GABA spillover from single inhibitory axons suppresses low-frequency excitatory transmission at the cerebellar glomerulus

Abstract

GABA type B receptors (GABA(B)-Rs) are present on excitatory terminals throughout the CNS, but surprisingly little is known about their role in modulating neurotransmission under physiological conditions. We have investigated activation of GABA(B)-Rs on excitatory terminals within the cerebellar glomerulus, a structure where glutamatergic excitatory and GABAergic inhibitory terminals are in close apposition and make axodendritic synapses onto granule cells. Application of the GABA(B)-R agonist baclofen depressed evoked mossy fiber EPSCs by 54% at 1 Hz. The amplitude of miniature EPSCs recorded in tetrodotoxin was unchanged in the presence of baclofen, but the frequency was significantly reduced, indicating a purely presynaptic action of baclofen under our recording conditions. At physiological temperature (37 degrees C) presynaptic GABA(B)-Rs were not tonically activated by spontaneous GABA release from Golgi cells, which fire at approximately 8 Hz in slices at this temperature. However, tonic activation could be induced by blocking GABA uptake or by lowering temperature. GABA(B)-Rs were activated at physiological temperature when Golgi cell firing was increased above the basal level by stimulating a single inhibitory Golgi cell input at 50 Hz, suppressing the mossy fiber-evoked EPSC by 24% at 1 Hz. Furthermore, glutamate release was selectively inhibited at low-frequency mossy fiber inputs (<10 Hz) during Golgi cell stimulation. Our findings suggest that GABA spillover in the glomerulus modulates sensory input to the cerebellar cortex.

Fig. 1.

Pharmacological activation of presynaptic GABA_B-Rs depresses glutamate release from mossy fibers.A, Average non-NMDA receptor-mediated EPSCs evoked by mossy fiber stimulation at 1 Hz in control solution and in 100 μm baclofen. B, Bath application of 100 μm baclofen reduced the EPSC amplitude in a reversible manner (each point is the average of 10 EPSCs). The coefficient of variation (CV) of the EPSC amplitude (bottom panel) increased during depression, indicating reduced glutamate release. C, Superimposed average spontaneous miniature EPSCs recorded in tetrodotoxin (0.5 μm) in control solution and in the presence of 100 μm baclofen. D, Histogram showing mean frequency of miniature EPSCs in control and in 100 μmbaclofen (n = 6).

Fig. 2.

Frequency dependence of GABA_B-R-mediated EPSC depression. A, Averaged steady-state EPSCs from the same cell recorded at different stimulation frequencies in control solution (thin trace) and in the presence of 100 μm baclofen (thick trace). B, Steady-state frequency dependence of EPSC amplitude in control (open symbols) and in the presence of 100 μm baclofen (filled symbols). The relationship shows the average of eight cells normalized to the EPSC amplitude at 1 Hz mossy fiber stimulation (MFS) in control solution.

Fig. 3.

Tonic activation of GABA_B-Rs is temperature-dependent. A, Averaged EPSCs evoked at 1 Hz in control and in 500 μm CGP35348 at 37°C.B, Steady-state frequency dependence of EPSCs for control solution (open symbols) and in the presence of 500 μm CGP35348 (filled symbol) at 37°C. The relationship shows the average of three cells normalized to the 1 Hz EPSC amplitude in control solution.C, Averaged EPSCs evoked at 1 Hz mossy fiber stimulation (MFS) in control solution and in the presence of the GABA_B antagonist CGP35348 (500 μm) at room temperature. D, Histogram of normalized EPSC amplitudes during control, 50 μm baclofen, 500 μmCGP35348, and 500 μm CGP35348 plus 50 μmbaclofen at room temperature (n = 4).

Fig. 4.

Inhibition of GABA uptake induces tonic activation of GABA_B-Rs at physiological temperature. A, Averaged EPSCs evoked at 1 Hz in control solution and in the presence of the GABA uptake blocker NO711 (50 μm) at 37°C.B, Averaged EPSCs evoked at 1 Hz in the GABA_B antagonist CGP35348 (500 μm) and in the presence of both 500 μm CGP35348 and 50 μmNO711 at 37°C.

Fig. 5.

Stimulation of single Golgi cell input suppresses mossy fiber EPSCs. A, Recording configuration: whole-cell patch-clamped granule cell and stimulation of independent inhibitory and excitatory inputs. B, Average non-NMDA EPSC waveforms from control period and during Golgi cell stimulation (GoS). C, Traces show average non-NMDA EPSCs amplitudes (normalized to pre-GoS baseline) evoked by 1 Hz mossy fiber stimulation in the presence of ionotropic GABA_Areceptor antagonist. Bar shows timing of 5 sec burst of 50 Hz GoS. Dashed lines show extrapolated single exponential fit to pre-GoS frequency-dependent depression for all data.Top trace shows average of all 31 cells. Middle trace shows average of 19 cells that responded to GoS (Responsive). Bottom trace shows average of 12 cells that did not respond to GoS (Non-responsive).D, Dashed line shows distribution of mean EPSC amplitudes during GoS for all cells. Solid lineshows distribution for responsive cells.

Fig. 6.

Golgi cell-induced suppression of mossy fiber EPSCs is presynaptic. A, Top panel,Normalized EPSC amplitudes for responsive cells during 1 Hz Mossy fiber stimulation in presence of ionotropic GABA_A receptor antagonist (same data as Fig. 5C, middle trace).Bar shows timing of 5 sec burst of 50 Hz GoS.Dashed lines show time course of frequency-dependent depression from all data (Fig. 5C, top trace).Bold line shows fit of single exponential function to EPSC recovery (τ = 3 sec). Bottom panel, CV during EPSC depression (n = 19). Dashed line shows pre-GoS baseline. B, Relationship between CV and normalized peak EPSC. Negative correlation (Spearman correlation coefficient; r = −0.84) indicates a reduction in release probability during EPSC depression.

Fig. 7.

GABA_B-Rs mediate Golgi cell stimulation-induced EPSC depression. A, Simultaneous 50 Hz Golgi cell stimulation (GoS) and 1 Hz mossy fiber stimulation (MFS) in the presence of GABA_Areceptor blockers. EPSCs were evoked by MFS, whereas no currents were associated with the GoS. B, Stimulus averaged currents: MFS indicates EPSC evoked at 1 Hz, and GoS shows IPSCs were not evoked during 50 Hz GoS. C, Normalized evoked EPSC amplitudes from the same cells before, during, and after 50 Hz GoS for control and in the presence of the GABA_B-R antagonist CGP35348 (500 μm; n = 5). Dashed lines show time course of frequency-dependent depression from all data (Fig. 5C, top trace).

Fig. 8.

Physiologically activated GABA_B-Rs suppress low-frequency excitatory transmission. A, Averaged EPSCs from the same cell evoked at 1 and 10 Hz mossy fiber stimulation (MFS) during 50 Hz Golgi cell stimulation (GoS). GABA_B-R-mediated EPSC depression was more pronounced at 1 Hz than at 10 Hz. B, Histogram of mean EPSC inhibition during GoS for four cells.