Clampex version 10

Dissection of spinal cords and recoding were done as described^{51 (link),52 (link)}. Briefly, after perfusion, spinal cords from P6 newborn pups were removed and hemisectioned in artificial cerebrospinal fluid (aCSF) containing: NaCl (127 mM), KCl (1.9 mM), KH₂PO₄ (1.2 mM), CaCl₂ (2 mM), MgSO₄ (1 mM), NaHCO₃ (26 mM) and d-glucose (20.5 mM) with oxygenated air (95%O₂/5%CO₂). After dissection, spinal cords were moved to aCSF-containing bath. Using tightly fitting glass pipets, dorsal roots were stimulated (0.3–0.6 mA, S88X, SIU-C, Grass technologies). Extracellular potentials are recorded from ventral roots (L4 and L5) using tightly fitting glass pipets with pre-amplifier (P55, Astro-Med) and digitizer (Digidata 1440A, Molecular Devices) and a data acquisition programme (Clampex version 10, Molecular Devices). Recorded traces are averages of 20 individual stimuli applied at 0.1 Hz.

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 monitor. A narrow-beam infrared LED (L850D-06; Marubeni, Tokyo, Japan, emission peak, 850 nm) was positioned outside the bath, as previously described [10 ]. Projection neurons in lamina I were identified by DiI fluorescence following 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 K-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 version10, (Molecular Devices).

Neurons were visualized using a fixed stage upright microscope (BX51WI Olympus microscope, Tokyo, Japan) equipped with a 40x water immersion objective, a CCD camera (ORCA-ER Hamamatsu Photonics, Hamamatsu City, Japan) and monitor screen. A narrow beam infrared LED (L850D-06 Marubeni, Tokyo, Japan, emission peak, 850 nm) was positioned outside the solution meniscus, as previously described [22 (link),45 (link),48 (link)]. Projection neurons in lamina I were identified by DiI fluorescence following injection into the lateral parabrachial nucleus. Whole-cell patch-clamp recordings were made with a pipette constructed from thin-walled single-filamented borosilicate glass using a microelectrode puller (PC-10; Narishige International, East Meadow NY). Pipette resistances ranged from 6 to 12 MΩ. Electrodes were filled with an intracellular solution containing the following (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl2, 5 EGTA, 5 HEPES, 5 MgATP, pH 7.2. Alexa fluor 488 (Invitrogen; 25 µM) was added to confirm recording from the target cell. Signals were acquired with an amplifier (Axopatch 200B, Molecular Devices, Sunnyvale CA). The data were low-pass filtered at 2 kHz and digitized at 10 kHz with an A/D converter (Digidata 1322A, Molecular Devices) and stored using a data acquisition program (Clampex version 10, Molecular Devices). The liquid junction potential was not corrected.

For optogenetic stimulation, a blue light pulse (GFP filter, centered around 485 nm, Lambda DG-4, Sutter instruments) was applied through the objective (40x) of the microscope for 5 ms using a shutter that was controlled by Clampex software (Clampex version 10, Molecular Devices). The light power on the sample was 1.3 mWmm^-2. The peak amplitude of outward current induced by blue light stimulation was measured in voltage clamp mode at −40 mV.