Choroid Plexus Rb+ Efflux Assay

Choroid plexus was isolated as above, initially in cold aCSF (in mM: 120 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.3 MgSO₄, 1 NaH₂PO₄, 25 NaHCO₃, 10 glucose, pH 7.4, equilibrated with 95% O₂/5% CO₂) but allowed to recover at 37 °C for 5–10 min before beginning of the experiment. Choroidal isotope accumulation was performed by a 10 min incubation in equilibrated (95% O₂/5% CO₂) aCSF-based isotope medium (2 μCi ml^{−1 86}Rb⁺, NEZ07200 (congener for K⁺ transport) and 8 μCi ml^{−1 3}H-mannitol, NET101 (extracellular marker), both from PerkinElmer), followed by 15 s wash prior to incubation in 0.5 ml equilibrated (95% O₂/5% CO₂) efflux medium (aCSF containing 20 μM bumetanide, 1 mM furosemide or vehicle (DMSO), each choroid plexus randomly assigned to each group). 0.2 ml of the efflux medium was collected into scintillation vials every 20 s (time points: 0, 20, and 40 s) and replaced with fresh aCSF. At the end of the experiment, choroid plexus was solubilized at room temperature with 1 ml Solvable (6NE9100, PerkinElmer) in the leftover efflux medium. The isotope content was determined by liquid scintillation counting with Ultima Gold^TM XR scintillation liquid (6013119, PerkinElmer) in a Tri-Carb 2900TR Liquid Scintillation Analyzer (Packard). The choroid plexus ⁸⁶Rb⁺ content corrected for ³H-mannitol (extracellular background) was calculated for each time point, and the natural logarithm of the choroid plexus content A_t/A₀ was plotted against time⁴⁷ . Slopes indicating the ⁸⁶Rb⁺ efflux rate constants (s⁻¹) were determined from linear regression analysis.