Excitatory actions of endogenously released GABA contribute to initiation of ictal epileptiform activity in the developing hippocampus

Abstract

In the developing rat hippocampus, ictal epileptiform activity can be elicited easily in vitro during the first three postnatal weeks. Changes in neuronal ion transport during this time cause the effects of GABA(A) receptor (GABA(A)-R) activation to shift gradually from strongly depolarizing to hyperpolarizing. It is not known whether the depolarizing effects of GABA and the propensity for ictal activity are causally linked. A key question is whether the GABA-mediated depolarization is excitatory, which we defined operationally as being sufficient to trigger action potentials. We assessed the effect of endogenous GABA on ictal activity and neuronal firing rate in hippocampal slices from postnatal day 1 (P1) to P30. In extracellular recordings, there was a strong correlation between the postnatal age at which GABA(A)-R antagonists decreased action potential frequency (P23) and the age at which ictal activity could be induced by elevated potassium (P23). In addition, there was a strong correlation between the fraction of slices in which ictal activity was induced by elevated potassium concentrations and the fractional decrease in action potential firing when GABA(A)-Rs were blocked in the presence of ionotropic glutamate receptor antagonists. Finally, ictal activity induced by elevated potassium was blocked by the GABA(A)-R antagonists bicuculline and SR-95531 (gabazine) and increased in frequency and duration by GABA(A)-R agonists isoguvacine and muscimol. Thus, the propensity of the developing hippocampus for ictal activity is highly correlated with the effect of GABA on action potential probability and reversed by GABA(A) antagonists, indicating that GABA-mediated excitation is causally linked to ictal activity in this developmental window.

Fig. 1.

Effects on neuronal firing rate of suppression synaptic excitation and inhibition in control conditions.A, Glutamate receptor antagonists NBQX (10 μm) and d-APV (50 μm) reduced neuronal firing rate in P5 (left traces) and P15 (right traces) rat hippocampal slices. Subsequent application of the GABA_A-R antagonist bicuculline (10 μm), in the presence of NBQX and d-APV, diminished neuronal firing at P5 further and increased the firing rate at P15. Examples of extracellular recordings from the CA3 pyramidal cell layer filtered for the MUA (400 Hz high-pass filter). Detected spikes are marked by vertical bars.Insets, Averaged spikes from extracellular recordings.B, The frequency of MUA recorded from the CA3 pyramidal cell layer after the application of NBQX and d-APV and then after the application of bicuculline. C, Age dependence of the effect on MUA of suppression synaptic excitation and inhibition at 3.5 mm [K⁺]_o.

Fig. 2.

Effects on neuronal firing rate and epileptiform activities of suppression synaptic excitation and inhibition at 8.5 mm [K⁺]_o.A, Glutamate receptor antagonists NBQX (10 μm) and d-APV (50 μm) abolish 8.5 mm [K⁺]_o-induced interictal and ictal epileptiform activity in P15 (left traces) and interictal epileptiform discharges in P30 (right traces) rat hippocampal slices. GABA_A-R antagonist bicuculline (10 μm) diminishes neuronal firing at P15 and increases the firing rate at P30. Extracellular recordings from CA3 pyramidal cell layer.B, The frequency of MUA (in sec⁻¹) recorded from the CA3 pyramidal cell layer after the application of NBQX and d-APV and then after the application of bicuculline. C, Age dependence of the effect on MUA of suppression synaptic excitation and inhibition at 8.5 mm[K⁺]_o.

Fig. 3.

Effects of extracellular potassium and synaptic transmissions on neuronal firing rate and the occurrence of ictal epileptiform patterns in the P10–P11 rat hippocampal slices.A, Extracellular potassium concentration dependence of the effect of bicuculline (10 μm) on neuronal firing rate. Ordinates, MUA frequency (sec⁻¹) in NBQX (10 μm),d-APV (50 μm), and CGP55845 (CGP) (1 μm) normalized to MUA frequency (sec⁻¹) in bicuculline (10 μm) at various concentration of [K⁺]_o. B, Occurrence of ictal epileptiform activities at different concentrations of extracellular potassium (number of slices).

Fig. 4.

Epileptiform activities induced by 8.5 mm extracellular potassium in hippocampal slice of a P5 rat. A, Extracellular field potential recordings (wide band, 0.1 Hz to 10 kHz) in the pyramidal cell layer of the CA3b (top trace) and CA3a (bottom trace) subregions. Bath application of 8.5 mm[K⁺]_o induced interictal epileptiform discharges, ictal-sustained and -intermittent discharges, and postictal depression. B, Examples of interictal epileptiform discharges on an expanded time scale before filtering (left traces) and after filtering (right traces) for population field activity (1–100 Hz bandpass filter) and MUA (400 Hz high-pass filter). C, Hypersynchronous ictal-sustained and ictal-intermittent epileptiform discharges on an expanded time scale.

Fig. 5.

Age-dependent alterations of ictal epileptiform patterns induced in rat hippocampal slices by 8.5 mmextracellular potassium. A, Example of extracellular field recording from electrode placed in CA3a pyramidal cell layer in P2 hippocampal slice. Bath application of high K⁺(8.5 mm) induced interictal epileptiform discharges, followed by ictal-intermittent discharges and postictal depression.B, At P16, ictus-like activities were characterized by high-frequency ictal-sustained discharges. C, At P28, only brief epileptiform discharges were observed. The events marked byasterisks are shown on extended time scale.D, Developmental profile of 8.5 mm[K⁺]_o induced epileptiform activities in rat hippocampal slices.

Fig. 6.

Effects of GABA_A receptor antagonists on high K⁺-induced epileptiform activities in rat hippocampal slices. A, GABA_A receptor antagonist bicuculline (10 μm) abolished ictal epileptiform discharges and reduced the frequency of interictal epileptiform discharges. The events marked by small letters (a–c) are shown on extended time scale.B, Low concentration (200 nm) of the GABA_A receptor antagonist SR95531 [gabazine (GBZ)] increased ictal activity interval and decreased their duration. High concentration (10 μm) of SR95531 abolished high K⁺-induced ictal activity in rat hippocampal slices. A, B, Extracellular field potential recordings from the CA3b pyramidal cell layer in P11 and P12 rat hippocampal slices. ^**At thep = 0.05 level, the two means are significantly different.

Fig. 7.

Effects of GABA_A receptor agonists on epileptiform activities in rat hippocampal slices. A, GABA_A receptor agonist muscimol (200 nm) decreased interval between ictus-like activities in P13 rat hippocampal slice. The events marked by asterisks are shown on extended time scale. B, Low concentration of the GABA_A receptor agonist isoguvacine (2 μm) decreased ictal activity interval and increased ictal activity duration in P12 rat hippocampal slice. Higher concentration of isoguvacine (10 μm) abolished ictal-sustained discharges and periodically suppressed interictal epileptiform discharges. A,B, Extracellular field potential recordings from the CA3b pyramidal cell layer in rat hippocampal slices. ^**At the p = 0.05 level, the two means are significantly different.