Participants
For the present study, 26 healthy participants were recruited via flyers that were distributed at the University Hospital Frankfurt. Participants were included in the study when they met the following inclusion criteria: age between 18 and 55 years, absence of psychiatric or neurological disorders in subjects or their first-degree family members, right-handedness, no head-related eczema and implants in the head or cranial region, no untreated thyroid dysfunction and no visual and/or hearing impairments. Pregnancy, drug use in the last 48 h, excessive alcohol consumption on the previous day or intake of medications that impair the ability to concentrate led to exclusion. Finally, if German was not the native language, extreme caffeine consumption and unusually short sleep duration also led to exclusion.
Mental health was assessed with the German Version of the Mini-International Neuropsychiatric Interview (M.I.N.I.) 36 and to obtain an approximate IQ measure, the Multiple choice vocabulary intelligence test (Mehrfachwahl-Wortschatz-Intelligenztest; MWT-B) 37 was performed. Handedness was determined with the German Version of the Edinburgh Handedness Inventory 38. Vision was checked with Tests for Colour Blindness by Ishihara Shinobu 39 and visual acuity with the Fernvisus-Test 40 by Edmund Landholt.
Three participants were excluded from the analysis after data collection because of uni- or bivariate outlier values in startle or PPI data. After exclusion, 23 participants remained in the analysis; 59.1% women (age in years M = 24; SD = 2.64) and 40.9% men (age in years M = 23.7; SD = 4.03). All participants were university students with a high school diploma as the highest level of education completed.
According to the study protocol approved by the Ethics Committee of the University Hospital Frankfurt (ID = 501/17), all participants had to sign a written consent form after receiving all relevant information in order to participate in the study. The anonymity of the participants was ensured by storing all data pseudonymized, while the personal data of the participants appeared only on the informed consents. Participants were informed that they were free to withdraw from the study at any time without giving reasons and were offered a monetary incentive of €10 per hour, which they received at the end of the second test session. Test-retest interval was M = 27.1 days (SD = 2.28, range: 21–32).
Working Memory assessment
In the n-back task 41 subjects viewed a series of digits (1–4) presented sequentially for 500 ms (inter-stimulus interval = 1500 ms). One of the numbers in each frame is highlighted and represents the target number to be maintained in memory. As the sequence progresses, the subject must indicate via a button press the highlighted number corresponding either to the currently displayed frame (0-back, control condition) or two frames previously (2-back, experimental condition). The stimuli are presented in a block design; each block lasts 28 s and four blocks are presented for each condition. The conditions are alternated, and the total run length is 4 min 16 s. All subjects practiced the task until they gave 60% correct answers in the 2-back condition.
WM performance is calculated as 2-back performance minus 0-back performance. Furthermore, we calculated 2-back residuals by regression out the 0-back performance from 2-back performance.
In the canonical change detection task (CDT) three red bars with different orientations are displayed on a computer monitor. Subjects are instructed to memorize the exact orientation of these three red bars. After a variable delay (2800-3200ms), subjects again saw three red bars. In 50% of the cases, these correspond exactly to the previously shown bars. In the remaining cases, the orientation of one bar was changed. Subjects were asked to decide whether the orientation of bars has changed compared to the previously shown bars or not. If no change in the orientation of the three red bars was noticed, the left mouse button should be pressed. If the participants notice that the orientation of one of the three red bars has changed, the right mouse button should be pressed. During the entire duration of the test, a small black cross is visible in the center of the computer screen, which the test participants should fixate with their eyes the entire time. Before the start of each new task, the fixation cross briefly turns red to announce the impending start of the next task. CDT working memory performance was calculated based on the number of correct answers.
PPI Testing
Acoustic stimulation was delivered binaurally through wireless headphones (Bose® Quiet Comfort® 25). All sound levels were calibrated by using an artificial ear (Brüel & Kjaer, type 4153). Subjects were continuously presented with 55 dB background noise (broadband white noise). Before the PPI main experiment subjects were presented six startle stimuli (40 ms; 98 dB broadband noise) at intervals of 8–12 s each. In the PPI main experiment, the following stimuli were presented in a pseudo-randomised order (not more than one of the same type consecutively) with a variable interval of 10–20 s: 10 x startle stimulus, 10 x pre-pulse (20 ms broadband noise 64, 68, 72, and 76 dB, i.e. 9, 13, 17, and 21 dB above background) followed by a startle stimulus (100 ms after pre-pulse onset), 10 x pre-pulse alone (76 dB) and 10 x no stimulus (3000 ms). After the main PPI experiment six successive startle stimuli at 8–12 ms intervals were presented. The intensity of the startle response was determined by measuring the electromyogram activity of the orbicularis oculi using two electrodes under the right eye. The test session lasted for 20 min.
Surface EMG was recorded using BrainAmp ExG16 amplifier with a sampling rate of 5000 Hz. The startle response was measured from the orbicularis oculi muscle with two 6mm Ag/AgCL cup electrodes with Elefix paste (Nihon Kohden) placed below the participant’s right eye approximately 1 cm under the pupil and 1 cm below the lateral canthus. Relevant skin surface areas were previously treated with a slightly abrasive gel (Nuprep skin preparation gel). All resistances were less than 6 kOhm.
To preprocess and analyze the EMG data, BrainVisionAnalyzer 2.2 Software (Brain Products, 2019) was used. Having collected the raw data for all participants, the first step in the preprocessing was manual artifact rejection, in which all major artifacts were marked and excluded from the further analysis. To filter the data, Butterworth Zero Phase Filters (low cutoff: 28 Hz, high cutoff: 450 Hz) and a notch filter (50 Hz) were used. The data was then rectified in order to obtain the same polarity across the dataset, and the 40 Hz lowpass filter was used to smooth the data. The data was segmented per condition, from − 100 to 300 ms. After segmentation, the data was baseline corrected (-100 to -500 ms), in order to make sure that the values are comparable across different participants and conditions.
After preprocessing, trial-wise peak responses were extracted in a time window between stimulus onset to 150 milliseconds after stimulus onset. Trials +/- 3 SD of baseline activity were excluded from further analysis. Averages calculated from the remaining trials were exported to IBM SPSS (version 25) for further analyses.
Statistical analysis
Startle responses (without prepulse) were analyzed by a repeated measure analysis of variance (ANOVA) with the within-subject factors session (t1and t2) and position (before, during and after the PPI main experiment). Effects of prepulse intensities were analyzed by a repeated measure analysis of variance (ANOVA) with the within-subject factors session (t1 and t2) and prepulse intensities (64, 68, 72 and 76 db). Alpha was 0.05 and Greenhouse-Geiser correction was used whenever necessary.
Test-retest reliability was assessed using two variants of the ICC, namely ICC(2,1) and ICC(3,1), defined by Shrout and Fleiss (1979) as:
ICC(2,1) = BMS-EMS/(BMS+(k-1)*EMS + k*(JMS-EMS)/N)
ICC(3,1) = BMS-EMS/BMS+(k-1)*EMS
where BMS = between-subjects mean square; EMS = error mean square; JMS = session mean square (the original terminology of “J” is “Judge”); k = number of repeated sessions and n = number of subjects. Thus, in the current study, k = 2 and n = 23.
The calculation of both these variants allowed us to determine the reliability in terms of relative (consistent measures = ICC(3,1)) or absolute agreement (ICC(2,1)). Both forms of the ICC estimate the correlation of the PPI between sessions, modeled by a two-way ANOVA. In the case of ICC(2,1), both effects (subjects and sessions) are assumed to be random, while for ICC(3,1) the effect of sessions is assumed to be fixed. Following Fleiss (1986), we denote ICC values < 0.4 as poor, 0.4–0.75 as fair to good and > 0.75 as excellent. For further assessment of reliability we also applied average measure ICC corresponding to an estimate of reliability in case of doubling number of trials 42.
Correlation analyses were calculated using rank correlation coefficient according to Spearman (two-tailed, alpha = 0.05).