Patch clamp experiments
Patch clamp experiments were performed on rat sensory neurons (DRGs) 24–48 h after their dissection, following a modified protocol from (Walwyn et al., 2007). First, cell viability was evaluated by an automated cell counter (Luna, Villeneuve, France) using acridine orange/propidium iodide dyes. During patch clamp recordings, cells were superfused with ECS buffer and visualized using a Zeiss Axiovert 200 inverse microscope (Zeiss, Jena, Germany). Patch clamp pipettes (resistance 3.5–8 MΩ) were produced using the Sutter P-97 puller (Sutter Instruments, Novato, CA, USA) from Borosilicate glass capillaries (Harvard Bioscience, MA, USA). Before the experiment, the glass pipettes were filled with an ICS buffer. Currents were amplified by an EPC-10 patch amplifier and recorded using Pulse software (HEKA, Lambrecht, Germany). Using a pressurized application system (Perfusion Pressure Kit VPP-6; Warner Instruments, Hamden, CT, USA), ECS buffer was added in a steady flow of 800–1,000 µl/min. After reaching the “giga-seal” at −60 mV, the membrane patch was breached to achieve the whole-cell configuration. The currents were initially recorded at a holding potential of −80 mV in ECS buffer in the absence of test compounds. Subsequently, the cells were depolarized to +10 mV (for 100 ms) for eight times following 20 s intervals. During the first five cycles, only ECS buffer continued to flow. On the sixth cycle, an opioid agonist (fentanyl, NFEPP) was added to the solution. During the last two cycles, the opioid antagonist naloxone was used to block/confirm opioid receptor-mediated effects. Neurons were selected for analysis after the 8th cycle. For experiments using PTX or gallein, the blockers were added to culture media overnight before the day of the experiment (Lu and Ikeda 2016). Opioid ligands were administered via a perfusion valve system (VC-6; Warner Instruments). All recordings were performed at room temperature.