Slice preparation and whole-cell patch-clamp recording

WT and ShcB-KO mice were sacrificed by cervical dislocation under deep anesthesia with diethyl ether. The cerebellum was excised, and parasagittal cerebellar slices (250 μm thick) were prepared from the vermis^50,51,58. Whole-cell recordings were obtained from visually identified PCs under an upright microscope (BX51WI, OLYMPUS, Tokyo, Japan) using a 40 × water-immersion objective at room temperature (23–25 °C), except for the experiments using dantrolene (36 ± 1 °C). The resistance of patch pipettes was 2.5–4.0 MΩ when filled with an intracellular solution composed of (in mM) 120 K-gluconate, 5 KCl, 5 NaCl, 1 EGTA, 4 ATP, 0.4 GTP and 10 HEPES (pH 7.3; adjusted with KOH). For the voltage-clamp recording of CF-EPSCs, a pipette solution with the following composition was used (in mM) 60 CsCl, 10 Cs D-gluconate, 20 TEA-Cl, 20 BAPTA, 4 MgCl2, 4 ATP, 0.4 GTP and 30 HEPES (pH 7.3). The standard bathing solution was composed of (in mM) 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgSO₄, 1.25 NaH₂PO₄, 26 NaHCO₃ and 20 glucose, which was bubbled continuously with a mixture of 95% O₂ and 5% CO₂. Bicuculline (10 μM) was always present in the saline to block spontaneous inhibitory postsynaptic currents. Square pulses were applied for focal stimulation (duration, 0.1 ms; amplitude, 0–90 V for CF stimulation, 0–10 V for PF stimulation) through a glass pipette with a 5 to 10 μm-diameter tip and filled with the standard bath solution. The membrane potentials were held at − 90 to − 80 mV for recording PF-EPSCs, and at − 20 to − 10 mV for recording CF-EPSCs, after the compensation for liquid junction potential. Ionic currents were recorded using a patch-clamp amplifier (EPC-9, HEKA, Lambrecht/Pfalz, Germany). Stimulation and on-line data acquisition were performed using PULSE software (HEKA) on a Macintosh computer. Signals were filtered at 3 kHz and digitized at 20 kHz. The fitting of the decay phases of EPSCs was performed with PULSE-FIT software (HEKA).

For the LTD experiments, the intensity of the stimulus was adjusted to evoke PF-EPSCs whose initial amplitudes were between 80 and 150 pA. After obtaining a stable initial recording for at least 10 min, a conjunctive stimulus was applied to induce LTD. The conjunctive stimulation protocol is composed of 300 single PF stimuli in conjunction with depolarization pulses (− 80 to 0 mV, for 50 ms) repeated at 1 Hz. Series resistance and membrane resistance were monitored throughout the experiments, and the data were discarded if either of these resistances varied by more than 10%. The data were also discarded if the slope of the PF-EPSC amplitude averaged every minute during the initial recording for 10 min was larger than 2% or if the amplitude did not stabilize within 20 min after the onset of the whole-cell conditions^34,36,59. The PF-EPSC amplitude was normalized by the mean value observed for 10 min before the CJS.