All experiments were performed in adult (∼8–10 years old) rhesus macaques (Macaca mulatta), ranging from 5 to 13 kg. All procedures followed the guidelines of the MIT Animal Care and Use Committee and the US National Institutes of Health. In total, 4 females and 2 males were trained in this study. They were pair-housed, and on a normal 12-hr diurnal schedule. In the Category-Saccade task, one of the animals had been previously trained on a conditional association task. In the Category-Match task, one of the animals was being actively treated with cyclosporine daily. All animals spent approximately 1-2 years of training on their respective tasks.

Method Details

Task Details Object-Match task have been presented previously ( Brincat and Miller, 2015 Brincat S.L.

Miller E.K. Frequency-specific hippocampal-prefrontal interactions during associative learning. The details of thetask have been presented previously (). In each session, six novel objects were chosen from an image database (Hemera Photo-Objects). Four were randomly designated as cue objects, and the remaining two as associate objects. In turn, each cue object was randomly paired with an associate object. The monkeys’ task throughout each session was to learn, through trial-and-error, which associate was paired with each cue. To initiate a trial, each monkey fixated on a central white dot for 0.5 s. After this fixation period, a cue object (foveal, 3° wide) was presented for 0.5 s, followed by a blank delay of 0.75 s. Two associate objects were then presented in a randomly-ordered series. Each object presentation lasted 0.5 s, and was then followed by another a brief delay of 0.6 s. To indicate that an object was a match, the monkey had to saccade to a subsequently presented visual target, a white dot presented 7.5° to the left or right of fixation. And if it did so, the animal received juice and a new trial began within 3 s. If incorrect, instead of juice, a red “error screen” flashed on for 1.5 s, and the animal had to wait 6 s for the subsequent trial. The location (left versus right) of the response target after each associate was randomized and unrelated to task performance. Category-Saccade task have also been presented previously ( Antzoulatos and Miller, 2011 Antzoulatos E.G.

Miller E.K. Differences between neural activity in prefrontal cortex and striatum during learning of novel abstract categories. Antzoulatos and Miller, 2014 Antzoulatos E.G.

Miller E.K. Increases in functional connectivity between prefrontal cortex and striatum during category learning. The details oftask have also been presented previously (). In this task, animals had to learn to classify a number of category exemplars generated from two different prototypes into two categories. Each category was directly tied to a specific saccadic target (right or left). To start a trial, animals first fixated within 1.5-2° of a red, central target (0.4° in diameter) for 0.7 s. After this fixation period, a randomly chosen category exemplar (6° by 6°) from either category was presented for 0.6 s. Trials from both categories were randomly interleaved throughout the session. One second after the end of the exemplar period, two saccade targets (a green dot, 0.6° in diameter) appeared on the left and right of the center of fixation (5° from the center). In order to indicate a response, the animals had to make a single, direct saccade within 1 s of target presentation and maintain fixation on it for 0.2 s. If the animal chose correctly, it was rewarded with drops of juice. If the animal did not, it was punished with a 5 s timeout, during which the cue was presented again at the location of the corresponding target. In each session of the Category-Match, animals had to classify a number of category exemplars generated from two novel different prototypes into two categories. Each category here, however, was neither tied to any particular saccade nor saccade location. Instead at the test period, the animal had the opportunity to freely investigate two exemplars, one of which matched the category of the sample exemplar, and then had to choose by fixating on this match. To initiate each trial, each animal had to fixate within 2.5° of a centrally located, red dot (0.2° in diameter) for 0.5 s. After this fixation, an exemplar of one of the two categories was presented at the center of the screen (7° by 7°) for 1 s. If the animal continued to fixate through this sample period, and a subsequent delay of 0.85 s (with an additional jitter of 0.4 s), then the central fixation dot disappeared and two new exemplars were presented on the left and right side of the screen (9° from the center of the screen). Once the test exemplars appeared, the animal had the opportunity to freely view both of the exemplars presented and make the correct choice. To indicate this choice, the animal had to fixate on one of the two peripherally presented exemplars for 0.7 s. If it made the correct choice, the white dots of the chosen exemplar turned green and the animal received juice. If the animal did not make the correct choice, the chosen exemplar turned red and no juice was given. Depending on the animal, the length of timeout incurred on error trials varied from 5-16 s.

Neurophysiology and Hardware In both the Category-Saccade and Object-Match task stimulus presentation and reward delivery were controlled by Cortex (NIMH, Laboratory of Neuropsychology) and presented on a 100 Hz CRT monitor. Eye movements and pupil size were monitored and recorded using an infrared eye tracking system (Eyelink I & Eyelink II, SR Research @ 500 Hz). In these tasks, up to 16 electrodes were lowered in PFC, HPC, or STR acutely. All recordings from PFC and STR, and most from HPC, were performed with epoxy-coated tungsten microelectrodes (FHC). On some HPC recordings, 24-channel linear probes with 300-um spacing between adjacent platinum iridium contacts were used (U-probes, Plexon). For targeting, the animals’ implanted chambers were co-registered with structural MRI images. For all of the PFC, STR, and some HPC recordings, these electrodes were lowered daily though the dura using custom-built, screw micro-drives. The exact location on the grid and orientation of the grid were varied to limit cortical damage and maximize coverage of the intended regions. For the linear probes, electrodes were lowered through a 25-gauge transdural cannula using a motorized drive system (NAN-S4, NAN instruments). The electrodes would be lowered until spiking was detected, and then electrodes were allowed to sit for about an hour to limit apparent neural drift. Neural activity was amplified, filtered, digitized and stored using an integrated multichannel recording system (Multichannel Acquisition Processor, Plexon). The signal from each electrode was amplified by a high input–impedance, unitary gain headstage (HST/8o50-G1, Plexon), referenced to ground, filtered from 0.7–300 Hz, and amplified 1000-fold. LFPs were recorded continuously at 1 kHz. Only electrodes with cells present on them were included for these analyses and, after trial cutting, evoked potentials were subtracted out from each individual trial. In the Category-Match task, stimulus presentation and reward delivery were controlled by custom software written in MATLAB using PsychToolbox. All stimuli were presented on a LCD screen at 144 Hz (ViewSonic VG2401mh 24” Gaming Monitor). Eye movements and pupil size were monitored using EyeLink II at 1000 Hz sampling. Four 8x8 channel Blackrock Cereport arrays with 1mm long electrodes were implanted in dorsomedial prefrontal cortex (dmPFC), dorosolateral prefrontal cortex (dlPFC), and ventrolateral prefrontal cortex (vlPFC). Each electrode was separated by 400 um. vlPFC, dlPFC, and dmPFC were all defined by anatomical landmarks following the craniotomy. The vlPFC array was placed 1 mm ventral to the principal sulcus and was centered at 9-12 mm anterior to the genu of the arcuate sulcus. In contrast, the dlPFC array was positioned slightly more rostral, 12-15 mm anterior to the genu of the arcuate and 1 mm dorsal to the principal sulcus. Finally, we placed the dmPFC (dorsomedial prefrontal cortex) array in the vicinity of where others have reported to identify the supplementary eye fields. The medial edge of the array was placed 5mm from the midline, and 5mm anterior to the genu of the arcuate sulcus. We recorded using Blackrock headstages (Blackrock Cereplex M and Cereplex E). Signals were sampled at 30 kHz, band-passed between 0.3 Hz and 7.5 kHz (1st order Butterworth high-pass and 3rd order Butterworth low-pass), and digitized at a 16-bit, 250 nV/bit. All LFPs were recorded with a low-pass 250 Hz Butterworth filter, referenced to ground, sampled at 1 kHz, and AC-coupled. In Monkey G, an error in the design of the Cereplex E head-stage made the system susceptible to ground loops and to DC-drifts in the signal. This required us to apply a high-pass, 0.5 Hz FIR filter in both directions on the whole dataset to avoid any phase distortions. All arrays had units present on at least 5, if not typically a large proportion of channels. All channels were included in this analysis, and for all synchrony analyses the evoked potentials averaged across trials were subtracted from each individual trial.

Prototype and Exemplar Generation Posner et al., 1967 Posner M.I.

Goldsmith R.

Welton Jr., K.E. Perceived distance and the classification of distorted patterns. Vogels et al., 2002 Vogels R.

Sary G.

Dupont P.

Orban G.A. Human brain regions involved in visual categorization. Antzoulatos and Miller, 2011 Antzoulatos E.G.

Miller E.K. Differences between neural activity in prefrontal cortex and striatum during learning of novel abstract categories. In both the Category-Match and Category-Saccade tasks, the visual stimuli were composed of 7 randomly located dots on a black background. To construct the categories, we followed previously published procedures (). Every day, two novel prototypes were created at random. These prototypes (as would be the exemplars) were generated as 7 arbitrarily positioned, 7-pixel dots on a grid of 140 by 140 pixels. In order to control for difficulty and ease, these arbitrarily constructed prototypes had to obey a number of rules: (1) They had no dot centers that fell within 14 pixels of one another. (2) The average dot position of the prototype was at the center of the grid. (3) No dots from each exemplar fell within a 10-dot margin on the edge. And, (4) the minimum Euclidean distance between all pairs of dots between each prototype was no greater than 200 pixels. Each of these 140 × 140 pixel exemplars subtended 6-7 degrees of visual angle. Posner et al., 1967 Posner M.I.

Goldsmith R.

Welton Jr., K.E. Perceived distance and the classification of distorted patterns. In order to generate the exemplars, the prototype dot patterns were jittered according to a procedure first established by Posner and colleagues (). To determine this jitter, we first defined 5 concentric annular regions. These annuli were centered around each dot, and spaced apart radially by 7 pixels. Region 1 refers to the annulus immediately surrounding the dot center, 1 dot-diameter away, and region 5 refers to the annulus 5 dot-diameters away from this dot. Next, each dot from each prototype was shifted away from its prototypical location by at least 1 region; no exemplar was identical to the prototype. Whether any particular dot was moved to regions 2 to 5 depended on the distortion level desired. Each exemplar had to be unique, different from any other exemplar, and, to ensure such, no more than 2 dots from each exemplar could be less than 10 pixels away from any other exemplar’s dots. Posner et al. defined 9 distinct levels of distortion, based on the probability of a dot to shift to each of these 5 concentric regions. Two of these 9 distortions were used in this task. At distortion level 1, 88% of dots were shifted to region 1, 10% to region 2, 1.5% to region 3, 0.4% to region 4, and 0.2% to region 5. At distortion 2, 75% of dots were shifted to region 1, 15% to region 2, 5% to region 3, 3% to region 4, and 2% to region 5. In the Category-Saccade task, the generated exemplars were largely at distortion level 2. In the Category-Match task, which appeared more difficult for animals to acquire, distortion level 1 exemplars were used for both animals. As a side note, in order to rule out that any of the reported effects were a result of the level distortion, we repeated 3 sessions in one of the two monkeys at distortion level 2. The results were similar; the monkey showed an enhancement of synchrony in the beta band on correct trials and theta band on incorrect trials. Overall, the use of these visual stimuli in both tasks provided for us a number of advantages: (1) These categories were not imbued with any overt meaning to the subject, for they held no apparent relationship to objects seen in daily life. (2) The exemplars which could, in fact, look distinctively different from one another were always perceptually related and averaged out to the original prototype. And (3), these categories could not be distinguished by any simple rule.

Block Design To facilitate learning, in both the Category-Match and Category-Saccade task, each learning session was organized into blocks. The blocks were defined by a progressively growing pool of available exemplars from which any could be used for a given trial and any task period (sample or test). In any given block, with the exception of block 1 in the Category-Saccade task, there were a total of 2block number of exemplars for each category. In the Category-Saccade task, in the first block, there was a single exemplar per category. The pool of available exemplars grew by accretion, “new” exemplars were added to a bank of “familiar” ones, so that the total available exemplar was equal to 2block. The terms novel and familiar are not an indication for how familiar any exemplar was to an animal, but simply a reflection of when it became available in the pool of potentially usable exemplars. As the blocks progressed, the chances for only seeing novel exemplars increased substantially, and performance on these novel exemplars suggested successful categorization. In fact, block transition was not possible without successful categorization, and the overlap of available exemplars between blocks favored a smoother learning process. In order to pass from one block to another, each animal had to achieve a particular behavioral criterion. The criteria diverged somewhat between the two category tasks. In the Category-Saccade task, a block transition occurred when the animal had correctly responded to 80% of the previous 20 trials. In the Category-Match task, both animals had a tremendous capacity for being biased in either choosing a particular location and/or a particular category. In order to limit these behavioral biases, each animal had to successfully complete 70% of the previous 10 trials for each potential condition (Category A – on left, Category A – on right, Category B – on left, and Category B – on right). Because of these behavioral criteria in both tasks, not all available exemplars were presented in each block. In the Category-Match task, an additional restraint was imposed on the pool of available exemplars presented in block 1. Because both animals struggled to pass block one, in which two exemplars from each category were presented, the exemplars from each category had to have a Euclidean distance of less than 20 pixels apart. This constraint reduced the difficulty of the first block, promoted rapid block passage, and ultimately favored category abstraction. Following block one, there was no limitation on the presented exemplars.