Cerebellar Purkinje Cell Electrophysiology

All experiments were approved by the Institutional Animal Care Unit Committee in Administration Office of Laboratory Animals Beijing China (B10831). Cerebellar sagittal slices (400 μm) were prepared from Wistar rats in postnatal day 14 ~ 15 that were anesthetized by injecting chloral hydrate (300 mg/kg) and decapitated by a guillotine. The slices were cut by a Vibratome in the modified and oxygenized (95% O₂ and 5% CO₂) artificial cerebrospinal fluid (mM: 124 NaCl, 3 KCl, 1.2 NaH₂PO₄, 26 NaHCO₃, 0.5 CaCl₂, 5 MgSO₄, 10 dextrose and 5 HEPES; pH 7.4) at 4°C, and were held in the normal oxygenated ACSF (mM: 126 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 2 CaCl₂, 2 MgSO₄ and 25 dextrose; pH 7.4) at 35°C for 1–2 hours. A slice was transferred to a submersion chamber (Warner RC-26G) and perfused by normal ACSF for electrophysiological experiments [57 (link),68 (link)-70 (link)].
Cerebellar Purkinje cells were identified based on their morphology and functions. Purkinje cells (somata above 40 μm) in the slices for whole-cell recording were located at the border between molecular layer and granule cells, and infused by fluorescence Alex-488 (5 μM in pipettes) under a DIC/fluorescent microscope (Nikon, FN-E600). The excited fluorophore showed the typical dendrites and axonal branches of Purkinje cells, through which we traced their main axons for recording spikes by loose-patch. Purkinje cells also were labeled by neurobiotin (Figure 1A). Purkinje cells showed fast spiking and no adaptation in amplitude and frequency [18 (link),42 (link),71 (link)-75 (link)].