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 (EC(50)) 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 ranged from 6.0 to 43.8 mM (n = 28).