Nahco3 26

Slices were prepared from P17–P90 male CD1 EAAT4-GFP or EAAT4-GlyT2-GFP mice. This strain was obtained by crossing EAAT4-GFP mice with a line expressing GlyT2-GFP (gift from H.U. Zeilhofer) (Zeilhofer et al., 2005 (link)), in which some GoCs express GFP. Mice were anesthetized by inhalation of isoflurane, and then killed by decapitation. Transverse slices were prepared as previously described (Valera et al., 2012 (link)). The cerebellum was dissected out and placed in cold artificial cerebrospinal fluid (ACSF) bubbled with carbogen (95% O₂, 5% CO₂), containing (in mM): NaCl 120, KCl 3, NaHCO₃ 26, NaH₂PO₄ 1.25, CaCl₂ 2.5, MgCl₂ 2, glucose 10 and minocycline 0.00005 (Sigma-Aldrich, USA). Then 300 µm-thick transverse slices were prepared (Microm HM 650V, Microm, Germany) in potassium-based medium, containing (in mM): K-gluconate 130, KCl 14.6, EGTA 2, HEPES 20, glucose 25, minocycline 0.00005 and D-AP5 0.05 (Sigma-Aldrich). After cutting, slices were soaked in a sucrose-based medium at 34°C, containing (in mM): sucrose 230, KCl 2.5, NaHCO₃ 26, NaH₂PO₄ 1.25, glucose 25, CaCl₂ 0.8, MgCl₂ 8, and minocyclin 0.00005 (Sigma-Aldrich) and maintained in a water bath at 34°C in bubbled ACSF.

To mitigate other anesthetic-driven confounding factors in our synaptic studies, we used Iso for acute induction of anesthesia in experimental mice (8 per group) previously exposed to sham or Iso prior to brain extraction. Following anesthesia, the brains were extracted rapidly and chilled in ice-cold dissection solution (in mM: sucrose 206, Na-Pyruvate 2, KCl 2, NaH₂PO₄ 1.25, NaHCO₃ 26, glucose 10, MgCl₂ 4, MgSO₄ 2, ascorbic acid 0.4, CaCl₂ 0.5; All from Sigma-Aldrich, USA). Transverse hippocampal slices (400μm) were cut with a VF300 microtome (Precisionary Instruments, USA). Intact slices were placed in a holding chamber containing aCSF (in mM: NaCl 124, KCl 2, NaH₂PO₄ 1.25, NaHCO₃ 26, glucose 10, CaCl₂ 2.5, MgSO₄ 1) at 32°C and oxygenated with 95% O₂/5% CO₂ for at least 2h before recordings. The osmolarity of all solutions was measured at 300–310 mOsm and the pH was maintained at ~7.3 under constant carbogenation.