WHOLE-CELL AND SINGLE-CHANNEL CURRENTS ACTIVATED BY GABA AND GLYCINE IN GRANULE CELLS OF THE RAT CEREBELLUM

1. Patch-clamp methods have been used to characterize GABA- and glycine-activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7- to 20-day-old rats. 2. All granule cells responded to 10 mu M GABA, while similar to 60% responded to 100 mu M glycine. With repeated agonist application, whole-cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg-ATP. 3. Whole-cell concentration-response curves gave EC(50) values of 45.2 and 99.6 mu M and Hill slopes of 0.94 and 2.6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole-cell current-voltage (I-V) relationships of GABA- and glycine-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with F- shifted the GABA reversal potential to a more negative value. 'Instantaneous' I-V relationships produced by ionophoretic application of GABA were linear, while 'steady-state' I-V relationships produced by ramp changes in potential showed outward rectification. For glycine, 'steady-state' I-V plots were linear. 5. Responses to GABA were blocked by the GABA(A) receptor antagonists bicuculline (15 mu M), SR-95531 (10 mu M) and picrotoxinin (100 mu M), while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside-out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine-activated single-channel currents had conductances of 32, 55 and 104 pS. Single-channel I-V relationships were linear. 7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 mu M) or SR-95531 (10 mu M) and were greatly reduced in frequency by tetrodotoxin (TTX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents. 8. In many cells a bicuculline-sensitive 'background current noise' was seen in the presence of glutamate antagonists and TTX. Power spectra of this background noise (following subtraction of noise recorded in the presence of bicuculline) were fitted with the sum of two Lorentzian curves and gave an estimated single-channel conductance of 13.7 pS, comparable to the value of 15.3 pS obtained from spectra of noise produced by externally applied GABA (10 mu M). 9. Our results indicate that granule cells express distinct GABA- and glycine-activated ion channels. Although both amino acids are known to be present in the terminals of Golgi neurones that form inhibitory inputs to granule cells, spontaneous inhibitory postsynaptic currents (IPSCs) in granule cells appear to be mediated exclusively by GABA.