Nah2po4

Triticale grains (Triticum aestivum L. emend. Fiori et Paol) were obtained from local commercial suppliers. Wheat grains with known seed-borne Fusarium infections were obtained from the Human Microbiology Institute (New York, NY, USA). Potato dextrose agar (PDA; Thermo Fisher Scientific Inc., Waltham, MA, USA) was used as a growth medium for seed germination.
Grains in the control group were sterilized according to Htwe et al. (2011) (link), with minor modifications. Briefly, uniformly sized grains were washed in a solution of 4.5% (w/v) NaOCl:water (1:1 ratio) for 20 min. For the experimental groups, uniformly sized grains were treated with different concentrations of three different solutions: 1) 0.1% and 0.05% M451 (a water-based solution of TGV-28 [Figure 1]; Human Microbiology Institute) in combination with 0.01% NaH₂PO₄ (Sigma Aldrich, St. Louis, MO, USA) for 30 min, 2) 0.01% NaH₂PO₄ for 30 min (Sigma Aldrich), and 3) Maxim XL v/v1:10 dilution in water for 12 h according to the manufacturer’s instructions (Syngenta Canada Inc., Guelph, ON, Canada) (Costa et al., 2019 (link); Pfeiffer et al., 2021 (link)). Subsequently, the grains from control and M451-treated groups were washed three times with autoclaved distilled water and then air-dried on filter paper. The grains treated with Maxim XL were left unwashed.

Rats were deeply anesthetized with isoflurane (Kent Scientific) and euthanized by decapitation. The brain was rapidly dissected and glued on a platform submerged in an ice-cold oxygenated (95% O₂/5% CO₂) cutting solution containing (in mM): 206 sucrose (Sigma-Aldrich), 10 D-glucose (Sigma-Aldrich), 1.25 NaH₂PO₄ (Sigma-Aldrich), 26 NaHCO₃ (Sigma-Aldrich), 2 KCl (Fisher Chemical), 0.4 sodium ascorbic acid (Sigma-Aldrich), 2 MgSO₄ (Sigma-Aldrich), 1 CaCl₂ (Sigma-Aldrich), and 1 MgCl₂ (Sigma-Aldrich). A mid-sagittal cut was made to divide the two hemispheres, and coronal brain slices (300 μm) were cut using a vibrating blade microtome (Leica VT1200). The brain slices were transferred to a holding chamber with oxygenated artificial CSF (ACSF) containing (in mM): 119 NaCl (Sigma-Aldrich), 2.5 KCl (Fisher Chemical), 1 NaH₂PO₄ (Sigma-Aldrich), 26.2 NaHCO₃ (Sigma-Aldrich), 11 D-glucose (Sigma-Aldrich), 1 sodium ascorbic acid (Sigma-Aldrich), 1.3 MgSO₄ (Sigma-Aldrich), and 2.5 CaCl₂ (Sigma-Aldrich; ∼295 mOsm, pH 7.2–7.3) at 37°C for 20 min and then room temperature for at least 40 min of rest. The slices were kept submerged in oxygenated ACSF in a holding chamber at room temperature for up to 7–8 h after slicing.

Mice were killed 3 h following water or saccharin consumption by decapitation, following anesthesia with isoflurane. Brain slices were cut at 300 μm in coronal sections using a vibratome (Campden-1000) in ice chilled cutting solution (110 mm sucrose, 60 mm NaCl, 3 mm KCl, 1.25 mm NaH₂PO₄, 28 mm NaHCO₃, 0.5 mm CaCl₂, 7 mm MgCl₂, 5 mm D-glucose, and 0.6 mm ascorbate, Sigma-Aldrich). The slices were placed in artificial CSF (ACSF; 125 mm NaCl, 2.5 mm KCl, 1.25 mm NaH₂PO₄, 25 mm NaHCO₃, 25 mm D-glucose, 2 mm CaCl₂, and 1 mm MgCl₂, Sigma-Aldrich) for a 30 min recovery period at 37°C and then kept at room temperature for at least an additional 30 min before electrophysiological recording. Throughout, ACSF was continually gassed, using carbogen (O₂ 95%, CO₂ 5%).