Participants

Thirty human individuals were invited to participate in the experiment. Behavioral measures were not acquired for the first participant due to technical start-up problems and another participant did not show up for the second session. The remaining 28 participants (age M = 19.50, SD = 2.05; 24 females; body mass index M = 22.50, SD = 2.69) had normal or corrected-to-normal vision, had no cardiac, hepatic, renal, neurological or psychiatric disorders, personal or family history of depression, migraine and no medication or drug use. Female participants that used contraception were only tested outside their menstrual period to minimize confounds of hormonal differences (for details, see39). Participants were naïve to the purpose of the experiment and were told that they had to drink orange juice to investigate the effects of vitamin C on behavior. As such, treatments were deceptive because participants were not aware of the choline administration. To prevent a potential overdose of choline we measured blood pressure at several moments during the experiment (see below). The experiments conformed to the ethical principles of the Declaration of Helsinki and were approved by the local ethical committee (Leiden University, Institute for Psychological Research). All participants were right-handed students and received study credit for participation. Furthermore, all gave informed written consent at the start of the first session of the experiment and participants were debriefed after the second session.

Apparatus and material

Depending on the session, participants were given 400 ml orange juice including 2 g dissolved choline bitartrate or a microcrystalline cellulose placebo, both consisting of a fine-grained, white powder that did not change the viscosity of the drink. Choline bitartrate contains 41.1% choline by molecular weight and 2 gram of choline bitartrate administration provides 800 mg of choline action, similar to the ingestion of approximately 5 hard-boiled eggs or 250 g beef liver. The given amounts were well below the established 3.5 g recommended upper intake level for adults (Food and nutrition board of the US institute of Medicine). Choline uptake peaks approximately thirty minutes after ingestion12 and acetylcholine levels in brains significantly raise after approximately forty minutes12 and remain high for at least ninety minutes11. In addition, choline bitartrate and lecithin (phosphatidylcholine) increase choline plasma levels in humans within one hour after ingestion40,41,42,43.

Stimuli were generated on an Asus Vivobook laptop computer with Windows 8 operating system (Microsoft) and MatLab (Mathworks). The computers desktop was extended to a presentation monitor that displayed 1280 by 1024 pixels at a 60-Hz refresh rate. Screen size was 30 cm in width and 23 cm in height (31 by 25 visual degrees) and the participant’s viewing distance to the screen was fixed with a chin and forehead rest at approximately half a meter. Heart rate (HR) and systolic and diastolic blood pressure (SBP and DPB) was measured from the non-dominant arm with an OSZ3 automatic digital electronic wrist blood pressure monitor (Welch Allyn). Choline supplementation did not significantly affect these measurements as compared to placebo (p’s > 0.05). Images (640 by 480 pixels) of the pupil were recorded with a tripod mounted RGB Flea3 USB3 camera (Point Grey, Richmond, BC, Canada) with a Tokina AT-X 90 mm macro-lens at 60 Hz while participant’s fixated a small dot at the center of a screen with a grey background.

Stimuli and procedure

We tested healthy students on a visuomotor task, while recording their pupil size, approximately 70 minutes after the administration of 2 gram of choline bitartrate or a placebo substance (for a flow-chart, see Fig. 1a). To control for choline uptake unaffected by other supplements, participants were restricted from drinking alcohol the day preceding the study and participants were not allowed to have breakfast, coffee, or cigarettes before the experiment. Only water and tea with sugar was allowed. The participants either started the experiment at 9:00 or 11:00 in the morning. At arrival in the laboratory, the participant’s pupil size, HR, SBP, DBP and subjective mood (indexed on a 9 by 9, pleasure by arousal grid; also see44) were assessed. Pupil measurements were repeated every twenty minutes before the visuomotor aiming task, again following immediately after the task and again approximately thirty minutes later. HR, SBP and DBP were assessed right before supplementation and 60 and 90 minutes after supplementation (i.e., before and after the task). After the first assessment, participants ingested 400 ml orange juice with dissolved choline or placebo at arrival in the laboratory. Fourteen participants took choline in session one and placebo in session two and the other fourteen participants vice versa. These sessions were separated by approximately 7 days (±1−3 days). The supplements were prepared in sealed containers by author MN and handed over to the naïve experimenter and none of the participants was able to determine a difference between the conditions.

Figure 1 Methods. (a) Participants were given a 2 g choline bitartrate or placebo supplement and assessed for their mood and arousal (MA), heart rate (HR) and blood pressure (BP) after arrival at the lab and right before and after the visuomotor task. Pupil size (P) was also measured at a 20 minute interval before the task and 30 minute interval after the task. Participants were allowed to consume one piece of fruit 30 minutes after supplementation and the visuomotor task was performed 40 minutes later. (b) During the task, participants had to move a mouse cursor and hit the center of a target as fast as possible (within one second) to accumulate scores for their accuracy and reaction time across trials. Scores were based on the hit distance to the target’s center and reaction times. (c) Pupil size was extracted from recorded images by determining the median radius of the pupil’s border (see white circle) from the center. Full size image

Half an hour after supplementation, participants were provided a maximum of two apples or mandarins to prevent hunger while waiting for the behavioral task. The task was timed seventy minutes after intake to ensure that choline was taken up in the participant’s system. As such, participants waited more than an hour before conducting the behavioral task.

In each session, participants performed a spatial working memory and a visuomotor task which took approximately twenty minutes to complete. Here, we only present the results of the latter task because the results from the working memory task were inconclusive. The visuomotor task is almost identical to CANTAB’s Motor Screening Task (Cambridge Cognition Ltd, Cambridge, United Kingdom) in which participants have to point their finger at a target presented on a touch screen as fast and accurate as possible. In our task, however, participants had to use a computer mouse to move a cursor to a target as fast as possible and click as close to the target’s center as possible to gain points in each trial. A trial started with the presentation of a blank grey screen (Fig. 1b depicts a white background for aesthetical purposes), a fixation dot and a mouse cursor for a duration randomly chosen from a uniform distribution within the range of 1 to 2 seconds (Fig. 1b). Next, the fixation dot disappeared and a circular target was shown at a random screen position. The target was 100 pixels in diameter (i.e., ~2.5 visual degrees) and consisted of a bulls-eye with 10 rings that alternated black and white as a function of eccentricity (the bulls-eye in Fig. 1b depicts less rings for aesthetical purposes). The target’s appearance released the mouse cursor and participants had to move the cursor to the target as fast as possible. Once the cursor arrived at the target, participants had to click the left mouse button with their right hand to hit the target as close to its center as possible. Participants could receive a maximum of 100 points when their reaction time was as fast as 250 ms (50 points) and accuracy right at the center (50 points). Reaction times slower than 250 ms decreased the score by 0.067 points per millisecond. A trial was automatically aborted when a participant did not hit the target within 1000 ms. Score also decreased as a function of hit distance to the target’s center (one point per pixel). A penalty of −100 points was given when target was missed or when the response was too late. After the response, participants received visual feedback for 1.5 s about their score in the current trial and their total score accumulated in previous trials. The task consisted of a total of 128 trials and each trial automatically started after the feedback.

Analysis

Reaction times (RT) were based on the median time between target onset and target hit across all correct trials per participant (i.e., RT’s faster than one second and hits on the target). Hit distance was the mean Euclidian distance in visual degrees between the mouse cursor and target’s center at the time of the mouse click (i.e., the hit) across all correct trials. Number of misses was based on the amount of trials in which participant’s made an inaccurate movement and clicked the mouse while the cursor was off the target.

Pupil size was computed with custom software through several image processing steps. First, a manually set region of interest in the recorded images of the eye was transformed to binary image patches (i.e., two bit images with only zero or one values) by setting a luminance threshold per participant. Second, the center of the pupil was calculated by taking the median of x and y positions of all pixels in the darker region of the binary image (i.e., image locations with a zero value). Third, the pupil’s border was detected with a contrast analysis of 72 linear image lines (“rays”) that were extracted from the original image regions. The lines were radially aligned around the pupil’s center and extended from here to the periphery with a radius of 255 pixels (i.e., well beyond the pupil’s border). A strong increase in contrast in the image line indicated the pupil’s border. Last, pupil diameter was computed by taking the median of all radial distances from pupil center to the detected borders, resulting in an accurately fitted circle around the pupil (Fig. 1c). Due to strong camera zooming, which allowed accurate pupil size extraction, the depth of view got quite narrow and the proportional area of image covered by the pupil got large. These factors increased the likelihood that the pupil would move out of image borders or camera’s focus. Pupil size measurements therefore failed for two participants (both 18 years old; 1 female; body mass index 25.3 and 29.8; 1 took a placebo and the other choline in the first session) because they were unable to maintain their head fixed. For the same reason pupil measurements of two and three additional participants failed during the second-last and last recording, respectively.

We tested for the effects of choline on reaction times and hit distance with paired samples, two-sided t-tests. The percentage change in pupil size was calculated by subtracting and subsequently dividing pupil size in subsequent measurements by pupil size in the first measurement. Effects on pupil size were tested with a two-way repeated measure ANOVA with supplementation (choline vs. placebo) and measurement time after the first assessment at the start of the session as factors. Post-hoc one-tailed paired samples t-tests indicated at which time point choline significantly decreased pupil size as compared to placebo. We computed two-sided Pearson correlations coefficients to assess the relationship between changes in pupil size and differences in reaction time and accuracy between choline and placebo conditions across several time points during the experiment. Of particular interest was the pupil measurement right after the assessment of visuomotor performance because these correlations were subject to less noise due to the shorter time lapse between the measurements of the two variables. Missing data of the last pupil assessments of three participants were linearly interpolated for the ANOVA but not for the post-hoc comparisons and correlations. A false discovery rate correction was applied to the p-values of the correlations per assessment during the experiment45. We additionally conducted a bootstrap with replacement procedure on the correlation data to calculate the confidence intervals on 1000 newly acquired coefficients per data set.