Activation of presynaptic GABA(A) receptors induces glutamate release from parallel fiber synapses

Abstract

The parallel fibers relay information coming into the cerebellar cortex from the mossy fibers, and they form synapses with molecular layer interneurons (MLIs) and Purkinje cells. Here we show that activation of ionotropic GABA receptors (GABA(A)Rs) induces glutamate release from parallel fibers onto both MLIs and Purkinje cells. These GABA-induced EPSCs have kinetics and amplitudes identical to random spontaneous currents (sEPSCs), but, unlike sEPSCs, they occur in bursts of between one and five successive events. The variation in amplitude of events within bursts is significantly less than the variation of all sEPSC amplitudes, suggesting that the bursts result from repetitive activation of single presynaptic fibers. Electron microscopy of immunogold-labeled alpha-1 subunits revealed GABA(A)Rs on parallel fiber terminals. We suggest that the activation of these receptors underlies the increased amplitude of parallel fiber-evoked Purkinje cell EPSCs seen with application of exogenous GABA or after the release of GABA from local interneurons. These results occur only when molecular layer GABA(A)Rs are activated, and the effects are abolished when the receptors are blocked by the GABA(A)R antagonist gabazine (5 microM). From these data, we conclude that GABA(A)Rs located on parallel fibers depolarize parallel fiber terminals beyond the threshold for Na+ channel activation and thereby induce glutamate release onto MLIs and Purkinje cells.

Figure 1.

GABA_AR activation induces EPSCs in MLIs. A, Spontaneous activity recorded in a molecular layer interneuron during the application of 50 μm GABA (black line). The expansion shows the presence of a flurry of fast inward currents recorded during the first seconds of GABA application. B–D, Histograms of the average frequency of inward events recorded during the same time period shown in A, with the black bar signifying the application of either 10 μm muscimol or 50 μm GABA. B, Under control conditions (n = 19 cells). C, In the presence of 5 μm gabazine (n = 3 cells). D, In the presence of 2 μm NBQX (n = 4 cells).

Figure 2.

GABA_AR activation produces bursts of EPSCs. A, Individual 8 s samples of synaptic activity recorded during four separate applications of muscimol. Note that EPSCs occur in bursts of between one and four events. B, Average decay times and amplitudes of sEPSCs and EPSCs evoked by muscimol. Open circles are individual cells, and black squares are the averages of all the cells. Dashed lines connect data from the same cell. C, Histograms of the average interevent intervals for sEPSCs and EPSCs evoked by muscimol application (n = 4 cells). The histogram of the control events is fit by a monoexponential equation with τ of 241 ms, and the histogram of the events induced by muscimol is fit by a biexponential equation with τ₁ of 207 ms (39%) and τ₂ of 19 ms (61%). D, Synaptic activity recorded in one cell before (left) and after (right) the bath application of 0.2 μm TTX. The downward deflections are EPSCs. The bars indicate the application of 10 μm muscimol. Spontaneous EPSCs happened to be on average larger than muscimol-evoked EPSCs in these traces, but this was not a general rule.

Figure 3.

Induction of GABA_AR-activated EPSCs shows long-lasting fatigue. A, Histogram of the frequency of EPSCs recorded after waiting 1.5 min (crosses), 5 min (circles), or 10 min (diamonds) between muscimol applications. The black line represents the application of 10 μm muscimol. B, Peak response recorded in each cell as a function of the three different time intervals. Symbols are the same as in A, and black boxes correspond to the averages. Lines connect data from individual cells. *p < 0.05, paired Student's t test.

Figure 4.

GABA_AR activation occurs in the molecular layer. A, Photographs of the placement of the puffer pipette relative to the interneuron that was recorded. In all three photos, the recording pipette is on the left and the puffer pipette is on the right. The left photo shows the puffer pipette in the control position. In the center photo, the puffer pipette (indicated by the white arrow) is moved away from the Purkinje cells (PC) to the outer molecular layer (ML), and, in the right photo, the puffer pipette (black arrow) is moved toward the Purkinje cells. B, Histograms of the average EPSC frequency recorded from the three positions of the puffer pipette. The biggest response is recorded when the pipette is in the control position (gray circles), and a diminished and delayed response is recorded when the puffer pipette is in the outer molecular layer (white circles) or the Purkinje cell layer (black circles). C, Representative traces of responses recorded when the puffer pipette is in the three different positions. Note that an outward current appears in all three traces but is much reduced and delayed when the puffer pipette is in the outer molecular layer or granule cell layer. This indicates that the agonist is eventually diffusing back to the interneuron from which we are recording, and it is not until this occurs (and the outward current appears) that the EPSC frequency begins to increase.

Figure 5.

GABA_AR activation induces EPSCs in Purkinje cells. A, Spontaneous activity recorded in a Purkinje cell held at −60 mV during the application of 10 μm muscimol (black line). The expected reversal potentials for IPSCs and EPSCs are −70 and 0 mV, respectively. The expansion shows the presence of bursts of fast inward currents as recorded in the MLIs. Note that, whereas the frequency of (inward) EPSCs is greatly increased, the (outward) IPSCs are abolished after several seconds of muscimol application, probably attributable to desensitization of GABA_ARs. B–E, Histograms of the average EPSC frequency recorded during the time period shown in A. B–D, Black bar again signifying the application of muscimol. B, Under control conditions (n = 13 cells). C, In the presence of 5 μm gabazine (n = 4 cells). D, In the presence of 2 μm NBQX (n = 4 cells). E, In control conditions, with the gray bar representing the application of 20 mm K⁺ instead of muscimol.

Figure 6.

Localization of α₁ GABA_AR subunit. A1–A4, Examples of gold particles (arrows) associated with the presynaptic element at the border of the presynaptic active zone (A1, A2) or within the presynaptic active zone (A3, A4). B1, B2, Examples of gold particles associated with the postsynaptic differentiation (arrows) or with the extrasynaptic membrane (crossed arrows). C, Histogram of the distances of particles (100 particles from 82 boutons) from the cleft center (0, dotted line). Note that immunolabeling predominates on the presynaptic side. D1, D2, Histograms of the distances from the synaptic border of the gold particles observed in the presynaptic (D1) and the postsynaptic element (D2). Distances were measured from the nearest border of the synapse (0, dotted line). The left portion of the graph indicates the distance outside of the synapse (extrasynaptic), and the portion on the right indicates distance within the synapse (intrasynaptic).

Figure 7.

GABA_AR activation increases the amplitude of EPSCs evoked in Purkinje cells. A, Time course of evoked EPSC amplitudes normalized to the average amplitude recorded in each cell during the 30 s before the application of 10 μm muscimol (black bar). EPSCs were recorded in Purkinje cells and were evoked by giving an extracellular stimulus to the parallel fibers every second. B, Representative traces of average EPSCs evoked in control conditions, in the presence of muscimol, and after muscimol was washed from the bath. C, Traces from B normalized to show no change in the voltage clamp during or after muscimol application.

Figure 8.

Interneuron activity increases the amplitude of EPSCs evoked in Purkinje cells. A, Illustration of a stimulus paradigm in which two individual sets of parallel fibers were stimulated using two separate stimulation electrodes to separate the fibers that were activated by two test stimuli (stim i and stim ii) from the fibers that were activated by a train of 10 stimuli at 100 Hz (see Materials and Methods). stim i was timed to produce an EPSC in the postsynaptic Purkinje cell 5 s before the train of stimuli given to nearby parallel fibers, and then stim ii was delivered 100 ms after the termination of the train. The entire sequence was repeated every 30 s for 10 min before 5 μm gabazine was washed onto the slice. B, Sample traces of averaged EPSCs from the control period before gabazine was perfused. The average EPSC produced from stim ii (right, solid line) has an increased amplitude compared with the average EPSC produced by stim i (left and right, dotted line). C, Sample traces of the average EPSCs recorded after gabazine had been perfused for 5 min onto the same cell as in B. The average EPSC produced from stim ii (right, solid line) is now identical to the average EPSC produced by stim i (dotted line, left and right). D, Group data from four cells showing that perfusion of gabazine onto the slice systematically abolishes the increase in EPSC amplitude caused by the train of stimuli given to nearby parallel fibers. Note the absence of any facilitation in gabazine indicating that the parallel fibers stimulated with the train are indeed distinct from the parallel fibers stimulated by the two test stimuli. All recordings were made in the presence of a blocker of GABA_BRs (2 μm CGP 55845).