Electrophysiology

Mice aged 10–52 days postnatally were decapitated following CO₂ anesthesia, and brains were rapidly removed and placed into chilled external solution (0–4 °C) containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1.25 mM NaH₂PO₄, 26 mM NHCO₃, and 20 mM glucose bubbled with 95% O₂ and 5% CO₂ (pH 7.4). Parasagittal cerebellar slices (250 μm thick) were prepared by using a vibratome slicer (VT1200s, Leica). The slices were recovered for 1 h at room temperature by incubating in a reservoir chamber bathed in the external solution. One slice was transferred to a recording chamber located on the stage of an upright microscope (BX51WI, Olympus). The recording chamber was continuously perfused with the oxygenated external solution supplemented with picrotoxin (0.1 mM, TOCRIS) to block inhibitory synaptic transmission. Whole-cell recordings were made from visually identified or fluorescent protein-positive PCs using an upright and fluorescent microscopes at 32 °C. The resistance of the patch pipettes was 1.5–2.5 MΩ when filled with the intracellular solution composed of 60 mM CsCl, 10 mM Cs d-Gluconate, 20 mM TEA-Cl, 20 mM BAPTA, 4 mM MgCl₂, 4 mM Na₂-ATP, and 0.4 mM Na₂-GTP (pH 7.3, adjusted with CsOH). The pipette access resistance was compensated by 70%. For recording mIPSCs, 10 μM NBQX, 5 μM R-CPP, and 1 μM TTX (tetrodotoxin) were added. Ionic currents were recorded with an EPC9 patch clamp amplifier (HEKA-Electronik). The signals were filtered at 3 kHz and digitized at 20 kHz. On-line data acquisition and off-line data analysis were performed using PULSE software (HEKA-Electronik). Stimulation pipettes were filled with the standard saline.

CFs were stimulated in the GL at 20–100 μm away from the PC soma through a patch pipette filled with the normal external solution with stimuli (duration, 0.1 ms; amplitude, 0–100 μA) repeated at 0.2 Hz. When a CF was stimulated, EPSCs were elicited in an all-or-none manner in the majority of control PCs, indicating that such PCs were innervated by single CFs. CF-mediated EPSCs (CF-EPSCs) displayed prominent paired-pulse depression with inter-stimulus interval of 50–100 ms, which enabled us to distinguish them from PF-EPECs that displayed clear paired-pulse facilitation⁶⁴. In some PCs, more than one discrete CF-EPSCs could be elicited when the stimulus intensity was increased gradually as exemplified in Supplementary Fig. ⁴. The number of CFs innervating the PC was estimated by counting the number of discrete CF-EPSC steps. To confirm the number of CFs innervating PC under recording, the stimulation electrode was moved systematically around the PC soma within the GL. Amplitude of CF-EPSCs was measured at the holding potential of −10 mV. The holding potential was corrected for liquid junction potential. For the CF-EPSCs whose amplitudes were smaller than 100 pA, they were evoked again at −70 mV to confirm that they represented discrete EPSC steps and paired-pulse depression. To examine the effects of knocking down BDNF in PCs, PCs were sampled at locations in which virus-infected PCs were rich (mOrange2-positive regions) and from locations in which transfected PCs were absent (mOrange2-negative regions). CF innervation patterns were compared between mOrange2-positive (BDNF KD) and mOrange2-negative (Control) regions in the same slices. To examine the effects of knocking down TrkB or p75^NTR in CFs, PCs were sample at locations in which virus-infected CFs were rich (EGFP-positive regions) (Fig. 5c) and from locations in which transfected CFs were absent (EGFP-negative regions). CF innervation patterns were compared between EGFP-positive (TrkB KD/p75^NTR KD) and EGFP-negative (Control) regions in the same slices. PFs were stimulated in the ML, and PF-EPSCs were recorded at the holding potential of −70 mV with gradually decreasing stimulus intensity from 10 to 1 μA.