Monoamine neurotransmitters (MAs), including dopamine (DA) and serotonin (5-HT), regulate brain functions such as behavior, memory, and learning. Neonicotinoids are pesticides that are being used more frequently. Neonicotinoid exposure has been observed to produce neurological symptoms, such as altered spontaneous movements and anxiety-like behaviors, which are suspected to be caused by altered MA levels. However, current neurotoxicity tests are not sufficiently sensitive enough to make these determinations. In this study, we performed some behavior tests, and derivatization reagents to improve the ionization efficiency, which was applied to liquid chromatography mass spectrometry (LC-MS/MS) to reveal the effect of neonicotinoid administration on MAs in the brain. We orally administered the neonicotinoid imidacloprid (0, 10, and 50 mg/kg body weight) to C57BL/6NCrSlc mice. In the behavior tests, a decrease in activity was observed. The LC-MS/MS quantification of MAs in various brain regions showed a decrease in some MA levels in the olfactory bulb and the striatum. These results showed, for the first time, that even a low dose of imidacloprid could alter MA levels in various parts of the brain.

Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of prion protein (PrP^Sc) after conversion of cellular prion protein (PrP^C). The glycosylphosphatidylinositol (GPI) anchor of PrP^C is involved in prion disease pathogenesis, and especially sialic acid in a GPI side-chain reportedly affects the PrP^C conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrP^C. Moreover, the sialic acid linkage–type in the GPI side-chain has not been determined for any GPI-anchored protein. Here, we report GPI-glycan structures of human PrP^C isolated from human brain and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. Liquid chromatography–electrospray ionization MS (LC–ESI–MS) analysis of human PrP^C from both biological sources indicated that Gly-229 is the ω site in PrP^C to which GPI is attached. Gly-229 in human PrP^C does not correspond to Ser-231, the previously reported ω site of Syrian hamster PrP^C. We found that approximately 41% and 28% of GPI anchors in human PrP^Cs from human and mouse knock-in brains, respectively, have N-acetylneuraminic acid in the side-chain. Using a sialic acid linkage–specific alkylamidation (SALSA) method to discriminate α2,3-linkage from α2,6-linkage, we found that N-acetylneuraminic acid in PrP^C’s GPI side-chain is linked to galactose through an α2,3-linkage. In summary, we report the GPI-glycan structure of human PrP^C, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.