Fmrfamide

To measure responses to FMRFamide, PSEM^89S, GABA, and muscimol, HCs were recorded with the perforated patch configuration using gramicidin as the perforating agent. The bathing solution contained the following: 120 mm NaCl, 2.5 mm KCl, 1.2 mm MgSO₄, 2.2 mm CaCl₂, and 3.0 mm HEPES, pH 7.7. A stock solution of gramicidin in ethanol (5 mg/ml) was prepared and diluted into the pipet solution (130 mm KCl, 3 mm NaCl, and 10 mm HEPES, pH 7.4) at a ratio of 6 μl/ml. Pipets were pulled to a resistance of ∼10–12 MΩ using a vertical puller (PC-100, Narishige Scientific Instruments). Following seal formation, the presence of a membrane potential in the current clamp recording configuration confirmed successful perforation, usually within the first minute. PSEM^89S (500 μm), GABA (1 mm), and muscimol (100 μm) were applied with 100-ms pulses of positive pressure (1–2 psi) from a second pipet positioned ∼100 μm from the cell. FMRFamide (30 μm) was bath applied for 5 min before measuring changes in membrane potential. FMRFamide was purchased from Sigma-Aldrich. Other agonists were purchased from Tocris Biosciences. Recordings were made using the MultiClamp 700b patch clamp amplifier, and pClamp software (Molecular Devices). All reported values were corrected for junction potentials, calculated using pClamp software.