Synchronous GABA-mediated potentials and epileptiform discharges in the rat limbic system in vitro

Abstract

Application of 4-aminopyridine (4AP, 50 microM) to combined slices of adult rat hippocampus-entorhinal cortex-induced ictal and interictal epileptiform discharges, as well as slow field potentials that were abolished by the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAGO, 10 microM) or the GABAA receptor antagonist bicuculline methiodide (BMI, 10 microM); hence, they represented synchronous GABA-mediated potentials. Ictal discharges originated in the entorhinal cortex and propagated to the hippocampus, whereas interictal activity of CA3 origin was usually recorded in the hippocampus. The GABA-mediated potentials had no fixed site of origin or modality of propagation; they closely preceded (0.2-5 sec) and thus appeared to initiate ictal discharges. Only ictal discharges were blocked by the antagonist of the NMDA receptor 3,3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP, 10 microM), whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished all epileptiform activities. The GABA-mediated potentials continued to occur synchronously in all regions even after concomitant application of CNQX and CPP. [K+]o elevations were recorded in the entorhinal cortex during the ictal discharge (peak values = 13.9 +/- 0.9 mM) and the synchronous GABA-mediated potentials (peak values = 4.2 +/- 0.1 mM); the latter increases were presumably attributable to postsynaptic GABAa-receptor activation because they were abolished by DAGO or BMI. Their role in initiating ictal activity was demonstrated by using DAGO, which abolished both GABA-mediated synchronous potentials and ictal discharges. These data indicate that NMDA-mediated ictal discharges induced by 4AP originate in the entorhinal cortex; such a conclusion is in line with clinical evidence obtained in temporal lobe epilepsy patients. 4AP also induces GABA-mediated potentials that spread within the limbic system when excitatory transmission is blocked and may play a role in initiating ictal discharge by increasing [K+]o.

Fig. 8.

A, Schematic drawing of a hippocampus–entorhinal cortex slice; the entorhinal region where recordings were made is indicated by the dotted area.B, Simultaneous field potential (top trace in each pair) and [K⁺]_orecordings (bottom trace in each pair) performed at different depths of the entorhinal cortex as indicated by thenumbers to the left of each pair. C, Plots of the maximal values of the [K⁺]_o increases and of the amplitude of the population spikes and of the DC shift associated with the ictal discharges recorded at different depths in three slices.

Fig. 1.

Field potential recordings performed simultaneously in the entorhinal cortex (middle layers) and in different hippocampal areas (apical dendrites of CA1 and CA3, and dentate granule layer) during application of 4AP demonstrate the occurrence of three different types of activity. The first (continuous line in A) is recorded synchronously in all areas and consists of a sustained ictal-like epileptiform discharge. The second type (arrows in A) is seen in the hippocampal regions only and consists of continuous interictal-like events. The third type of synchronous activity, referred to as GABA-mediated potential, is recorded in all areas and is characterized by a slow field potential that is of negative polarity in all areas except dentate gyrus (asterisk in B).A and B are continuous recordings.

Fig. 2.

Expanded traces of simultaneous field potential recordings performed in the entorhinal cortex and in different hippocampal areas (same experimental conditions as in Fig. 1) show the modalities of onset and spread of ictal discharges (A), hippocampal interictal events (B), and GABA-mediated potentials (C). Note that the onset of the ictal discharges occurs first in the entorhinal cortex (a and b inpanel A), and different sites of origin characterize the different examples of GABA-mediated potentials (a–d inpanel C).

Fig. 3.

Effects induced by sectioning the perforant pathway (A) or both the perforant pathway and the subicular area to achieve a complete separation between the entorhinal cortex and the hippocampus proper (B). The location and the extent of the cuts are shown at the top of each panel. A, Lesioning the perforant pathway only abolishes ictal discharges in all hippocampal areas; under these conditions, the interictal activity of hippocampal origin continues to occur, and GABA-mediated potentials can be recorded synchronously in both entorhinal cortex and hippocampus.B, The complete surgical separation of the entorhinal cortex and hippocampus makes the GABA-mediated potentials occur asynchronously in entorhinal cortex and hippocampal areas.

Fig. 4.

Effects induced by the NMDA receptor antagonist CPP (10 μm) on the spontaneous field potential activity induced by 4AP. Note that the ictal discharges are abolished and replaced by a sequence of interictal events (arrowheads) that appear synchronously in the entorhinal cortex and hippocampus proper; by contrast, the interictal activity recorded in the hippocampus proper continues to occur at a higher rate than in control. Note also that the synchronous GABA-mediated potentials are not affected by CPP application.

Fig. 5.

Effects induced by the non-NMDA receptor antagonist CNQX on the spontaneous synchronous activity induced by 4AP.A, CNQX abolishes all types of epileptiform discharges in both entorhinal cortex and hippocampus, whereas GABA-mediated field potentials continue to occur synchronously in most of the cases, even after further addition of the NMDA receptor antagonist CPP.B, Plot of the changes induced by CNQX on the amplitude and rate of occurrence of the GABA-mediated potential analyzed in the entorhinal cortex and CA3 of six slices. C, Effects induced by CNQX on the epileptiform activity recorded in the entorhinal cortex at different depths (as indicated by the numbers on theleft) from the pia; in this experiment, both interictal and ictal discharges were recorded under control conditions, whereas after CNQX application only a synchronous event reminiscent of the GABA-mediated potential is seen.

Fig. 6.

Effects induced by DAGO on the synchronous activity recorded in the entorhinal cortex and CA3 area during application of 4AP. Note in A that during DAGO, both GABA-mediated field potentials and ictal discharges disappear, whereas interictal discharges propagating to the hippocampus proper appear in the entorhinal cortex. This effect is accompanied by a decrease in the rate of occurrence of hippocampal interictal discharges. B, Plot of the changes induced by DAGO on the rate of occurrence of GABA-mediated potentials, ictal discharges, and interictal events in five slices.

Fig. 7.

Field potential (top trace) and [K⁺]_o (bottomtrace) recordings in the middle layers of the entorhinal cortex during application of 4AP. The GABA-mediated field potentials are associated with transient increases in [K⁺]_o (left panel in A), while a sustained elevation is seen during the tonic phase of the ictal discharge; note that the ictal discharge is initiated by an increase in [K⁺]_o that is larger than the elevations associated with the isolated GABA-mediated field potential. Note in B that distinct increases in [K⁺]_o accompany clonic discharges during return of [K⁺]_o toward baseline values (asterisk) and that a pronounced undershoot follows the termination of the ictal discharge.

Fig. 9.

A, Effects induced by the excitatory amino acid receptor antagonists CNQX and CPP on the 4AP-induced synchronous activity recorded in the entorhinal cortex with simultaneous field potential and [K⁺]_o recordings.B, Field potential and [K⁺]_o recordings performed in the entorhinal cortex at different depths from the pia (as indicated by the numbers at top of each panel) during application of 4AP, CNQX, and CPP. C, Plots of the peak values and duration of the [K⁺]_o increases as well as of the amplitude of the GABA-mediated field potentials recorded at different depths in five entorhinal cortex slices during application of 4AP, CNQX, and CPP.