Edited by: Preston E. Garraghty, Indiana University Bloomington, United States
Reviewed by: Natasha Sigala, University of Sussex, United Kingdom; Benjamin J. Clark, The University of New Mexico, United States
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Previous research indicated that monkeys with neonatal perirhinal lesions (Neo-PRh) were impaired on working memory (WM) tasks that generated proactive interference, but performed normally on WM tasks devoid of interference (
A recent study reported that adult monkeys with neonatal lesions of the perirhinal cortex (Neo-PRh) had working memory (WM) impairments that were characterized by a tendency to make perseverative errors on tasks that generated proactive interference (
Resolving proactive interference requires suppressing behavioral responses based on “old” information, and flexibility to shift cognitive resources toward learning/remembering “new” information (
Lesion studies in monkeys have already demonstrated a double-dissociation between behavioral inhibition supported by the orbitofrontal cortex (OFC), and cognitive flexibility supported by the ventrolateral prefrontal cortex (vlPFC) (
The Institutional Animal Care and Use Committee (IACUC) at Emory University in Atlanta, GA, United States, approved all experimental protocols. All guidelines specified in the NIH Guide for the Care and Use of Laboratory Animals (
Eleven adult rhesus macaques, aged 9–16 years, participated in this experiment (7 females, 4 males). Between 7 and 12 days postnatal, 6 monkeys received bilateral injections of ibotenic acid to the perirhinal cortex (group Neo-PRh; 3 females, 3 males), and 2 received sham surgeries (group Neo-C; 1 female, 1 male). One animal did not undergo any surgical or anesthetic procedures (Neo-UC; 1 female). All of these subjects received the same rearing conditions, which included extensive socialization opportunities with age-matched peers and human caregivers (for detailed description of rearing procedures see
Two additional monkeys received sham operations in adulthood (Adult-C; 1 female; 1 male) and were available to participate in behavioral testing. These Adult-C animals were mother-raised in a large colony of macaques at the Yerkes NPRC Field Station under a semi-naturalistic environment (see
At the time of this experiment, all monkeys were housed individually in rooms with 12-h light/dark cycles (7 AM/7 PM), fed Purina Old World Primate chow (formula 5047) and supplemented with fresh fruit enrichment. During testing, the food ration was given once daily following testing, and adjusted individually to ensure that the animals were motivated to perform on the task and maintained their weight at 85% or above of their free-feeding weight. Water was given
Between 10 and 12 days of age, subjects in groups Neo-PRh and Neo-C underwent surgery to create excitotoxic lesions of the perirhinal cortex using ibotenic acid, or sham operations, respectively. Animals in the Adult-C group were between 6 and 12 years of age at the time of their sham surgeries.
The brain was imaged with a 3T Siemens Magnetom Trio system (Siemens Medical Solutions, Malvern, PA, United States at YNPRC) using a 5 cm surface coil. Both pre-surgery and 1 week post-surgery, two sets of images were obtained: (1) high-resolution structural T1 images [3D T1-weighted fast spoiled gradient (FSPGR)-echo sequence, TE = 2.6 ms, TR = 10.2 ms, 25° flip angle, contiguous 1 mm sections, 12 cm FOV, 256 × 256 matrix]; and (2) Fluid Attenuated Inversion Recovery (FLAIR) images, [TE = 140 ms, TR = 1000 ms, inversion time (TI) = 2200 ms, contiguous 3 mm sections, 12 cm FOV, 256 × 256 matrix; image sequences acquired in three series offset 1 mm posterior]. The T1-weighed images were used to calculate the injection sites pre-surgery and the FLAIR images were used to estimate the extent of PRh damage as well as damage to adjacent structures, as described in the section below.
Throughout the duration of the pre-surgical MRI scans, subjects were sedated (10 mg/kg of 7:3 Ketamine Hydrochloride, 100 mg/ml, and Xylazine, 20 mg/ml, administered i.m.) and intubated to allow inhalation of isoflurane (1–2%, v/v) and maintain in an appropriate plane of anesthesia. The subject’s head was restrained in a stereotaxic apparatus and an IV drip (0.45% NaCl and dextrose) was used to maintain normal hydration. Vital signs (heart and respiration rates, blood pressure, body temperature and expired CO2) were constantly monitored during the scan and surgical procedures that followed.
Following the pre-surgical scans, animals were immediately transported to the operating room and maintained under deep anesthesia with Isoflurane gas (1–2%, v/v, to effect) throughout the surgical procedures, which were performed using aseptic conditions. The scalp was shaved and cleaned with chlorhexidine diacetate (Nolvasan, Pfizer). Bupivacaine Hydrochloride (Marcaine 25%, 1.5 ml), a long-lasting local anesthetic, was injected along the planned midline incision of the scalp, which extended from the occipital to the orbital ridges. Bilateral craniotomies (1 cm wide × 2.5 cm long) were made above the areas to be injected. The Neo-PRh group was given injections 2 mm apart along the rostral-caudal length of the perirhinal cortex using 0.4 μl ibotenic acid (Biosearch Technologies, Novato, CA, United States 10 mg/ml in PBS, pH 7.4, at a rate of 0.2 μl/min).
Animals in the Neo-C and Adult-C groups underwent the same procedures, except that the injection needles were not lowered into the brain. At completion of surgery, the dura, galea, and skin were closed in anatomical layers and the animals removed from isoflurane, extubated, and closely monitored until complete recovery from anesthesia. Analgesic (acetaminophen, 10 mg/kg PO) was given QID for 3 days after surgery. Additionally, animals received dexamethazone sodium phosphate (0.4 mg/kg IM) to reduce edema and Cephazolin (25 mg/kg IM) SID starting 12 h prior to surgery and ending 7 days after to prevent infection.
Lesion extent was estimated using MRI images (coronal FLAIR) acquired 1-week post-surgery. In this post-surgical scan, edema caused by cell death after the excitotoxin injections is visible as hypersignals. Lesion extent was evaluated with methods described in detail by
Summary of lesion extent.
Subjects | PRh |
ERh |
TE |
|||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
L% | R% | X% | W% | L% | R% | X% | W% | L% | R% | X% | W% | |
Neo-PRh-1 | 89.76 | 79.91 | 83.34 | 69.04 | 28.51 | 2.28 | 15.39 | 0.65 | 4.53 | 9.70 | 7.11 | 0.44 |
Neo-PRh-2 | 68.16 | 70.58 | 69.37 | 48.11 | 17.72 | 20.65 | 19.19 | 3.36 | 0.14 | 0.06 | 0.10 | 0.00 |
Neo-PRh-3 | 65.45 | 81.02 | 73.23 | 53.02 | 7.72 | 3.12 | 5.42 | 0.24 | 0.26 | 3.39 | 1.82 | 0.01 |
Neo-PRh-4 | 59.40 | 74.73 | 67.06 | 44.39 | 11.55 | 17.84 | 14.69 | 2.06 | 0.72 | 2.62 | 1.67 | 0.02 |
Neo-PRh-5 | 75.90 | 66.81 | 71.35 | 50.71 | 38.60 | 29.86 | 34.32 | 11.53 | 0.72 | 0.41 | 0.57 | 0.00 |
Neo-PRh-6 | 74.12 | 80.31 | 77.22 | 59.53 | 25.34 | 43.64 | 34.49 | 11.06 | 0.37 | 2.93 | 1.65 | 0.01 |
Neo-PRh-1 | 0.00 | 0.00 | 0.00 | 0.00 | 8.24 | 10.86 | 9.55 | 0.89 | 0.13 | 2.39 | 1.26 | 0.00 |
Neo-PRh-2 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 2.76 | 1.38 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Neo-PRh-3 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.27 | 0.14 | 0.00 |
Neo-PRh-4 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Neo-PRh-5 | 7.02 | 3.93 | 5.47 | 0.28 | 0.00 | 0.00 | 0.00 | 0.00 | 3.37 | 0.00 | 1.68 | 0.00 |
Neo-PRh-6 | 0.00 | 0.00 | 0.00 | 0.00 | 3.78 | 4.17 | 3.97 | 0.16 | 3.32 | 0.32 | 1.77 | 0.01 |
Pre- and post-Surgical MR Images from a representative case (Neo-PRh-6). MR images shown at three rostro-caudal levels through the perirhinal cortex are pre-surgical structural T1 weighted coronal images (left column) and 1 week post-surgical coronal FLAIR images (right column) for a representative case. Visible in the post-surgical images are regions of hypersignal (white areas) that are indicative of edema and cell damage resulting from the ibotenic acid injection. Arrows point to the rhinal sulcus (left column) and to areas of hypersignal (right column). See
Prior to participating in this study, all subjects had experience with cognitive tests including concurrent discrimination learning, reinforcer devaluation, object reversal learning, safety signal learning, and emotional regulation (
The ID-ED task was conducted in a soundproof testing chamber with an automated testing apparatus. This apparatus consisted of a 3M Microtouch Touch Screen monitor and MedAssociates mini M&M dispenser controlled by a custom-written program using Presentation software. Before beginning the ID-ED task, monkeys were acclimated to the testing chamber, the touch screen, and the sound of the reward (M&M) dispenser in 15-min sessions for 3 consecutive days. After these sessions, the animals readily triggered the screen and ate the rewards as they were dispensed.
The Interdimensional-Extradimensional (ID-ED) set shifting task was based on the Wisconsin Card sort paradigm and closely resembled the version in the CANTAB battery of tasks (
Intradimensional-extradimensional (ID/ED) Set Shifting Task Schematic. In the ID-ED paradigm, monkeys learned the series of discrimination problems and reversals illustrated here. They first learned two simple discrimination problems using blue shapes (SD1 and SD2). After reaching criterion on discrimination SD2, they received 3 successive reversals of SR1 to SR3. They were then given a compound discrimination CD in which the blue shapes of the last SR3 discrimination was overlayed with orange lines, but monkeys had to continue to respond to the blue shapes and ignore the orange lines. After reaching criterion on CD, they received 3 successive reversals of this discrimination problem (CR1 to CR3) following which they were moved to the Intradimensional shift (IDR), in which new blue shapes and orange lines were used but monkeys continued to respond to blue shapes. After three successive reversals of IDS (IDS1 to IDS3), they were moved to the Extradimensional Shit (EDS) in which new blue shapes and orange lines were used but this time the animals had to respond to the orange lines and ignore the blue shapes. Criterion was set at 10 correct choices in a row before moving to the next stage. Plus indicates stimulus rewarded and minus indicates unrewarded stimulus for each discrimination and reversal.
The first stage was a simple discrimination (SD1) between two blue shapes (S1+ and S2-). This stage was repeated a second time (SD2) using novel stimuli (S3+ and S4-) to ensure that the animals had fully acclimated to the testing chamber and were sufficiently motivated to complete 60 trials each session. SD2 was followed by a series of 3 reversals (SRs) using the same SD2 stimuli but with the reward contingency between S3+ and S4- switching for each reversal. Performance on reversals assessed behavioral inhibition by requiring subjects to learn to suppress responses toward a previously reinforced stimulus and to switch to a previously non-reinforced stimulus. Once monkeys completed three reversals, a second dimension was introduced to the stimuli; the blue shapes were overlaid with orange lines (LA and LB). This third stage involved discrimination between compound (shape+line) stimuli (compound discrimination, CD). Importantly, on half the trials LA overlay S3 and LB overlay S4, and on the other half LA overlay S4 and LB overlay S3. Therefore, in the CD stage monkeys learned to respond selectively to the S+ shape regardless of which line (LA or LB) was associated with the shape. When the monkeys learned this new discrimination, they completed another series of three reversals (compound reversals, CR). Following the CR stage, an Intradimensional Shift (IDS) was given, in which a new set of compound shape-line stimuli were introduced, and monkeys transferred the rule of responding to shape (S+) and ignoring the lines. Upon completing the IDS, there was another series of three reversals between the S+/S- (Intradimensional reversals, IDR). The final stage was an Extradimensional Shift (EDS) in which a new set of compound shape-line stimuli were introduced, but now monkeys were rewarded for choosing a specific line stimulus (L+) rather than the shape. Performance on the EDS stage assessed cognitive flexibility.
The errors of Adult-C and Neo-C groups were compared on all stages of the task using independent-sample
To assess group differences in the ability to learn the reversal contingencies across stages, we compared the total number of errors to complete each series of reversals using a Group x Reversal type ANOVA with repeated measures for the second factor. Planned comparisons between groups for each reversal type were run using independent-sample
Similarly, to assess group differences in the ability to learn the simple (SD) and compound discrimination (CD) problems as well as the intradimensional (ID) and extradimensional (ED) discrimination problems, the numbers of errors across all discrimination problems were analyzed using a Group X Stage ANOVA with repeated measures for the second factor. Additional planned independent-sample
All analyses were also run using sex as a second independent factor to determine whether there were any female/male differences among the groups. None of the analyses revealed significant sex effects, and so both sexes were combined for all analyses reported in the Results section.
Finally, bivariate Pearson correlations were run to determine if the extent of PRh damage, or unintended damage in the adjacent entorhinal cortex (ERh), was related to the number of errors to reach criterion at each stage of the ID/ED task.
The numbers of errors required to complete each reversal stage are illustrated in
Reversal Stages. Total number of errors to complete the Simple Reversal (SR), Compound Reversal (CR), and Intradimensional Reversal (IDR) Stages for animals in the Neo-PRh group (shaded bars) and the Control group (open bars). Vertical lines represent ±1 SE.
Summary of intradimensional-extradimensional (ID-ED) task performance.
Groups | Discrimination stages |
Reversal stages |
||||||
---|---|---|---|---|---|---|---|---|
SD1 | SD2 | CD | EDS | SR | CR | IDR | ||
Neo-PRh-1 | 2 | 68 | 56 | 26 | 304 | 634 | 549 | 284 |
Neo-PRh-2 | 74 | 64 | 24 | 19 | 274 | 591 | 296 | 181 |
Neo-PRh-3 | 7 | 25 | 24 | 15 | 127 | 501 | 432 | 75 |
Neo-PRh-4 | 80 | 64 | 20 | 1 | 29 | 509 | 267 | 29 |
Neo-PRh-5 | 26 | 18 | 53 | 43 | 144 | 283 | 137 | 51 |
Neo-PRh-6 | 21 | 189 | 34 | 11 | 325 | 1033 | 634 | 126 |
Neo-C-1 | 7 | 19 | 14 | 3 | 30 | 170 | 77 | 54 |
Neo-C-7 | 243 | 14 | 25 | 10 | 76 | 365 | 391 | 263 |
Neo-C-9 | 133 | 2 | 22 | 12 | 80 | 542 | 602 | 300 |
Adult-C-3 | 15 | 27 | 27 | 14 | 83 | 887 | 526 | 214 |
Adult-C-4 | 22 | 93 | 105 | 9 | 103 | 618 | 265 | 84 |
The numbers of errors that the Neo-PRh and Control groups required to complete each discrimination stage are illustrated in
Discrimination Stages. Total number of errors to learn the Simple Discrimination (SD1 and SD2), Compound Discrimination (CD), Intradimensional Shift (IDS), and Extradimensional Shift (EDS) for animals in the Neo-PRh group (shaded bars) and the Control group (open bars). Vertical lines represent ±1 SE, and ∗indicates significant group differences (
The extent of PRh damage was not significantly correlated with the number of errors on any stage [SD1:
This is the first study to date that has investigated the impact of neonatal PRh lesions on cognitive flexibility and behavioral inhibition using the ID-ED set-shifting task. The results indicated that Neo-PRh lesions had little impact on the ability of adult monkeys to acquire novel visual discriminations in the SD, CD, and IDS stages, or to complete the reversal stages, but significantly impaired performance on the EDS stage. These results revealed that mechanisms important for visual discrimination learning and behavioral inhibition functioned in the normal range following the early lesions, whereas mechanisms mediating cognitive flexibility were significantly impaired. These findings are discussed in turn.
Visual discrimination learning involves the formation of stimulus-response associations. In the SD, CD, and IDS stages, monkeys learned which of two stimuli to respond to in order to obtain a reward, and which one to avoid. Monkeys with Neo-PRh lesions completed the visual discrimination stages of the ID/ED task as quickly and accurately as controls. These data confirmed similar findings from the same animals when tested on the 60-pair concurrent discrimination task (personal communication, J. Bachevalier), and indicated that Neo-PRh lesions do not impair simple discrimination learning. Monkeys with PRh lesions incurred in adulthood are also able to perform similar discrimination tasks normally (
In the Reversal stages, monkeys learned to switch their response strategies, that is avoid the stimulus previously rewarded and select the previously unrewarded stimulus. This kind of learning involves inhibition of previously acquired stimulus-reward associations. In the current study, monkeys with Neo-PRh lesions were unimpaired on all reversal stages of the ID-ED task. This finding corroborates data from an earlier study with the same Neo-PRh animals in which they were unimpaired in learning 5 concurrent object discrimination reversal problems (personal communication, J. Bachevalier). Previous research has already indicated that the OFC is important to support behavioral inhibition during reversal learning (
It is also noteworthy that the spared performance of our Neo-PRh group contrasts with the impaired performance of monkeys with adult-onset PRh lesions on similar reversal tasks (
Cognitive flexibility involves the ability to switch attention to different sources of information, especially when behavioral responses become unsatisfactory or inadequate. The EDS stage requires flexibility to ignore the previously attended-to dimension of the stimuli (shape) and shift attention to the previously ignored dimension of the stimuli (line). Neo-PRh monkeys had significant difficulty shifting their response strategies during the EDS stage, as indicated by their high error rates. Compared with the normal performance on reversal learning (behavioral inhibition) reported above, these data suggest that Neo-PRh lesions impaired mechanisms of cognitive flexibility.
Given the critical anatomical connections of the PRh with the lateral prefrontal cortex, it is possible that the deficits resulted from direct damage to the PRh or from downstream effects of the neonatal PRh lesions on the normal maturation of other neural structures, especially those with protracted anatomical and functional development, such as the PFC (
A previous report indicated that the performance of the same Neo-PRh monkeys on WM tasks that generated proactive interference was characterized by greater tendencies for perseverative errors, yet the same animals performed normally on a WM task that was devoid of interference (
The critical involvement of the dorsolateral PFC is well established in WM processes of monitoring/manipulation (
Infancy represents a stage of development characterized by increased levels of neural plasticity (for reviews see
AW, JW, and RR acquired the data. AW and JB designed the experiments, analyzed the data, and prepared the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The veterinary and animal husbandry staff at YNPRC provided expert care and handling of the animals during the course of this study. The image core facility provided support during the MR imaging. Members of the Bachevalier laboratory provided help with the surgical procedures and rearing of the monkeys during infancy.