Cerebrospinal Fluid

Animals were anesthetized with isoflurane. After decapitation, the brain was quickly transferred to the frozen cutting solution (containing, in mM, 15 KCl, 3.3 MgCl₂, 110 K-gluconate, 0.05 EGTA, 5 HEPES, 25 glucose, 26.2 NaHCO₃ and 0.0015 (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) with carbogen gas (95% O₂ and 5% CO₂). The brain was sliced into 250-µm thick sections using a vibratome (VT1200S, Leica), and transferred into artificial cerebrospinal fluid (aCSF, containing, in mM, 124 NaCl, 3 KCl, 2 MgCl₂, 2 CaCl₂, 1.23 NaH₂PO₄, 26 NaHCO₃, 25 glucose) with carbogen gas (95% O₂ and 5% CO₂) at 35 °C for at least 1 h, then at room temperature covered with aluminum foil to avoid light exposure. An amplifier (Multiclamp 700B, Molecular Devices) and a digitizer (Axon Digidata 1550B, Molecular Devices) were used for patch clamp recording. The recording chamber was perfused with aCSF saturated with carbogen gas (95% O₂ and 5% CO₂) at room temperature. A glass pipette (GC150-10; Harvard Apparatus) was made with a puller (P-1000, Sutter Instrument) and its resistance was between 2.8 and 7 MΩ. The pipette was loaded with K-gluconate-based pipette solution (in mM, 138 K-gluconate, 8 NaCl, 10 HEPES, 0.2 EGTA-Na₃, 2 Mg-ATP, and 0.5 Na₂-GTP, pH 7.3 with KOH) for whole-cell recording, or aCSF for loose cell recording. Under an epifluorescent microscope (BX51WI, Olympus), 505 nm LED illumination (74 µW/mm², 100 ms, Niji, Blue Box Optics) was used to visualize native EYFP fluorescence from ACR2-EYFP. We identified LC-NA neurons by a combination of anatomical location, triangular cellular shape, and fluorescence observed in the recording area. In Fig. 2, the membrane potential was held at − 60 mV for measuring current deflection. In Fig. 3, the membrane potential was held from − 120 to − 40 mV in 20 mV steps with a duration of 700 ms. The voltage deflection was evaluated at a current holding of 0 pA. Clampex 11.0.3 (Molecular Devices) was used to record the data.