Cerebellar recordings

The brains of 18–20-month-old mice were removed in ice cold protective cutting artificial cerebrospinal fluid (aCSF1) containing (in mM): 95 N-Methyl-D-glucamine, 30 NaHCO₃, 20 HEPES, 25 glucose, 2.5 KCl, 1.25 NaH₂PO₄, 2 thiourea, 5 sodium ascorbate, 3.0 sodium pyruvate, 10 MgSO₄, 0.5 CaCl₂, 12 N-acetylcysteine, adjusted to pH 7.3 and an osmolarity of 300–310 mOsmol, saturated with 95% O₂/5% CO₂)⁸³ and cut into halves. 300 µm thick coronal slices were made from the cerebellum with a vibratome (VT1200S; Leica, Wezlar, Germany). Slices were placed in an incubation beaker with aCSF1 at 34 °C for 10–15 min, then transferred into another incubation beaker with aCSF2 (in mM): 125 NaCl, 25 NaHCO₃, 25 glucose, 2.5 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 2 thiourea, 5 sodium ascorbate, 3 sodium pyruvate, 12 N-acetylcysteine, adjusted to pH 7.3 and an osmolarity of 300–310 mOsmol, saturated with 95% O₂/5% CO₂) until use.

Loose-Patch recordings from cerebellar Purkinje cells (PCs) were performed in aCSF3 containing (in mM): 125 NaCl, 25 NaHCO₃, 25 glucose, 2.5 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, saturated with 95% O₂/5% CO₂) as described previously⁸⁴, using thick-walled borosilicate glass recording electrodes (2.0 mm o.d., Science Products, Germany) filled with aCSF3 and a final resistance of 3–5 MOhm. PC spikes were recorded for 100 s. Evoked responses were elicited by stimulating parallel fibers about 200 µm away from the PC bodies using a monopolar stimulation with an additional pipette containing aCSF3 (DS3, Digitimer). Frequency, interspike interval and CV of ISI were analyzed with Neuromatic Plugin of IgorPro software (WaveMetrics, Portland, OR, USA).