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Current-clamp and voltage-clamp recordings of ZsGreen-expressing POMC neurons were performed at ~32 °C in the perforated patch-clamp configuration. Neurons were visualized with a fixed-stage upright microscope (BX51WI, Olympus) using ×40 and ×60 water-immersion objectives (LUMplan FL/N ×40, 0.8 numerical aperture, 2 mm working distance; LUMplan FL/N ×60, 1.0 numerical aperture, 2 mm working distance, Olympus) with infrared differential interference contrast optics51 and fluorescence optics. ZsGreen-expressing POMC neurons were identified by their anatomical location in the ARC and by their ZsGreen fluorescence that was visualized with an X-Cite 120 illumination system (EXFO Photonic Solutions) in combination with a Chroma 41001 filter set (ex: HQ480/×40; bs: Q505LP; em: HQ535/50m). Electrodes with tip resistances of between 4 and 6 MΩ were fashioned from borosilicate glass (0.86-mm inner diameter; 1.5-mm outer diameter; GB150-8P, Science Products) with a vertical pipette puller (PP-830, Narishige). All recordings were performed with an EPC10 patch-clamp amplifier (HEKA) controlled by the program PatchMaster (version 2.32; HEKA) running in Windows. In parallel, data were recorded using a micro1410 data acquisition interface and Spike 2 (version 7, both from CED). Current-clamp recordings were sampled at 25 kHz and low-pass filtered at 2 kHz with a four-pole Bessel filter. Voltage-clamp recordings were sampled at 5 kHz, smoothed (𝜏 = 0.2 s) and downsampled to 0.5 Hz. The calculated liquid junction potential of 14.6 mV between intracellular and extracellular solution was compensated or subtracted offline (calculated with Patcher’s Power Tools plug-in from for IGOR Pro 6; Wavemetrics).

Perforated patch experiments were conducted using protocols modified from previous studies52,53. Recordings were performed with pipette solution containing (in mM): 140 K-gluconate, 10 KCl, 10 HEPES, 0.1 EGTA and 2 MgCl2, adjusted to pH 7.2 with KOH. ATP and GTP were omitted from the intracellular solution to prevent uncontrolled permeabilization of the cell membrane54. The patch pipette tip was filled with internal solution and backfilled with internal solution, which contained the ionophore to achieve perforated patch recordings and 0.02% tetramethylrhodamine-dextran (3,000 MW, D3308, Invitrogen) to monitor the stability of the perforated membrane. Amphotericin B (A4888; Sigma) was dissolved in DMSO to a concentration of 40 µg µl−1 (D8418, Sigma) following the protocols of a previous study55. The used DMSO concentration (0.1–0.3%) had no obvious effect on the investigated neurons. The ionophore was added to the modified pipette solution shortly before use. The final concentration of amphotericin B was ~120–160 µg ml−1. Amphotericin solutions were prepared from undissolved weighted samples (stored at 4 °C protected from light) on every recording day. During the perforation process, access resistance (Ra) was monitored continuously and experiments started after Ra values reached steady state (~15–20 min) and the action potential amplitude was stable.

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