GRAMICIDIN PERFORATED PATCH-CLAMP TECHNIQUE REVEALS GLYCINE-GATED OUTWARD CHLORIDE CURRENT IN DISSOCIATED NUCLEUS SOLITARII NEURONS OF THE RAT

1. The inhibitory response of exogenously applied glycine was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole cell configuration using new perforated patch-clamp technique termed ''gramicidin perforated patch technique,'' which maintains intact intracellular Cl- concentrations. 2. Using the gramicidin perforated patch technique, at a holding potential (V-H) of -45 mV, glycine induced outward currents in a concentration-dependent manner with a EC(50) of 4.0 x 10(-5) M and at a Hill coefficient of 1.5. In contrast, using the nystatin perforated patch technique, glycine induced inward currents at the same V-H in a concentration-dependent manner with an EC(50) of 4.9 x 10(-5) M and at a Hill coefficient of 1.2. 3. The glycine-induced outward currents were blocked by strychinine in a concentration dependent manner with an IC50 of 2.2 x 10(-8) M. The blockade was competitive. 4. The current-voltage relationship for the 10(-5) M glycine response showed a dear outward rectification. 5. Ten-fold change of extracellular Cl- with a large impermeable anion resulted in a 65 mV shift of the reversal potential of glycine-induced currents (E(Gly)), indicating that the membrane behaves like a Cl- electrode in the presence of glycine. 6. The intracellular Cl- activity calculated from the E(Gly) ranged from 7.3 to 18.2 mM, with a mean value of 13.3 mM. 7. The values of E(Gly) in the individual neurons were significantly negative to the resting membrane potentials, suggesting the existence of active transport of Cl-.