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.