GABAB receptor-mediated inhibition of tetrodotoxin-resistant GABA release in rodent hippocampal CA1 pyramidal cells

Abstract

Tight-seal whole-cell recordings from CA1 pyramidal cells of rodent hippocampus were performed to study GABAB receptor-mediated inhibition of tetrodotoxin (TTX)-resistant IP-SCs. IPSCs were recorded in the presence of TTX and glutamate receptor antagonists. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs by a presynaptic action. The inhibition by (R)-(-)-baclofen was concentration-dependent, was not mimicked by the less effective enantiomer (S)-(+)-baclofen, and was blocked by the GABAB receptor antagonist CGP 55845A, suggesting a specific effect on GABAB receptors. The inhibition persisted in the presence of the Ca2+ channel blocker Cd2+. There was no requirement for an activation of K+ conductances by (R)-(-)-baclofen, because the inhibition of TTX-resistant IPSCs persisted in Ba2+ and Cd2+. Because the time courses of TTX-resistant IPSCs were not changed by (R)-(-)-baclofen, there was no evidence for a selective inhibition of quantal release from a subgroup of GABAergic terminals. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs in guinea pigs and Wistar rats, whereas the inhibition was much smaller in Sprague Dawley rats. In Cd2+ and Ba2+, beta-phorbol-12,13-dibutyrate and forskolin enhanced the frequency of TTX-resistant IPSCs. Only beta-phorbol-12, 13-dibutyrate reduced the inhibition by (R)-(-)-baclofen. We conclude that GABAB receptors inhibit TTX-resistant GABA release through a mechanism independent from the well known effects on Ca2+ or K+ channels. The inhibition of quantal GABA release can be reduced by an activator of protein kinase C.

Fig. 1.

Effect of (R)-(−)-baclofen on TTX-resistant IPSCs. A, Eight consecutive traces (2 sec each) showing TTX-resistant IPSCs before (left), 1 min after start of (R)-(−)-baclofen application (middle), and 7 min after commencing wash (right). B, Cumulative amplitude (B1) and frequency (B2) distributions are plotted for control, (R)-(−)-baclofen effect, and wash. The amplitude distributions were unchanged. The frequency distribution was shifted to the right by (R)-(−)-baclofen. The number of events used for the cumulative plots was 495 for control, 232 during (R)-(−)-baclofen, and 445 during wash.C, Concentration-dependent reduction of the frequency of TTX-resistant IPSCs by (R)-(−)-baclofen. Effects were calculated as the ratio (expressed in percent) of the frequency in the presence of a given (R)-(−)-baclofen concentration to the mean frequency before and after (R)-(−)-baclofen application. Squares are mean values from 5–14 cells for each concentration. Error bars represent SEM. The solid line is a fit (least-square method) to the Hill function, assuming a Hill coefficient of 1.0 and a maximal reduction of the frequency to 45% of the control. The calculated EC₅₀ is 1.78 μm. D, Block of the (R)-(−)-baclofen effect by a high-affinity GABA_B antagonist (CGP 55845A). The plot of the sum of the amplitudes sampled for 20 sec against time shows that (R)-(−)-baclofen inhibited TTX-resistant IPSCs. The effect was blocked by the GABA_B antagonist CGP55845A. The antagonist by itself had no effect. Horizontal barsindicate the drug application periods.

Fig. 2.

(R)-(−)-baclofen-induced reduction of the frequency of TTX-resistant IPSCs in the presence of Cd²⁺. A, Display of data as in Figure1A, B. (R)-(−)-baclofen reduced the frequency of TTX-resistant IPSCs in 100 μmCd²⁺. The effect of (R)-(−)-baclofen was fully reversible. B, (R)-(−)-baclofen did not affect the amplitude of TTX-resistant IPSCs (B1) but reduced their frequency (B2). The number of events used for the cumulative plots was 309 for control, 126 during (R)-(−)-baclofen, and 296 for recovery.

Fig. 3.

Kinetics of TTX-resistant IPSCs in (R)-(−)-baclofen. A, Distributions of the mono-exponential decay time constant τ in control solution (mean ± SD, 21.5 ± 7.0 msec; n = 177) and in the presence of (R)-(−)-baclofen (19.5 ± 6.4 msec; n = 99) were not significantly different (p > 0.1, paired Student’st test), whereas the frequency was reduced.Inset shows a mono-exponential fit (time constant, τ, 18.06 msec; amplitude coefficient, A, 26.8). Calibration: 5 pA, 20 msec. White and stippled bars are control data; black bars represent data obtained in (R)-(−)-baclofen. B, Distribution of the rise time (10–90%) of TTX-resistant IPSCs recorded in control solution (mean ± SD, 1.63 ± 0.68 msec;n = 267) and in the presence of (R)-(−)-baclofen (1.69 ± 0.70 msec;n = 113) were also not significantly different (p > 0.1). C, Plot of the amplitude of individual TTX-resistant IPSCs against their τ value in the absence (C1) and presence (C2) of (R)-(−)-baclofen. (R)-(−)-baclofen did not change the relation between the amplitude of TTX-resistant IPSC and τ. D, Plot of the rise time of individual TTX-resistant IPSCs against their peak amplitude in the absence (D1) and presence (D2) of (R)-(−)-baclofen. (R)-(−)-baclofen did not change the relation between rise time and amplitude when measured for individual events.

Fig. 4.

(R)-(−)-baclofen does not reduce the frequency of TTX-resistant IPSCs in a CA1 neuron of a Sprague Dawley rat but inhibits TTX-resistant IPSCs in a CA1 neuron of a Wistar rat. All recordings were performed in Cd²⁺ (100 μm). A, Eight consecutive traces (2 sec each) showing TTX-resistant IPSCs before (left) and 90 sec after start of the (R)-(−)-baclofen application (middle), and 7 min after commencing wash (right). B, Cumulative amplitude (B1) and frequency (B2) distributions are plotted for control, (R)-(−)-baclofen effect, and wash. (R)-(−)-baclofen did not significantly reduce the frequency or amplitude of TTX-resistant IPSCs in 100 μmCd²⁺ in this cell. The number of events used for the cumulative plots was 180 for control, 162 during (R)-(−)-baclofen, and 160 for recovery. C, D, (R)-(−)-baclofen reduced the frequency of TTX-resistant IPSCs in Wistar rats, whereas the amplitude distribution was unchanged. The number of events used for the cumulative plots was 248 for control, 151 during (R)-(−)-baclofen, and 240 for recovery.

Fig. 5.

Effects of (R)-(−)-baclofen on TTX-resistant IPSCs on stimulation of β-phorbol-12,13-dibutyrate (β-phorbol.) or forskolin (forskol.). TTX-resistant IPSCs were recorded in Ba²⁺ (1 mm) and Cd²⁺ (100 μm). A, GABA_Breceptor-mediated inhibition of TTX-resistant IPSCs in the presence of β-phorbol-12,13-dibutyrate. Consecutive traces (1 sec each) show that β-phorbol-12,13-dibutyrate (β-phorbol.; 1 μm) strongly increased the frequency of TTX-resistant IPSCs. (R)-(−)-baclofen reduced the frequency of TTX-resistant IPSCs. B, Cumulative amplitude and frequency distributions of TTX-resistant IPSCs reveal that neither (R)-(−)-baclofen nor β-phorbol-12,13-dibutyrate affected the amplitude distribution; however, β-phorbol-12,13-dibutyrate increased and (R)-(−)-baclofen thereafter reduced the frequency of TTX-resistant IPSCs. The number of events used for the cumulative plots was 443 for control, 780 for β-phorbol-12,13-dibutyrate, and 541 during β-phorbol-12,13-dibutyrate and (R)-(−)-baclofen. C, GABA_Breceptor-mediated inhibition of TTX-resistant IPSCs in the presence of forskolin (20 μm). Consecutive traces (1 sec each) show that forskolin increased the frequency of TTX-resistant IPSCs. (R)-(−)-baclofen in the presence of forskolin still reduced the frequency. D, Cumulative amplitude and frequency distributions of TTX-resistant IPSCs. Neither forskolin nor (R)-(−)-baclofen (traces are superimposed) significantly affected the amplitude distribution (D1). Forskolin increased the frequency of TTX-resistant IPSCs; (R)-(−)-baclofen reduced it (D2). The number of events used for the cumulative plots was 364 for control, 652 for forskolin, and 374 during forskolin and (R)-(−)-baclofen.

Fig. 6.

Graphic representation of the effects of β-phorbol-12,13-dibutyrate, forskolin, and (R)-(−)-baclofen on TTX-resistant IPSCs. All recordings were performed in the presence of Cd²⁺ (100 μm) and Ba²⁺ (1 mm).A, β-phorbol-12,13-dibutyrate (phorbol; 1 μm) or forskolin (forskol; 20 μm) increased the frequency of TTX-resistant IPSCs. B, In control (white column) and in the presence of forskolin (forskol), (R)-(−)-baclofen ((−)Bac) strongly reduced the frequency of TTX-resistant IPSCs. In the presence of β-phorbol-12,13-dibutyrate, the reduction was significantly smaller (phorbol; Student’s ttest; p < 0.05). Columns represent mean effect for all cells (n = 4 forA; n = 6 for B). Error bars represent SEM.