Slice Preparation and Patch-Clamp Recordings.

Procedures identical to those previously published (16, 77, 78) were used for slice preparation, electrophysiological recordings, and data analysis. Briefly, rats were rendered unconscious with carbon dioxide and then were decapitated. After brain removal, coronal cerebellar slices from the cerebellum were cut at 34 °C on a vibrating slicer (Leica VT1200S) with sucrose artificial cerebrospinal fluid (S-ACSF) containing (in mM) 200 sucrose, 2.5 KCl, 1.2 MgCl₂, 0.5 CaCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 20 dextrose. Slices were incubated for 1 h at 34 °C in 95% O₂-saturated and 5% CO₂-saturated ACSF containing (in mM) 125 NaCl, 3.0 KCl, 1.2 MgSO₄, 2.0 CaCl₂, 1.2 NaH₂PO₄, 26 NaHCO₃, and 10 dextrose and then were maintained at room temperature until electrophysiological recording. Vertical vibration of the blade was manually adjusted with a Vibrocheck device (Leica Biosystems) before slice preparation and was set to 0 µM.

A slice was placed in a modified recording chamber containing the bath solution (ACSF). DCN neurons were identified morphologically through a 40× water-immersion objective using differential interference contrast (DIC)-IR optics (Olympus BX50WI). PRV-infected neurons were identified by GFP under fluorescence optics. Whole-cell patch-clamp recordings were performed using an Axon MultiClamp 700B on cells with diameters of 15–20 µM in the interpositus and the medial portion of the lateral nucleus. These neurons are regarded as large glutamatergic projection neurons (26, 33). Generally, we made recordings in two to three cells of DCN slices from each rat. Patch pipettes made from borosilicate glass (1.5 mm o.d., 0.86 mm i.d.; catalog no. BF150-86-10; Sutter Instrument Company) were pulled with a P97 Brown–Flaming micropipette puller (Sutter Instrument Company). The final resistances of pipettes filled with the internal solution [containing (in mM) 140 potassium gluconate (C₆H₁₁O₇K), 4.6 MgCl₂·6H₂O, 10 Hepes, 10 EGTA, 4.0 Na₂ATP, pH 7.3 (KOH)] were between 5 and 8 MΩ. Data were low-pass filtered at 2 kHz and acquired at 20 kHz. Membrane properties were measured when the neuron had stabilized for 5 min after the whole-cell configuration was achieved. Quantitative analysis included resting membrane potential measured directly upon breakthrough in the whole-cell configuration, input resistance based on membrane potential changes due to depolarizing current injections immediately after whole-cell configuration, AP threshold (APH), current required for eliciting the first AP, the half-width of elicited AP (APD₅₀) including rising and falling phases, the amplitude of elicited AP, the number of elicited APs, latency to the first AP elicited by a 250-ms duration depolarizing current injection, peak amplitude of the AHP, S1S2 interval, the current required for hyperpolarization-induced RDs, and the properties of RDs. Recordings were accepted only if the resistance of the initial seal formations was greater than 1 GΩ and were rejected if their output was unstable or series resistance changed more than 20%. To obtain an accurate measurement of neuronal excitability independent of membrane potential changes, continuous direct current was applied through the recording electrode to hold the cell at a −70 mV baseline. All recordings were made at room temperature.

All electrophysiological data were recorded online using pCLAMP 10 software (Axon Instruments). Standard off-line analyses were conducted using Clampfit 10.0.

Data are presented as means ± SEM. One-way ANOVA followed by least significant difference post hoc comparisons was calculated in SPSS (Version 24; SPSS Inc.) with P < 0.05 as the criterion for significance.