In the developing rat hippocampus, endogenous activation of presynaptic kainate receptors reduces GABA release from mossy fiber terminals

Abstract

Presynaptic kainate receptors regulate synaptic transmission in several brain areas but are not known to have this action at immature mossy fiber (MF) terminals, which during the first week of postnatal life release GABA, which exerts into targeted cells a depolarizing and excitatory action. Here, we report that, during the first week of postnatal life, endogenous activation of GluK1 receptors by glutamate present in the extracellular space severely depresses MF-mediated GABAergic currents [GABA(A)-mediated postsynaptic currents (GPSCs)]. Activation of GluK1 receptors was prevented by treating the slices with enzymatic glutamate scavengers that enhanced the clearance of glutamate from the extracellular space. The depressant effect of GluK1 on MF-GPSCs was mediated by a metabotropic process sensitive to pertussis toxin. In the presence of U73122 (1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a selective inhibitor of phospholipase C, along the transduction pathway downstream to G-protein, GluK1 activation increased the probability of GABA release, thus unveiling the ionotropic action of this receptor. In line with this type of action, we found that GluK1 enhanced MF excitability by directly depolarizing MF terminals via calcium-permeable cation channels. Furthermore, GluK1 dynamically regulated the direction of spike time-dependent plasticity occurring by pairing MF stimulation with postsynaptic spiking and switched spike time-dependent potentiation into depression. The GluK1-induced depression of MF-GPSCs would prevent excessive activation of the CA3 associative network by the excitatory action of GABA and the emergence of seizures in the immature brain.

Figure 1.

AMPA receptors do not contribute to unitary postsynaptic currents evoked in CA3 principal cells by minimal stimulation of granule cells in the dentate gyrus. A, Unitary synaptic currents evoked in a CA3 pyramidal cell at P4 with different stimulation intensities before (Control) and during application of GYKI 52466 (30 μm). Each trace is the average of 15–20 responses (including failures). B, Peak amplitudes of synaptic currents represented in A are plotted as a function of stimulus intensities. The bars are SEM, and the dashed lines are the averaged amplitude of GPSCs. Latency (C) and rise time (D) distributions of individual currents evoked in a CA3 pyramidal cell by minimal stimulation of granule cells in the dentate gyrus in the absence (Control) and in the presence of GYKI 52466. Note that, in the presence of GYKI 52466, the unimodal distribution of latencies and rise times of individual responses did not change.

Figure 2.

Endogenous activation of presynaptic kainate receptors downregulates MF-GPSCs. A, Superimposed individual traces of MF-GPSCs evoked in the presence of GYKI 52466 (30 μm) and GYKI 52466 plus DNQX (50 μm). Note that, in this and in the following experiments, GYKI 52466 was always present in the bathing solution in control conditions. Below, Averaged traces (successes plus failures). Note that DNQX enhanced the amplitude of the first response and reduced the number of synaptic failures. B, Summary plot showing the mean amplitude of GPSCs obtained in eight cells in the presence of DNQX, l-AP4, and PTX. Vertical bars are SEM. The horizontal dashed line refers to the mean amplitude value measured before DNQX. C, Superimposed individual traces (above) and average traces (below) of MF-GPSCs evoked in the presence of GYKI 52466 (30 μm) and GYKI 52466 plus UBP 302 (10 μm). D, As in B, but in the presence of UBP 302, l-AP4, and PTX (n = 19).

Figure 3.

Bath application of the selective GluK1 agonist ATPA mimics the effects of endogenous glutamate on kainate receptors. A, Averaged traces of GPSCs evoked in a CA3 pyramidal cell by stimulation of granule cells in the dentate gyrus, in control condition and after addition of ATPA (1 μm). B, Summary plot for 11 cells. C–F, Amplitude (C), successes (D), PPR (E), and inversed square of CV (F) measured in individual cells before and after application of ATPA. **p < 0.01; ***p < 0.001.

Figure 4.

Ambient glutamate activates presynaptic GluK1 kainate receptors on mossy fibers terminals. A, Averaged traces of GPSCs evoked by stimulation of granule cells in the dentate gyrus before and after addition of the glutamate scavenger (GPT plus pyruvate) and the scavenger plus UBP 302. The broad-spectrum mGluR_1-8 antagonist LY341495 (100 μm) was present throughout the experiments. B, Pooled data from seven cells to show that the glutamate scavenger increased the mean peak amplitude of GPSCs and fully occluded the effects of UBP 302. Error bars indicate SEM. C–E, Successes (C), PPR (D), and inversed square of CV (E) measured in individual cells before and after application of the glutamate scavenger. **p < 0.01.

Figure 5.

The depressant effect of kainate on GPSCs involves a G-protein-mediated mechanism. A, Summary plot showing the effects of UBP 302 on the peak amplitude of GPSCs in controls (open symbols) and after overnight treatment with PTx (closed symbols). The insets above the graph represent average traces obtained in the presence of GYKI 52466 and GYKI 52466 plus UBP 302. B, As in A, but in the presence of the glutamate scavenger showing that also the effect of the scavenger is dependent on G-protein. C, Application of baclofen (20 μm; bar) induced an outward currents associated with a reduction of spontaneous synaptic events (top trace). Application of baclofen to a cell recorded with a patch pipette containing GDPβS failed to produce an outward current but was still able to depress the ongoing synaptic activity (bottom trace). D, E, GDPβS into the intrapipette solution did not affect UBP 302-induced GPSCs facilitation. D, Representative traces. E, Summary plot from five cells. Error bars indicate SEM.

Figure 6.

Blocking PLC with U73122 reveals the ionotropic action of GluK1 on MF-GPSCs. A, Averaged traces of MF-GPSCs evoked in a CA3 principal cell in the presence of U73122 (10 μm; Control) and U73122 plus UBP 302 (10 μm). B, Summary plot showing the mean amplitude of GPSCs obtained in six cells in the presence of U73122 before and during application of UBP 302 (bar). The amplitude of synaptic responses is normalized to those obtained before UBP 302 (dashed line). In the presence of UBP 302, the amplitude of the synaptic responses was significantly different from controls (p = 0.01). C–F, As in A and B, but in the presence of ATPA (1 μm) (C, D) and the glutamate scavenger (GPT plus pyruvate) (E, F). Note that both the ATPA-induced potentiation and the scavenger-induced depression of GPSCs amplitude were significantly different from controls (p = 0.01 and p < 0.001, respectively). Error bars indicate SEM.

Figure 7.

GluK1 receptors sensitive to philantotoxin control MF excitability. A, Consecutive traces showing antidromic spikes recorded in granule cells on stimulation of MF in stratum lucidum before and during application of UBP 302 (note that the stimulus strength was set to obtain >50% of successes). B, Summary plot of UBP 302 effects on success rate (n = 7). C, D, In cells with <50% of successes, ATPA enhanced MF excitability and the success rate (n = 7). E, F, PhTx (3 μm) mimicked the effects of UBP 302 (n = 7). G, H, In the presence of philantotoxin, UBP was not effective (n = 7). ***p < 0.001. Error bars indicate SEM.

Figure 8.

At immature MF–CA3 synapses, presynaptic kainate receptors control the direction of STDP. A, Schematic representation of the experimental design. B, The stimulation of granule cells in the dentate gyrus (pre) preceded the postsynaptic spike (post) by 15 ms (Δt). C, Summary plot of the mean peak amplitude of GPSCs recorded in the presence of GYKI 52466 before and after pairing (arrow at time 0; n = 11). The dashed line represents the mean amplitude of GPSCs before pairing. The insets represent averaged GPSCs obtained from a single neuron before and after pairing. Note that pairing induced synaptic depression. D, Summary plot of MF-GPSCs amplitude obtained in the presence U73122 versus time (n = 7). Note that blocking PLC with U73122 failed to produce any effect on GPSCs amplitude. E, As in C, but in the presence of UBP 302 (n = 9). In this case, pairing induced synaptic potentiation. Error bars indicate SEM.