Plasticity of GABA(B) receptor-mediated heterosynaptic interactions at mossy fibers after status epilepticus

Abstract

Several neurotransmitters, including GABA acting at presynaptic GABA(B) receptors, modulate glutamate release at synapses between hippocampal mossy fibers and CA3 pyramidal neurons. This phenomenon gates excitation of the hippocampus and may therefore prevent limbic seizure propagation. Here we report that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hr later by a loss of GABA(B) receptor-mediated heterosynaptic depression among populations of mossy fibers. This was accompanied by a decrease in the sensitivity of mossy fiber transmission to the exogenous GABA(B) receptor agonist baclofen. Autoradiography revealed a reduction in GABA(B) receptor binding in the stratum lucidum after status epilepticus. Failure of GABA(B) receptor-mediated modulation of mossy fiber transmission at mossy fibers may contribute to the development of spontaneous seizures after status epilepticus.

Figure 1.

Mossy fiber fEPSPs were identified by demonstrating marked frequency facilitation (Freq Facil.) and sensitivity to the group II metabotropic glutamate receptor agonist DCG-IV. A, fEPSPs (traces are averages of 5 trials, taken from experiment shown in B) recorded in the stratum lucidum while stimulating the stratum granulosum of the dentate gyrus at baseline frequencies of 0.05, 1, and 0.05 Hz in the presence of the group II metabotropic glutamate receptor DCG-IV (1 μm). B, Left graph, Example of an experiment demonstrating the identification of mossy fiber fEPSPs by frequency facilitation (arrows, change in stimulation frequency from 0.05 to 1 Hz) and DCG-IV sensitivity. Right graph, Same experiment showing the effect of an increase in frequency plotted against stimulus number. C, Summary data (n = 8). The fEPSP amplitude increased to at least 200% of baseline (20th/1st response) when the stimulus frequency was increased from 0.05 to 1 Hz, and the mGluR agonist DCG-IV (1 μm) decreased fEPSP amplitude to <30% of baseline.

Figure 2.

Heterosynaptic depression evoked in control slices is mediated by GABA_B receptors. A conditioning train was applied to the conditioning pathway preceding every 10th stimulus delivered to the test pathway. A, Example of one experiment from a control slice. Heterosynaptic depression was observed as a reduction in amplitude of the fEPSPs that were preceded by a train (open circles) compared with fEPSPs not preceded by a train (each filled circle represents the average of the 9 unconditioned fEPSPs in each cycle). The opioid antagonist naloxone (10 μm) had no effect on the magnitude of depression. The GABA_B receptor antagonist SCH50911 (20 μm) abolished the depression. fEPSPs (averages of 5 trials each) are shown in the absence (1) and presence (2) of SCH50911. B, Summary of the ratio of conditioned/unconditioned fEPSP amplitudes in 8 control slices showing the abolition of heterosynaptic depression by blocking GABA_B receptors. C, Example of presynaptic fiber volleys from one experiment showing that the conditioned presynaptic fiber volley [preceded by a conditioning train (c)] is not different in amplitude or shape from the unconditioned fiber volley [not preceded by a train (u)]. Traces are averages of responses in the presence of 50 μm NBQX from two cycles of nine unconditioned stimuli and one conditioned stimulus.

Figure 3.

GABA_B receptor-mediated heterosynaptic depression was absent after SE. A, Mossy fiber fEPSPs in slices after pilocarpine-induced SE (n = 12) showed frequency facilitation and DCG-IV sensitivity, which was not significantly different from that of control slices. B, Heterosynaptic depression was absent after pilocarpine-induced SE: example of one experiment. fEPSPs are shown in the absence and presence of SCH50911 (each trace is the average of 5 trials; filled circles, conditioned; open circles, unconditioned). The GABA_B receptor antagonist SCH50911 had no significant effect. C, There was no significant heterosynaptic depression in animals after pilocarpine-induced (Pilo) or perforant path (PP) stimulation-induced SE in contrast to the marked depression seen in control (Con) animals. D, Size of GABA_B receptor-mediated heterosynaptic depression, estimated from the effect of the GABA_B antagonists SCH50911 and CGP52432 on the fEPSP ratio (SE: both models combined; n = 18).

Figure 4.

Sections through GABA immunogold-labeled mossy fiber terminals in SE (A, B) and control (C, D) animals. Tissue from animals after SE have significantly higher density of immunogold particles (arrows). Arrowheads, Synaptic sites; mft, mossy fiber terminal; s, spine. Scale bar: A, B, 100 nm; C, 120 nm; D, 60 nm.

Figure 5.

Heterosynaptic depression could not be rescued after SE by blocking group II metabotropic glutamate receptors by blocking GABA uptake or by increasing the number of conditioning stimuli. A, Example of one experiment. The group II metabotropic glutamate receptor antagonist LY341495 (LY; 500 nm) failed to restore heterosynaptic depression in a slice obtained after pilocarpine-induced SE. Application of the GABA transporter GAT1 blocker NO711 (20 μm) also failed to affect the fEPSP ratio. Sample traces, fEPSPs in control conditions and in the presence of LY34195, NO711, and CGP52432 (each trace is the average of 5 trials). B, Summary of effects of NO711 (n = 4) and LY341495 (n = 5) on the fEPSP ratio in animals after pilocarpine-induced SE. C, A 100 Hz train did not rescue heterosynaptic depression in slices after SE. The fEPSP ratio did not change significantly when the train was increased from 50 to 100 Hz (p = 0.4; paired t test).

Figure 6.

Mossy fiber fEPSPs become less sensitive to the GABA_B receptor agonist baclofen after SE. A, fEPSPs (averages of 5 traces) illustrating reduced sensitivity to baclofen 24 hr after SE. Left to right, Traces are shown at baseline and in 0.1, 1, and 10 μm baclofen. B, Baclofen has a less potent effect on mossy fiber fEPSPs 24 hr after SE (p < 0.05; unpaired t test on IC₅₀ values; 6 control slices and 7 post-SE slices), and the I_max was significantly reduced in slices 3 weeks after SE (p = 0.007; n = 6).

Figure 7.

Reduction of binding of [³H]CGP62349 to GABA_B receptors in the stratum lucidum after pilocarpine-induced SE. A, Example of an autoradiograph from an animal after pilocarpine-induced SE. We measured binding in the stratum lucidum, (black arrow). The stratum pyramidale of CA3 is marked with a white arrow. B, The binding parameter B_max, which reflects maximal binding of the radioactive ligand to GABA_B receptors, was significantly lower than that of the control after SE (control, n = 5; SE, n = 4; p = 0.033 for difference).