Acute tryptophan depletion
ATD inhibits serotonin synthesis by lowering the availability of the serotonin precursor, tryptophan. This is achieved by participants consuming a tryptophan-free amino acid drink, which results in a substantial decline in plasma tryptophan (Trp) levels, with peak depletion occurring approximately 4–7 h later (Williams et al. Venous blood samples (taken pre- and 4-h post-oral consumption) were separated by centrifugation for 10 min at a relative centrifugal force (RCF) of 2500 g. Concentrations of free Trp, tyrosine (TYR), phenylalanine (PHE), valine (VAL), leucine (LEU), and isoleucine (ILE) in plasma were analyzed by Biochrom 30 Plus amino acid analyzer (Biochrom Ltd., UK). VAL, LEU, and ILE levels were analyzed to calculate the ratio of plasma Trp to other large neutral amino acids (LNAAs). 5-HT depletion magnitude was calculated using the ratio of TRYP/ΣLNAAs before ATD at baseline minus T1 (after 4 h, immediately before fMRI experiment) (ATD baseline: TRYP/ΣLNAAs − ATDT1: TRYP/ΣLNAAs). The 5-HT supplementation magnitude was calculated using the ratio of TRYP/ΣLNAAs after TRYP supplementation at baseline minus T1 (TRYP + baseline: TRYP/ΣLNAAs − TRYP + T1: TRYP/ΣLNAAs).