Slice preparation, electrophysiology, and uncaging

Coronal 250 μm slices containing NAc were prepared in ice-cooled artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 120, KCl 2.5, MgCl₂ 1.0, CaCl₂ 2.5, Na₂HPO₄ 1.25, NaHCO₃ 26.0, glucose 14.6 and HEPES 5.0, bubbled with 95% O₂ and 5% CO₂. Slices were kept at 34 °C for 10–20 minutes and then kept at room temperature until recordings. In the recording chamber, slices were superfused with aCSF at 30–32 °C. Visualized whole-cell patch-clamp recording techniques were used to measure the response to the uncaging of orexin. Patch pipettes (3–7 MΩ) were pulled from borosilicate glass pipettes (GC150T-10, Harvard Apparatus). The internal solution contained (in mM): potassium gluconate 130, MgCl₂ 4, MgATP 3.4, Na₃GTP 0.1, creatine phosphate 10, HEPES 5 and EGTA 1.1. The membrane potential was measured at 0 pA current injection using EPC 10 USB Patch Clamp Amplifiers (HEKA). The access resistance was monitored by a hyperpolarizing step of -10 mV. The data were excluded if the access resistance was above 20 MΩ. To evaluate the effect of uncaging of photo-OXB, UV LED light was applied with or without caged orexin (300 nM) to the brain slices on an upright Axio Examiner A1 microscope (Zeiss) using a Plan-Apochromat 63x/1.0 M27 objective (light source: Colibri 7, Zeiss; 385 nm wavelength with 30 nm bandwidth) at 2 mW/mm² intensity for 2 seconds with or without photo-OXB (300 nM). After the application of the UV, the slices were perfused with at least 5 mL of the aCSF to wash out uncaged photo-OXB before starting the recording of another neuron in the same brain slice. WT OXB (300 nM) or suvorexant (1μM) were applied to the slice by perfusion in aCSF.