Salivette collection device

The salivary enzyme alpha-amylase which is mainly involved in the digestion of starch in the oral cavity has received increasing attention as an indicator of sympathetic activity within the past years [37] (link). Pharmacological studies have shown that its release is dependent on β-adrenergic transmission [38] (link). Using Salivette collection devices (Sarstedt, Nümbrecht, Germany), we sampled alpha-amylase at 10 min before and at 10 and 20 min after the onset of CPT and TSST to fully capture peak activity and return to baseline. Participants were instructed to place the saliva collection swabs in their mouths and to refrain from chewing for exactly 2 min [39] (link). Samples were stored at −30°C until analysis. Alpha-amylase activity (calculated and expressed in U/ml) was determined using an enzyme kinetic method [40] (link),[41] (link).

To assess stress objectively salivary samples were collected for the analyses of cortisol and alpha-amylase (Salimetrics, High Sensitivity Salivary Cortisol EIA kit 1-3002 and Salivary a-amylase kinetic enzyme assay kit 1-1902). The detailed protocol for salivary cortisol collection can be found in (Weber et al., 2022 (link)). We calculated an estimate of the cortisol awakening response (CAR, i.e., the rapid increase in cortisol in the morning (Hellhammer et al., 2009 (link), Wust et al., 2000 )) using the area-under-the-curve with respect to ground (Pruessner et al., 2003 (link), Stalder et al., 2016 (link)). In comparison to cortisol, which represents a slow response to hypothalamus–pituitary–adrenal (HPA)-axis activity, alpha-amylase can be used as an indicator for the rapid sympathetic – adrenal medullary response and has previously been found to be elevated in FND patients (Apazoglou et al., 2017 (link)). Saliva samples for analysis of alpha-amylase were collected before entering the MRI scanner using Salivette collection devices (Sarstedt, Germany).

Saliva samples were obtained at four time points throughout the session (T₋₅, T₊₁₅, T₊₃₀, T₊₆₅ with respect to stress onset) by placing a cotton swab in participants’ mouths for 45–60 s using Salivette collection devices (Sarstedt, Nümbrecht, Germany). Samples were stored at −20 °C until analysis. After thawing, saliva samples were centrifuged for 10 min at 4000 rounds per minute (rpm) to remove particulate material. Cortisol concentrations from saliva samples were assayed using a solid-phase enzyme-linked luminescence immunoassay at the Cognitive Psychology Department, University of Hamburg, Germany (Director: Prof. Lars Schwabe). The assay was conducted with 50 µL of saliva according to the specification and protocols of the manufacturer (LIA; IBL/Tecan, Hamburg, Germany). Cortisol values were log transformed prior to analysis to reduce skewness.

Saliva was sampled with Salivette collection devices (Sarstedt, Nümbrecht, Germany). Participants were instructed to place the collection swabs in their mouths and to refrain from chewing for 2 min. They were asked to take nothing by mouth other than water, to refrain from brushing their teeth for 10 minutes, and from smoking for 30 minutes prior to sampling. If deviating from this guideline, they were asked to thoroughly rinse their mouth with water before taking a sample. Participants otherwise followed their normal daily routine. To control for confounding effects of female hormonal status on cortisol^{66 (link)}, hormonal status was assessed via self-report prior to the sampling days (i.e., no menstrual cycle because of menopause or polycystic ovary syndrome, hormonal contraceptives, natural menstrual cycle, male). Salivettes were stored in participants’ freezers until returned to the laboratory where they were stored at −30 °C until assay (Department of Biological and Clinical Psychology, University of Trier, Germany). Cortisol levels (expressed in nmol/l) were determined using a time-resolved fluorescence immunoassay with intra-/interassay variabilities of <10%/12%^{67 (link)}.

Participants will be provided with a package containing written instructions for saliva collection and Salivette collection devices (Sarstedt, Nümbrecht, Germany). Three saliva samples will be collected on one morning: one immediately upon awakening without rinsing the mouth with water, one 15 min and one 30 min after. To avoid contamination of saliva, participants will be asked not to brush their teeth, drink or eat at least 60 min before sampling.
Saliva samples will be centrifuged at 4 °C (1,000 rpm, 2 min; Hettich MR22) and stored at − 80 °C until assayed. The samples will be assayed for salivary-free cortisol as a duplicate in a single assay batch per participant via commercially available enzyme immunoassay (IBL International GmbH).