands from the prefrontal cortex. These results suggest that the activity of

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most insular neurons reflects that of the areas projecting to the insular cortex. Therefore, a primary function of the insula might be the monitoring of cerebral activity during cognition and behavioral manifestation, and integration of this information. These characteristics of the insula further suggest that the insula is located in a strategic position for memory formation. Consistent with this idea, activity changes in the insula have been reported during recognition memory tasks, retrieval of autobiographical memory, and mental navigation along memorized routes (Grady et al., 1994; Fink et al., 1996; Ghaem et al., 1997; Reed et al., 2004). These studies suggest an involvement of the insula in memory formation and retrieval, which is consistent with the idea that the insula is a limbic integration cortex (Augustine, 1996).

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(Accepted 4 August 2006) (Available online 18 September 2006)