Whole-cell current noise produced by excitatory and inhibitory amino acids in large cerebellar neurones of the rat.

Abstract

1. Membrane noise and current changes produced by glutamate and related excitatory amino acids have been examined in cultured large cerebellar neurones (including Purkinje cells), with whole-cell patch-clamp methods. The sensitivity of these neurones to the inhibitory amino acids .gamma.-aminobutyric acid (GABA) and glycine has also been studied. 2. The neurones formed inhibitory synapses in culture, and displayed spontaneous synaptic currents. Reducing the pipette Cl- concentration (i.e. intracellular concentration) caused a negative shift in their reversal potential, and the currents could be blocked with bicuculline (10 .mu.M), suggesting that they were mediated by GABAA receptors. Spontaneous synaptic activity was also considerably reduced in the presence of 3 .mu.M-tetrodotoxin. 3. Analysis of the increase in whole-cell current noise produced by the application of GABA (3 .mu.M) gave noise spectra that were fitted by two Lorentzian components with slow and fast time constants of 23.6 and 1.9 ms at a membrane potential (Vm) of -110 mV. The mean single-channel conductance estimated from GABA noise was .gamma.noise = 12 pS. Glycine (10 .mu.M) whole-cell current responses were Cl-mediated and reversibly abolished by 1 .mu.M-strychnine. 4. Bath application of excitatory amino acids gave whole-cell current changes accompanied by an increase in synaptic activity. Postsynaptic responses to the excitatory amino acids were more redily seen after the inhibitory synaptic currents had been abolished by bicuculline. Membrane current changes were obtained in response to the putative transmitters glutamate and aspartate, and the agonists NMDA (N-methyl-D-aspartate), ibotenate, quisqualate and kainate. Their reversal potential was approximately -5 mV. 5. A majority of noise spectra produced by the various glutamate receptor agonists were fitted by two Lorentzian components; the rest were fitted with a single Lorentzian component. The noise time constants were apparently not dependent on the type of glutamate agonist used to activate the receptor channels. Pooling data for all agonists gave a mean time constant for single-component spectra of .tau.noise = 4.8 .+-. 0.3 ms; for two-component spectra the time constants were .tau.1 = 22.7 .+-. 1.8 ms and .tau.2 = 2.2 .+-. 0.12 ms (Vm = -110 to -50 mV). It is likely that the two components present in whole-cell noise spectra reflect complex kinetics of glutamate receptor channels. 6. The mean single-channel conductance was estimated from whole-cell noise for the various excitatory amino acids. The estimates obtained were .gamma.(glutamate) = 21.8 pS, .gamma.(aspartate) = 30.6 pS, .gamma.(NMDA) = 34.3 pS, .gamma.(ibotenate) = 25.6 pS, .gamma.(quisqualate) = 5.6 pS, .gamma.(kainate) = 3.6 pS. 7. The fact that the estimates of the conductances and time constants differ from values previously reported in some other types of neurone suggests differences in the channel properties, or relative proportions of the various types of glutamate channels present, in different types of neurones.