Materials and Methods

Participants
Eighteen young adults (age 18–36, M = 24.0, 9 female), 18 elderly adults (age 64–85, M = 69.4, 9 female) and 20 PD patients (age 45–79, M = 61.8, 9 female) participated in the experiment after giving written informed consent. They were paid an amount of € 37 for participation and 5 cents for every correct reward trial with a maximum of € 6.40 for two blocks. Young adults were students at the University of Amsterdam. Elderly were recruited from a database of healthy elderly subjects1 who had previously expressed their interest in participation in cognitive aging research. Patients were recruited through Dutch national websites dedicated to PD's disease. Participants had normal or corrected-to normal vision, measured with the Landolt ring chart and were matched on IQ (crystallized IQ as measured by the “Nederlandse Lees Test Volwassenen” (NLV) and fluid IQ as measured by the “Raven Complex Forms”). None of the young and senior participants had a history of neurological or psychiatric disorders, mild cognitive impairment, eye movement or vision problems, or was taking any drugs influencing the central nervous system. The PD patients fulfilled formal diagnostic criteria for PD according to the Unified Parkinson's Disease Rating Scale (UPDRS), had a mean disease duration of 7 years (range of onset 2–13 years) and a mean estimated motor subscore of 16.7 (range 6–38) on the UPDRS which could be labeled as moderately affected when compared to other studies using this measure (Morgante et al., 2006; Harrison et al., 2009). None of the patients suffered from tremors in the neck or head. Patients were asked to continue taking their medication at the required time on the day of testing, and tests were planned 60–90 min after medication intake. Nine patients were receiving dopamine precursors (levodopa/carbidopa), six were taking a monoamine oxidase inhibitor, three were on catechol-O-methyl transferase (COMT)-inhibitors and 14 patients were receiving one or more dopamine agonists. One patient did not take medication on a regular basis.

Procedures
The experiment was divided into a training session and a main experimental session. During training, participants were presented with a series of trials to familiarize themselves with the stimulus-reward associations and antisaccade response requirements. Participants completed a 25-min training block and a 25-min experimental block, each subtending 128 trials, with a 10-min break between blocks. All experimental procedures were approved by a local ethics committee, and conducted in accordance with the Helsinki Declaration, international laws, and institutional guidelines.

Task

Temporal order of stimulus presentation
Each trial (see Figure 1) started with a central fixation dot surrounded by two square outlines (each subtending 3.8° visual angle) on the left and right side of the fixation dot (distance 12.4°). After the fixation display a central visual instruction cue was presented (for 600 ms) followed by a variable cue-target interval of 4.5–6 s, terminated by a peripheral antisaccade target (a white asterisk subtending 2°, displayed for 500 ms). The antisaccade target was presented for 500 ms pseudorandomly in the center of the left or the right square outline. The target indicated that participants should make an immediate eye movement to the opposite side of the screen. Their response was immediately followed by presentation of a feedback image (presented for 500 ms).
Figure 1  Temporal order of stimuli in the antisaccade task. In reward anticipation trials the instruction cue was a gold circle; in no reward trials the instruction cue was a silver circle. In specific spatial preparation trials, an arrow was displayed, indicating where the subsequent antisaccade target would appear; in neutral non-specific preparation trials, a bar was presented. The length of the preparation interval between the instruction cue and the antisaccade target was varied between 4.5 and 6 s. Immediately after the antisaccade a golden coin was displayed when a reward had been won; a silver circle was presented after a correct response on a non-reward trial, and after an incorrect or slow response a silver ring was displayed.

Feedback: reward, no reward, and error
The feedback image indicated to participants whether the response was correct or whether the trial was rewarded. On rewarded trials, the reward was symbolically represented as an image of a golden Euro coin. On non-rewarded trials, a silver blank disk of the same size, shape, and luminance was displayed. After an incorrect or too slow response a silver ring with a black circle in the middle was presented. Colors of rewarded, non-rewarded, and error feedback were calibrated to equal luminance using Colorfacts 7 and the color calibration system EyeOneMonitor2. Participants were informed that they would receive a monetary reward on golden reward trials in which they performed fast and correctly but not on the silver non-reward trials. In line with some other investigations using monetary reward (Ramnani and Miall, 2003) the exact amount of gain was not displayed in the feedback to avoid mental calculation.

Instruction cues: reward prospect and spatial preparation
To investigate the effect of reward anticipation and specific preparation on antisaccade performance, we presented instruction cues before the appearance of the peripheral antisaccade target. In a 2 × 2 factorial design the instruction cues independently manipulated the level of reward expectation (two levels: reward and no reward expected) and the level of response preparation (two levels: specific preparation or non-specific preparation of the antisaccade response), by means of color and shape.
The level of reward expectation was manipulated by the color of the instruction cue: In reward trials the instruction cue was a gold circle; in no-reward trials the instruction cue was a silver circle. The colors of the reward and the no-reward cue were calibrated to equal luminance using Colorfacts 7 and EyeOneMonitor.
The level of response preparation was manipulated by the content of the instruction cue: in specific preparation trials, an arrow was displayed in the center of the circle, indicating where the subsequent target would appear; in the neutral non-specific preparation trials, a bar replaced the arrow. The arrow enabled subjects to prepare for the appearance of the peripheral antisaccade cue, while a bar would give no information on target location.

Eyetracking and stimulus delivery set-up
Subjects were seated 60 cm in front of a computer screen in a dimly lit room with their head stabilized in a chin rest. An infra-red camera was mounted in front of the right pupil in order to monitor eye movements. Eye movements were recorded with ViewPoint Eyetracker PC-60 (Version 2.7, Arrington Research Inc.3) software on a standard PC. Bidirectional communication between this PC and a second one responsible for the delivery of stimuli (using Presentation software4) ensured that stimulus onset times were registered in the eye movement data and that adequate feedback was provided to oculomotor responses on each trial. Eye movements were registered with a sampling rate of 60 Hz along with signals marking the stimulus onset times. Before task onset a 9-point calibration procedure was performed. To eliminate slow drift in eye tracking-signal during the task, calibrated eye position was manually corrected to the central fixation cross. Regions of interest were defined by two peripheral outer square outlines (the endpoints of the antisaccade eye movements) surrounding the central fixation dot. The PC which tracked eye movements, signaled to the stimulus presentation PC when an eye movement left the fixation region and entered one of the target regions. The Presentation PC recorded correct trials versus errors and presented feedback accordingly.

Analysis
Saccade parameters were detected with in-house Java-based software5 using minimum amplitude (>1.5°) and velocity (>30°/s) criteria and were subsequently visually inspected and double-checked for accuracy. In line with common definitions (Fischer et al., 1993) saccades with a latency of less than 80 ms after the display of the peripheral onset target were classified as anticipatory responses. Exclusion criteria applied to trials exceeding 800 ms (miss), trials with blinks during saccadic execution, trials in which participants failed to focus their eyes on the central cue or in which gaze was not at fixation 200 ms before target appearance. To avoid latency differences between correct and erroneous antisaccades, incorrect trials were not included in latency analysis. A trial without a premature eye movement toward the peripheral target but with a saccade landing at the location of the square outline on the opposite side of the screen executed within 800 ms was classified as a correct trial. To investigate premature eye movements, the interval between the preparatory cue and the display of the peripheral antisaccade target was examined for fixation breaks. Fixation breaks were defined as saccades or antisaccades executed after exposure to the cue and before appearance of the target, with an amplitude at least equal to the mean antisaccade response across trials minus one standard deviation, such that fixation break amplitudes fell within the range of the largest 84.13% of all saccadic responses (Brown et al., 2007). Antisaccade latencies and accuracy as well as fixation breaks were analyzed using a mixed 3 × 2 × 2 × 2 ANOVA design with one between-subjects variable (young, elderly, or patient) and three within-subjects variables (reward or no reward; specific- or non-specific cue; and delay interval of 4500–5000 or 5500–6000 ms). Additional analyses were conducted using a similar ANOVA design, but now with the between-subjects factor consisting of healthy elderly, mild PD patients, and severe PD patients.