Diminished presynaptic GABA(B) receptor function in the neocortex of a genetic model of absence epilepsy

Abstract

Changes in GABA(B) receptor subunit expression have been recently reported in the neocortex of epileptic WAG/Rij rats that are genetically prone to experience absence seizures. These alterations may lead to hyperexcitability by downregulating the function of presynaptic GABA(B) receptors in neocortical networks as suggested by a reduction in paired-pulse depression. Here, we tested further this hypothesis by analyzing the effects induced by the GABA(B) receptor agonist baclofen (0.1-10 microM) on the inhibitory events recorded in vitro from neocortical slices obtained from epileptic (>180 day-old) WAG/Rij and age-matched, non-epileptic control (NEC) rats. We found that higher doses of baclofen were required to depress pharmacologically isolated, stimulus-induced IPSPs generated by WAG/Rij neurons as compared to NEC. We also obtained similar evidence by comparing the effects of baclofen on the rate of occurrence of synchronous GABAergic events recorded by WAG/Rij and NEC neocortical slices treated with 4-aminopyridine + glutamatergic receptor antagonists. In conclusion, these data highlight a decreased function of presynaptic GABA(B) receptors in the WAG/Rij rat neocortex. We propose that this alteration may contribute to neocortical hyperexcitability and thus to absence seizures.

Fig. 1

A–C Intracellular responses to focal electrical stimuli recorded in NEC and WAG/Rij neocortical cells in the presence of control ACSF (a) and during application of glutamatergic receptor antagonists (CNQX+CPP) (b). Samples were obtained at different membrane potentials by injecting pulses of intracellular current. Note that under control conditions the WAG/Rij cell shown in C generates a burst of action potentials as well as that during depolarizing commands the neurons in A and B produce a hyperpolarization characterized by fast and slow components. D, E Plots of the reversal potential values of IPSP fast and slow components recorded in control ACSF and in the presence of glutamatergic receptor antagonists from NEC (n = 9 and 10, respectively) and WAG/Rij (n = 7 and 12, respectively) cells. Statistical analysis of these values revealed no significant difference between the two types of tissue.

Fig. 2

A Effects induced by baclofen (0.1–10 μM) on the pharmacologically isolated IPSPs recorded intracellularly from a NEC and from a WAG/Rij neuron following single-shock stimuli. Note that the effects exerted in the WAG/Rij cell are less pronounced than in NEC as well as that this difference can be better appreciated during injection of steady hyperpolarizing current. B Percentage of reduction induced by increasing doses of baclofen on the peak amplitude of the stimulus-induced responses measured in the two types of tissue during injection of hyperpolarizing current (membrane values = −84 to −90 mV). Note that values of reduction are significantly (p < 0.01) different in NEC and WAG/Rij rats at low baclofen doses, but they became similar when approaching the plateau (2 μM) of baclofen effect. * p <0.05, ** p < 0.01 vs. the preceding (lower) baclofen dose; ^## p < 0.01 vs. the respective NEC group of baclofen treatment, Fisher’s LSD test for multiple comparisons.

Fig. 3

A Effects induced by baclofen (0.5 μM) on the pharmacologically isolated IPSPs recorded intracellularly with K-acetate+QX314 filled electrodes from NEC and WAG/Rij neurons following single-shock stimuli. Note that the effects exerted in the WAG/Rij cell are less pronounced than in NEC and that these effects are reversed by application of the GABA_B receptor antagonist CGP 55845; note also that the stimulus-induced event generated by the WAG/Rij neuron is abolished by bath application of picrotoxin. B Percentage of reduction induced by 0.5. and 5 μM of baclofen on the peak amplitude of the stimulus-induced responses measured in the two types of tissue. Note that values of reduction are significantly (^## p < 0.01) different in NEC and WAG/Rij rats at both baclofen doses. ** p < 0.01 vs. baclofen 0.5 μM, Fisher’s LSD test for multiple comparisons.

Fig. 4

A Intracellular and field potential recordings of the spontaneous activity induced by application of 4AP+CNQX+CPP in a WAG/Rij neocortical slice. Note that the negative-going field events are mirrored by a long-lasting depolarization at the intracellular level (arrow). B Intracellular recordings of the spontaneous events recorded at different membrane potentials from NEC and WAG/Rij neocortical neurons during application of ACSF containing 4AP+CNQX+CPP. Note that the long-lasting depolarization is preceded by an early hyperpolarization (arrowheads) and followed by a slow hyperpolarization (asterisks). Note also that the amplitude of these different components is modified by injection of intracellular current in a similar manner in both NEC and WAG/Rij tissue. C Intracellular responses obtained following electrical stimuli demonstrate similar changes during intracellular injection of depolarizing and hyperpolarizing current. B, C Note that small amplitude action potentials, which may represent ectopic spikes, occur in both types of tissue during spontaneous and stimulus-induced network-driven IPSPs (thick arrows).

Fig. 5

Effects induced by baclofen on the rate of occurrence of the synchronous field potentials induced by application of 4AP+CNQX+CPP in NEC and WAG/Rij neocortical slices. A Field potential recordings obtained under control conditions and during application of 0.5 and 5 μM of the GABA_B receptor agonist. Note that the effects are less pronounced in the WAG/Rij slice with both concentrations. B Percentage reduction in the occurrence of the synchronous field activity during increasing concentrations of baclofen. Note that values of reduction are significantly (p < 0.01) lower in NEC compared to WAG/Rij rats at every baclofen dose. ** p < 0.01 vs. the preceding (lower) baclofen dose; ^# p < 0.05, ^## p < 0.01 vs. the respective NEC group of baclofen treatment, Fisher’s LSD test for multiple comparisons.