Hepes 20

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.

Under the control of a binocular loupe (Zeiss, Oberkochen, Germany), the substantia nigra was cut out of the brain of adult animals at the coordinates–5.2 to 0 mm from Bregma, in accordance with the mouse brain atlas [72 ]. The obtained block of nervous tissue was mounted on a table placed in a chamber with cooled (4 °C) Krebs-Ringer buffer (KRB) of the following composition (mM): NaCl 120, KCl 4.8, CaCl₂ 2.0, MgSO₄ 1.2, NaHCO₃ 25, D-glucose 10.1, HEPES 20, and ascorbic acid 0.13 (all from Sigma-Aldrich, St. Louis, MO, USA), pH 7.4. Then, serial sections of the substantia nigra, 70 µm thick, were prepared using a vibratome (Microm HM 650 V, Thermo Scientific, Waltham, MA, USA).