GRAMICIDIN-PERFORATED PATCH RECORDING - GABA RESPONSE IN MAMMALIAN NEURONS WITH INTACT INTRACELLULAR CHLORIDE

1. By the development of a new perforated patch method using gramicidi n, the effects of GABA on neurones dissociated from the rat substantia nigra pars reticulata (SNR) were examined without disturbing the intr acellular chloride concentration. 2. Using the patch pipette solution containing gramicidin (100 mu g ml(-1)), the access resistance dropped to less than 20 M Ohm within 40 min after making the gigaohm seal. 3. Under current-clamp conditions, GABA caused a hyperpolarization accom panied by a blockade of spontaneous firing. Under voltage clamp at a h olding potential (V-h) of -50 mV, GABA evoked an outward current by wa y of bicuculline- and picrotoxin-sensitive GABA(A) receptors. 4. A 10- fold change of extracellular chloride concentration resulted in a 58 m V shift of the reversal potential of GABA-induced outward current (E(G ABA)), indicating that the membrane behaves like a chloride electrode in the presence of GABA. 5. The intracellular chloride activities (aCl (i)), calculated with the Nernst equation using both extracellular chl oride activity and E(GABA) values, ranged from 2.8 to 19.7 mM with a m ean value of 9.5 mM. The aCl(i) was not affected either by different p ipette solutions or by different holding potentials more hyperpolarize d than -40 mV. 6. In the recording from SNR neurones in brain slice us ing the gramicidin-perforated patch-clamp technique, the inhibitory an d excitatory postsynaptic currents were recorded in different current directions and the former was blocked by bicuculline. 7. In conclusion , the gramicidin-perforated patch method will disclose previously unkn own aspects of biological responses involving Cl-.