Chromeleon integration 6

Concentrations of monoamines (DA, 5-HT) and their metabolites (dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA)) were measured in the FCx, STR, and HPC by HPLC-EC, essentially as previously described [38 (link)]. Briefly, 600 µL of extraction buffer were added to the different brain structures, which were then homogenized in a TissuLyser system (3 × 1 min at 30 Hz, 4 °C; Qiagen, Courtaboeuf, France). After 20 min of centrifugation (16,000× g, 4 °C), the supernatant containing the analytes to be measured was collected and divided into two aliquots. The first was immediately frozen at −80 °C for protein analysis by Western blotting (WB), while the second was centrifuged further for 2 min in filter tubes (1600× g, 4 °C) before being stored at −80 °C until use for HPLC-EC. For this purpose, 20 µL of each sample were injected into a high-performance liquid chromatograph equipped with an electrochemical detector coupled to a Chromeleon integration 6.8 software (Dionex, Sunnyvale, CA, USA), which allows the detection of the different analytes based on their respective retention time. Final concentrations were calculated against external standards, which were injected twice daily, and expressed per g of fresh tissue.

Brain 5-HT, DA and their metabolites, the 5-hydroxyindoleacetic acid (5-HIAA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by HPLC-EC, essentially as previously described (30 (link)). Half structures of interest were lysed in 600 μL of fresh extraction buffer (4 × 1 min at 30 Hz) using a Tissue Lyser (Qiagen, Courtaboeuf, France). After homogenate centrifugation (16,000 g, 4°C, 20 min), the supernatants were centrifuged again into filtering tubes (1,600 g, 4°C, 2 min) and the final supernatants containing the monoamines stored at −80°C until use. For HPLC assay, 20 μL of the supernatant were injected into a chromatograph equipped with a 5 μm C18, 3×100 mm silica column (ACE, AIT France, Cormeilles-en-Parisis, France) and coupled to an electrochemical detection system (Antec Decade 2, CJ Lab, La Frette, France). Monoamines and their metabolites were identified through their retention times and quantified using the Chromeleon integration 6.8 software (Dionex, Sunnyvale, CA, US). Results were expressed in nmoles/g of tissue.