Whole‐cell and single‐channel currents activated by GABA and glycine in granule cells of the rat cerebellum.

Abstract

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 M GABA, while -60% responded to 100 /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 EC50 values of 45-2 and 99-6 /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 GABAA receptor antagonists bicuculline (15 uM), SR-95531 (10 /M) and picrotoxinin (100 uM), 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 singlechannel 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 uM) or SR-95531 (10 #M) and were greatly reduced in frequency by tetrodotoxin (ITX; 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 #M).

Pharmacological Characterization of Glycine-Gated Chloride Currents Recorded in Rat Hippocampal Slices

“…Thus the EC 50 concentration we observe in slices is higher than that reported for GlyRs recorded in dissociated hippocampal cultures (40 -72 vs. 270 M) (Shirasaki et al 1991;Ye et al 1999;Yoon et al 1998). This discrepancy in agonist EC 50 concentration between culture and slice preparations have been previously observed (Kaneda et al 1995). Additionally, the modest speed in which agonists can be applied to recorded neurons in slices and the varying distances these cells are from the slice surface means that receptor desensitization will likely take place during the rising phase of the response that will affect the amplitude of the current; however, the extent of the desensitization is unknown (Kaneda et al 1995).…”

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

“…Tonic inhibition mediated by extrasynaptic, δ subunit-containing GABARs has been identified in a number of neurons [31][32][33][34][35] and is thought to regulate neuronal excitability; however, the role of tonic inhibition in shaping behavior is unclear. The results reported here were obtained from cerebellar granule cells, a cell type for which there is strong evidence that receptors containing α6 and δ subunits are extrasynaptic 16,19,36 and are required for tonic GABA current 20,21 .…”

Molecular and synaptic organization of GABAA receptors in the cerebellum: Effects of targeted subunit gene deletions

“…Synaptic inhibition in the cerebellum is also mediated, to a lesser extent, by glycine receptors (38,39). A majority of Golgi and Lugaro cells have a dual GABAergic/glycinergic phenotype (40,41).…”

“…The specificity of transmission in their postsynaptic targets is ensured by the differential expression of receptors. Thus, spontaneous IPSCs in granule cells are mediated selectively by GABA A receptors (39), whereas Golgi cells, Lugaro cells, unipolar brush cells, and possibly other interneurons exhibit either glycinergic or mixed glycinergic/GABAergic currents ( Figure 1) (42,43). At least in Golgi cells, GABA A and glycine receptors can co-exist within a given postsynaptic site (42).…”