Electrophysiology.

Slices were placed in the well of a Plexiglas chamber attached to a modified stage on an Olympus BX51WI upright microscope and perfused with aCSF containing 5 mM CsCl to block the slow depolarizing afterpotential (Ghamari-Langroudi and Bourque 1998; Teruyama and Armstrong 2005, 2007) and 10 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX; Sigma-Aldrich, St. Louis, MO), 40 µM 2R-amino-5-phosphonovaleric acid (Tocris, Minneapolis, MN), and 100 µM picrotoxin (Tocris) to block fast synaptic currents. The aCSF was bubbled constantly with 95% O₂-5% CO₂, warmed to 32°C ± 1°C, and flowed at ~2 ml/min. For recordings in which we applied Cd²⁺ to the cells, we used phosphate-free aCSF consisting of the following (in mM): 121.0 NaCl, 1.3 MgCl₂, 3.0 KCl, 26.0 NaHCO₃, 20.0 D-glucose, and 2.5 CaCl₂ to prevent Cd²⁺ precipitation. Whole cell and current-clamp recordings were obtained using an Axon Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA). Traces were digitized using an Axon 1440A Digitizer at 10 kHz on a Dell desktop computer running Clampex 9 software (Molecular Devices).

Recording pipettes (4–8 MΩ) were pulled from borosilicate glass (1.5-mm OD) using a P-1000 flaming/brown horizontal micropipette puller (Sutter Instruments, Novato, CA). The pipette internal solution consisted of the following (in mM): 135.0 KMeSO₄, 8.0 NaCl, 10.0 HEPES, 2.0 Mg-ATP, 0.3 Na-GTP, 0.1 leupeptin, 6.0 phosphocreatine, and 0.2 EGTA with pH 7.2–7.4 and 285–295.0 mOsm (kg/H₂O). Biocytin (0.1%) (Sigma-Aldrich) was added on the day of the experiment for visualization during immunochemical identification of cell type (Horikawa and Armstrong 1988). The liquid junction potential for the KMeSO₄ internal was approximately −10 mV and was not corrected.

AHPs measured in current clamp were evoked from a resting membrane potential of −55 mV by injecting 300 pA of current via a 20-spike, 20-Hz pulse train. AHP amplitudes correlate with spike count, so, to isolate the mAHP, we injected a shorter train of 5 spikes of the same amplitude and frequency (Ghamari-Langroudi and Bourque 2004). For Ca²⁺ channels, reagents included 5.0 µM nifedipine (Nif; Sigma-Aldrich) to block L-type channels, 1.0 µM ω-conotoxin GVIA (CnTx GVIA; Peptides International, Louisville, KY or Alomone Laboratories, Jerusalem, Israel) to block N-type channels, 500.0 nM agatoxin-IVA (AgTx IVA; Alomone Laboratories) to block P/Q-type channels, and 0.3 μM SNX-482 (Alomone Laboratories) to block R-type channels. Because SNX-482 also blocks A-type K⁺ currents (Liu and Bean 2014), 4 mM 4-aminopyridine (4-AP; Sigma-Aldrich) was used to block A-current (I_A) and other voltage-gated K⁺ channels contributing to spike width before administering SNX-482. Cells whose series resistance exceeded 20 MΩ and/or changed by >20% during the recording were discarded. Traces were averaged over two or more runs for each individual cell.