Glycine response in acutely dissociated ventromedial hypothalamic neuron of the rat: New approach with gramicidin perforated patch-clamp technique

1. We investigated the glycine-induced response in ventromedial hypothalamic (VMH) neurons freshly dissociated from 8- to 12-day-old rats using the nystatin and gramicidin perforated patch recording modes. The nystatin-formed pores in the plasma membrane are permeable for both monovalent cations and anions, whereas those formed by gramicidin are permeable only to monovalent cations. Therefore, when the patch-pipette contains 150 mM Cl^- and gramicidin, the physiological intracellular Cl^- concentration ([Cl^-](i)) is undisturbed in the cell-attached condition of the pipette. 2. At holding potentials of -40 to -60 mV, glycine induced inward currents and outward currents in the nystatin and gramicidin perforated patch recording modes, respectively. The values of the half- maximum effective concentration (EC₅₀) and the Hill coefficient in the concentration-response relationships of the glycine responses were 2.9 x 10^{- 5} M, 1.1, and 4.2 x 10^-5 M, 1.4, respectively. These values were quite similar in both recording modes. 3. The reversal potentials of the glycine responses (E(Gly)) were -1.5 mV in the nystatin perforated patch recording and -75.0 to -24.8 mV in the gramicidin perforated patch recording. 4. Strychnine (3 x 10^-8 M) inhibited the glycine-induced outward currents in a competitive manner and the half-inhibition concentration (IC₅₀) of strychnine on the 10^-4 M glycine-induced response was 1.9 x 10^-8 M. 5. The physiological [Cl^-](i) in the VMH neurons calculated from the E(Gly) obtained by the gramicidin perforated patch mode range from 6.0 to 43.8 mM (n = 28).