Fluorocitric Acid Preparation and Use

DL-fluorocitric acid (FCA) barium salt (#F9634) and barium chloride (BaCl₂, #217565) were purchased from Sigma-Aldrich. Because FCA was commercially available as Ba²⁺ salt, which may interfere with neuronal physiological properties leading to confounding results, we decided (Chou et al., 2009 (link)) to use BaCl₂ salt in control patch-clamp experiments and (Furlan et al., 2006 (link)) to precipitate Ba²⁺ during drug preparation for in vivo stereotaxic microinjections. For electrophysiology experiments (see 2.6), BaCl₂ was dissolved in H₂O and FCA in HCl 0.1 M as a stock solutions and diluted (1:100) to the final concentration (100 µM) in artificial cerebrospinal fluid (ACSF, in mM: 117 NaCl, 4.7 KCl, 1.2 NaH₂PO₄, 2.5 CaCl₂, 1.2 MgCl₂, 25 NaHCO₃ and 11 glucose) on the day of the experiment (Khan et al., 2019 (link); Vizuete et al., 2019 (link)). For intra-CeA microinjection (see 2.7.1), the compound was dissolved in HCl 0.1 M and the Ba²⁺ was precipitated by the addition of (2–3 drops) Na₂SO₄ 0.1 M. This solution was buffered with Na₂HPO₄ 0.1 M and centrifuged at 800 g for 10 min. The supernatant was removed and diluted in ACSF to the final concentration (100 µM) and pH was adjusted to 7.3 (Paulsen et al., 1987 (link); Hayakawa et al., 2010 (link); Paquette et al., 2021 (link)).