Patch-Clamp Electrophysiology.

Coronal slices (200 μm) of 3- to 5-wk-old male mice were cut with a vibrating microtome in ice-cold cutting solution (KCl 2.5 mM, NaH₂PO₄ 1.25 mM, MgCl₂ 5 mM, CaCl₂ 0.5 mM, NaHCO₃ 26 mM, glucose 11 mM, sucrose 210 mM, pH 7.4). Slices were incubated for 30 min in oxygenated artificial CSF (NaCl 125 mM, KCl 2.5 mM, NaH₂PO₄ 1.25 mM, MgCl₂ 1 mM, CaCl₂ 2 mM, NaHCO₃ 26 mM, glucose 11 mM, pH 7.4) at room temperature, and then were recorded, submerged, and perfused with oxygenated artificial CSF (2 mL/min) maintained at room temperature. Patch-clamp recordings were performed in whole-cell, current-clamp mode by using a Multiclamp 700B amplifier (Molecular Devices) in current-clamp mode. Signals were low-pass filtered at 5 kHz and digitized at 50 kHz by using a Digidata1322A interface and acquired by using Clampex 10.2 software (Molecular Devices). Data were analyzed by using Clampfit 9.0 (Molecular Devices). Patch electrodes were filled with a pipette solution (K-gluconate 125 mM, KCl 10 mM, MgATP 4 mM, Hepes 10 mM, Na-GTP 0.3 mM, phosophocreatine-Na₂ 8 mM, EGTA 0.5 mM, pH 7.2). Neurobiotin (0.1%) was also added to the pipette solution to label the patched-clamp cells. We assessed pharmacological effects on the change of firing rate in the absence of tetrodotoxin (TTX, Nakarai Tesque) and on the change of resting potential in the presence of TTX (1 μM). YNT-185, EMPA, and orexin-A were dissolved with DMSO, and TTX was dissolved with distilled water and adjusted with artificial CSF. TMN neurons were identified by a depolarizing sag during hyperpolarizing pulses produced by activation of a hyperpolarization-activated current (16). After patch-clamp experiments, slices were fixed and the neurobiotin in the patched cells was identified with streptavidin conjugated with Alexa Fluor 488 (1:1,000; Invitrogen). These histaminergic neurons were identified with guinea pig anti-histidine decarboxylase (HDC) antibody (1:1,000; EuroProxima, catalog no. 2263B-GP265-1) and anti-guinea pig IgG antibody conjugated with Alexa Flour 594 (1:1,000; Invitrogen). Finally, slices were mounted with Vectashield H-1200 (Vector) and examined by using an LSM700 microscope (Zeiss).