Imaging and Electrophysiology of Projection Neurons

Neurons were visualized using a fixed-stage upright Olympus microscope equipped with a 40x water immersion objective, a CCD camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu City, Japan), and a monitor. A narrow-beam infrared light-emitting diode (LED) (L850D-06; Marubeni, Tokyo, Japan, emission peak, 850 nm) was positioned outside the bath, as previously described.^{10 (link)} Projection neurons in lamina I were identified by DiI fluorescence after retrograde labeling in the parabrachial nucleus. Whole-cell patch clamp recordings were made using borosilicate glass microelectrodes pulled using a PC-10 puller (Narishige International, East Meadow, NY). Pipette resistances ranged from 6 to 12 MΩ. Electrodes were filled with a solution containing the following (in mM): 135 potassium gluconate, 5 KCl, 0.5 CaCl₂, 5 EGTA, 5 HEPES, and 5 MgATP; pH 7.2. Alexa Fluor 488 (Invitrogen; 25 mM) was added to confirm recording from the targeted cell. Recordings were acquired using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The data were low-pass filtered at 2 kHz and digitized at 10 kHz using a Digidata 1322A (Molecular Devices) and stored using Clampex version 10 (Molecular Devices).