Activation of Presynaptic GABA_A Receptors Induces Glutamate Release from Parallel Fiber Synapses

GABA_AR activation induces EPSCs in interneurons

Using a pressure pipette, we applied 50 μm GABA to parallel fibers in cerebellar slices while monitoring spontaneous activity in MLIs under whole-cell voltage-clamp conditions. The application of GABA induced a slow outward current in the interneurons as predicted from the holding potential (−60 mV) and the [Cl⁻]_i (10 mm) (Fig. 1A). However, GABA also induced a flurry of fast inward currents that coincided with the onset of the application of the agonist (Fig. 1A; average time course in B). This phenomenon is not an artifact caused by the pressure pipette because it is blocked by the specific GABA_AR antagonist gabazine (5 μm). In three of three cells, the antagonist completely blocked both the slow outward current as well as the fast inward currents (Fig. 1C). In two of these three cells, gabazine was washed from the bath and both currents returned (data not shown).

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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).

To determine whether the effect also requires the activation of GABA_B in addition to GABA_A receptors, we made recordings in the presence of 1 μm CGP 55845 [2S)-3-[(15)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl)(phenylmethyl)phosphinic acid] and found that the fast inward currents remained (data not shown). The fast inward currents were also induced when GABA was replaced by the highly specific GABA_AR agonist muscimol (10 μm) (see Figs. 2, 4, 5). Therefore, the fast inward currents result from the specific activation of GABA_ARs.

In MLIs, IPSCs and EPSCs can be easily distinguished based on the decay kinetics of the two currents. The waveform of the IPSC decay is best fit by a biexponential equation whose fastest time constant is ∼8.7 ms, whereas the decay of EPSCs is best fit by a monoexponential equation with a time constant of ∼1.3 ms (Llano and Gerschenfeld, 1993). The fast inward currents that we elicited when puffing GABA were distinguishable from IPSCs because they were fit by monoexponential equations with time constants <2 ms and were absent when the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline(NBQX) (2 μm) was washed onto the slice. This concentration of NBQX completely blocked the fast inward currents and had no effect on the slow outward current, indicating that the fast currents were EPSCs originating from brief activations of AMPA receptors (Fig. 1D).

GABA_AR-induced EPSCs are similar to sEPSCs

GABA_AR-induced EPSCs (Fig. 2A) have properties that are nearly identical to spontaneous EPSCs recorded in the same cells under control conditions. The decays were fit by single exponentials with τ values averaging 1.0 ± 0.1 ms compared with 1.3 ± 0.2 ms in control conditions (p > 0.05, paired Student's t test; n = 4) (Fig. 2B) and amplitudes averaging 50.9 ± 6.3 pA compared with 54.5 ± 5.7 pA in control conditions (p > 0.05; n = 4) (Fig. 2B). However, unlike in control conditions, the GABA_AR-induced EPSCs appeared in short bursts (Fig. 2A). When the inter-EPSC intervals from data recorded under control conditions are plotted in a histogram, they are fit by a single-exponential equation (τ of 241 ms), whereas two exponentials were required to fit the data collected when muscimol was present (τ₁ of 207 ms, τ₂ of 19 ms, A₁ of 39%, and A₂ of 61%) (Fig. 2C). The need for a second exponential indicates that the EPSCs occurred in bursts, and the parameters of the fast component indicate that the average time in between events within the bursts is 19 ms. The burst pattern of Figure 2A, in which the response is clearly composed of several distinct bursts, is incompatible with schemes in which release would occur randomly in a large number of presynaptic fibers. It suggests instead that no more than one fiber was active at any given time and that each active fiber gave rise to a series of EPSCs, either because it fired repetitively or because it remained depolarized for some time (∼200 ms, the duration of a burst). To verify whether the individual bursts which compose the response originated from repetitive activation of the same synapse, we looked at the variability of the amplitudes within bursts and compared them with the variability of all sEPSCs combined, because EPSCs originating from the same synapse are predicted to have similar properties whereas EPSCs from different synapses should show greater variability (Forti et al., 1997; Auger and Marty, 2000). We found that the coefficient of variation of amplitudes recorded during bursts was significantly smaller than the total coefficient of variation from all EPSCs (0.36 ± 0.02 vs 0.61 ± 0.04; p < 0.05, paired Student's t test), consistent with bursts having originated from single synapses.

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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.

To determine whether repetitive presynaptic firing is part of the mechanism responsible for generating the bursts, we tested for the involvement of Na⁺ channels. To do this, we repeated the above experiments and washed 0.2 μm TTX onto the slice after inducing the effect under control conditions. In four of four cells, TTX completely abolished the GABA_AR-induced EPSCs (Fig. 2D), indicating that the effect requires Na⁺ channel activation and subsequent firing of presynaptic action potentials.

GABA_AR-induced EPSCs show long-lasting fatigue

Despite the prolonged presence of the GABA_AR agonist (15 s), the overall period of bursting was variable, lasting from ∼0.5 to 5 s (Figs. 1A, 2D), with each individual burst lasting only for a few hundred milliseconds (Fig. 2A). This indicates that GABA_AR activation is sufficient to induce the bursting but is not sufficient to maintain it. During activation, GABA_A receptors enter desensitized states (Jones and Westbrook, 1995), and this could explain why the increased EPSC frequency is not maintained despite prolonged presence of muscimol. To gain insight into the mechanism responsible for stopping the bursting and perhaps the mechanism responsible for producing it, we determined the amount of time that is required to fully recover the effect between agonist applications. We found that, when compared with using 5 min intervals between applications, repeating the agonist application every 1.5 min either abolished the response altogether or severely attenuated its magnitude (p < 0.05, Student's t test; n = 4) (Fig. 3A,B). However, waiting 10 min between agonist applications showed a trend toward increasing the response without producing a significantly different response than when 5 min intervals were used (p > 0.05, Student's t test; n = 4) (Fig. 3A,B). After long-lasting GABA pulses, GABA_ARs have been shown to recover from desensitization with time constants on the order of at most ∼13 s (Jones and Westbrook, 1995). Therefore, because the increase in EPSC frequency was not fully recovered after 90 s, GABA_AR desensitization is unlikely to provide the entire explanation for its transience. It has been noted in another context that responses to long GABA applications recover more slowly than expected from GABA_AR desensitization properties (Segal, 1993). An attractive explanation is that the responses involve changes in [Cl⁻]_i and take time to recover. Changes in [Cl⁻]_i are particularly likely because of the surface to volume ratio of the axon domain, but testing for [Cl⁻]_i redistribution with direct measurements from the axons will await additional study.

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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.

GABA_AR activation occurs in the molecular layer

It is not obvious how GABA_AR activation induces activity in parallel fibers, but one possibility is that the agonist that was released from our puffer pipette was not localized to the molecular layer and instead diffused into the granule cell layer. If this was the case, then several scenarios could explain why EPSCs are induced by the GABA_AR agonists. The cell bodies of granule cells contain GABA_ARs (Pirker et al., 2000), and, despite hyperpolarized E_GABA with respect to the threshold for voltage-gated Na⁺ channels (Brickley et al., 1996), it is feasible that prolonged activation of the GABA_ARs on the cell bodies could induce spiking in granule cells via a biphasic response to GABA_AR activation similar to that seen in other cell types (Alger and Nicoll, 1982; Staley et al., 1995; Kaila et al., 1997; Perkins and Wong, 1997; Price and Trussell, 2006). If diffusion of our GABA_AR agonists into the granule cell layer induced such a depolarization in the cell bodies of the granule cells, then it could have possibly generated a train of action potentials in granule cell somata and led to the burst of postsynaptic EPSCs we recorded in postsynaptic targets. In fact, Golgi cells, whose cell bodies are also in the granule cell layer, have been shown to increase their synaptic output in response to application of GABA (Kaneda et al., 1995), perhaps attributable to a similar mechanism.

To determine whether this or any other scenarios involving the diffusion of the agonist away from the tip of the pipette to the granule cell layer was causing the induction of EPSCs, we designed an experiment in which different locations of the puffer pipette were used to release muscimol. We reasoned that, if we moved the pipette closer to the site of induction of the EPSCs, then the muscimol would have a shorter distance to diffuse and the effect would be stronger and recorded after a shorter delay, whereas moving the pipette away from the site of induction would increase the delay and attenuate the response. Therefore, after inducing the effect in the control position with the puffer pipette next to the patch-clamped interneuron (Fig. 4, left), we used the laminar organization of the cerebellar cortex as a guide to move the pipette to two additional positions. We placed the puffer pipette in a position closer to the granule cells in the Purkinje cell layer (Fig. 4A, middle) as well as in a position the same distance away from the control position on the other side of the patch-clamped interneuron (Fig. 4A, right). A significantly larger response was recorded when the puffer pipette was placed at the control position in the center of the molecular layer (p < 0.05, Student's t test; n = 5) (Fig. 4B,C). The weak response recorded when the pipette was moved away from the control position coincided with a small, slow, outward current in the interneuron. This current occurred after a much longer delay from the beginning of the puffer application than the outward current that was recorded when the puffer pipette was in the control position (Fig. 4C). This delayed outward current is indicative of muscimol diffusion back to the interneuron in the molecular layer and was recorded when the pipette was either moved closer to or farther from the granule cell layer. Therefore, the small delayed EPSC response recorded when the pipette is moved away from the control position probably results from muscimol diffusion away from the tip of the puffer pipette back to the parallel fibers. Together, these data indicate that the site of induction is in the molecular layer and not on the granule cell somata.

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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.

GABA_AR activation induces EPSCs in Purkinje cells

Having determined that activation of GABA_ARs in the molecular layer induces release of glutamate from parallel fibers, we next performed whole-cell recordings from Purkinje cells to test whether the effect could also be recorded in other cell types targeted by parallel fibers. We found that the fast inward currents also occurred in Purkinje cells (Fig. 5A,B), and, as in the interneurons, the response was blocked with 5 μm gabazine (n = 4) (Fig. 5C) or 2 μm NBQX (n = 3) (Fig. 5D). Therefore, the response recorded in Purkinje cells can also be attributed to GABA_AR-induced activation of AMPA-mediated currents. Along with the results of the experiments in which the position of the puffer pipette was varied, these data indicate that GABA_AR activation in the molecular layer leads to release of glutamate from the parallel fibers.

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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.

EPSC induction does not result from K⁺-mediated volume transmission

The possibility remains that the GABA_ARs are not directly on parallel fibers but instead are on another class of cells in the molecular layer that cause indirect depolarization of parallel fibers. It has been demonstrated that GABA_AR stimulation can lead to increased [K⁺]_o of up to ∼8 mm (Barolet and Morris, 1991; Avoli et al., 1996; Kaila et al., 1997). Another explanation for the ability of GABA_AR agonists to activate parallel fibers in our study could be that, by applying GABA to the molecular layer, we activated GABA_ARs on other cells in the vicinity of the parallel fibers (e.g., interneurons, Purkinje cells, or glia) and caused the subsequent release of K⁺ from these cells, which then depolarized parallel fibers beyond the voltage-gated Na⁺ channel threshold. To test this hypothesis, we performed experiments in which we recorded from Purkinje cells and induced EPSCs using 10 μm muscimol and then attempted to reproduce the EPSC induction by exchanging the puffer pipette with one containing ACSF supplemented with 20 mm K⁺. We found that exchanging muscimol for 20 mm K⁺ produced a small inward current in the Purkinje cell but did not increase the frequency of sEPSCs (n = 4) (Fig. 5E). Therefore, a secondary pathway involving the release of K⁺ from neighboring cells does not appear to be responsible for the GABA_AR-induced EPSCs.

GABA_AR α₁ subunits are located on parallel fiber terminals

Having determined that GABA_ARs located on parallel fibers are responsible for the muscimol-induced activation of their synapses, we next investigated the localization of GABA_ARs on these fibers. Using electron microscopy of immunolabeled α₁ subunits, we imaged the molecular layer and found that 27% of all asymmetrical synapses had α₁ subunits associated with the plasma membrane at either or both the presynaptic (Fig. 6A1–A4) and postsynaptic (Fig. 6B1,B2) sides of the synapse. These asymmetric synapses were considered to be originating from parallel fibers because parallel fibers form >99% of the excitatory, asymmetric synapses in the molecular layer of the cerebellum (Palay and Chan-Palay, 1974; Silver et al., 1998; Foster et al., 2002). The immunogold particles were primarily associated with the active zone, although occasional membrane labeling occurred away from the synapse (Fig. 6B1,B2, crossed arrows). A particle density profile performed orthogonally with respect to the active zone indicated that most labeling occurred within 25 nm of the center of the synaptic cleft (Fig. 6C). This distribution is consistent with a direct association of the antibody to the presynaptic membrane in 74% of the cases and to the postsynaptic membrane in 26% of the cases. Although the presence of postsynaptic GABA_ARs at glutamatergic synapses in the cerebellum is consistent with a previous study (Nusser et al., 1998), their presence on the presynaptic membrane is a novel finding. A lateral distribution analysis further determined that gold particles were found more often within the synapse than in the extrasynaptic membrane (59 vs 33 particles, respectively, in the presynaptic membrane and 26 vs 17 particles, respectively, in the postsynaptic membrane) (Fig. 6D1,D2). Both for presynaptic and postsynaptic labeling, it is striking that the majority of particles were close to the edge of the synaptic densities. One possibility for this finding is that lateral diffusion of receptors on the cell membrane (Triller and Choquet, 2005) may be slower at the presynaptic terminals and at the postsynaptic density than in the extrasynaptic membrane, and this could underlie the enrichment of α1 subunits in these areas. Incomplete preservation of morphology after preparation of the tissue for EM precluded reliable identification of parallel fiber axons, but it is certainly possible that receptors exist on the axon as well. Altogether, these results indicate that α₁ subunits are targeted to the presynaptic side of parallel fiber synapses, and they support the hypothesis that functional GABA_ARs underlie the muscimol-induced depolarization of these synapses.

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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).

GABA_AR activation increases parallel fiber transmission

If GABA_ARs cause depolarization of the presynaptic terminal, then it would be predicted that transmission at parallel fiber synapses should be increased when the receptors are activated (Awatramani et al., 2005). To test whether transmission is affected, we evoked EPSCs in Purkinje cells by stimulating parallel fibers and compared EPSCs evoked in control conditions with EPSCs evoked in the presence of muscimol. To achieve this, EPSCs were evoked in coronal slices under control conditions by placing an extracellular stimulating electrode in the molecular layer ∼300 μm from the patch-clamped Purkinje cell. A puffer pipette was placed in the parallel fibers approximately half way between the stimulation electrode and the patch-clamped Purkinje cell, and 10 μm muscimol was puffed after baseline EPSCs were recorded for 30 s. Figure 7A shows that the amplitudes of the resulting EPSCs recorded in Purkinje cells were increased during application of muscimol and returned to control amplitudes after muscimol was washed away. Muscimol increased evoked EPSC amplitudes by 42.1 ± 6.6% (Fig. 7A,B), and the effect was completely blocked by 5 μm gabazine (data not shown). These results indicate that muscimol application increased the amplitude of parallel fiber EPSCs by activating presynaptic GABA_ARs.

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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.

Presynaptic receptors activated by synaptically released GABA potentiate parallel fiber transmission

The experiments above demonstrate that activation of the presynaptic receptors with exogenously applied GABA agonists potentiate parallel fiber transmission, but the concentrations and time course we used for the application of the agonist in those experiments are obviously different from those that the receptors would encounter in normal cerebellar function. We therefore wanted to determine whether activation of the receptors by GABA released from neurons has the same effect on parallel fiber transmission. Previously, Dittman and Regehr (1997) demonstrated that GABA_BRs on parallel fibers can be activated by GABA released from nearby interneurons. By recording evoked EPSCs in Purkinje cells using a stimulation paradigm identical to the one used in their study but using a GABA_BR antagonist (see Materials and Methods) (Fig. 8A), we show that the GABA released from interneuron activity also activates GABA_ARs. In Figure 8B, we show that EPSCs evoked 100 ms after a train of 10 stimuli at 100 Hz is given to the parallel fibers have amplitudes that are 14 ± 4% greater than EPSCs that are evoked before the train (n = 4; p < 0.05, paired Student's t test) (Fig. 8D). However, washing 5 μm gabazine onto the slice abolished the difference in amplitudes of the events evoked before and after the train (Fig. 8C,D). This effect is attributable to the sequential activation by the stimulus train of parallel fibers and their postsynaptic MLIs. The GABA released by the MLIs activates the GABA_ARs on the presynaptic parallel fibers and facilitates the EPSC recorded postsynaptically. However, when the presynaptic receptors are blocked by 5 μm gabazine, the GABA released by the interneurons has no effect and the postsynaptically recorded EPSC is unchanged by the stimulus train. These experiments demonstrate that activation of presynaptic GABA_ARs by GABA released from nearby MLIs induces a facilitation of parallel fiber transmission similar to that recorded when receptors are activated with exogenously applied agonists.

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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).