Synchronous GABA-receptor-dependent potentials in limbic areas of the in-vitro isolated adult guinea pig brain

Abstract

Epileptiform discharges are known to reflect the hypersynchronous glutamatergic activation of cortical neurons. However, experimental evidence has revealed that epileptiform synchronization is also contributed to by population events mediated by GABA(A) receptors. Here, we analysed the spatial distribution of GABA(A)-receptor-dependent interictal events in the hippocampal/parahippocampal region of the adult guinea pig brain isolated in vitro. We found that arterial perfusion of this preparation with 4-aminopyridine caused the appearance of glutamatergic-independent interictal potentials that were reversibly abolished by GABA(A) receptor antagonism. Laminar profiles and current source density analysis performed in different limbic areas demonstrated that these GABA(A)-receptor-mediated events were independently generated in different areas of the hippocampal/parahippocampal formation (most often in the medial entorhinal cortex) and propagated between interconnected limbic structures of both hemispheres. Finally, intracellular recordings from principal neurons of the medial entorhinal cortex demonstrated that the GABAergic field potential correlated to inhibitory postsynaptic potentials (membrane potential reversal, -68.12 +/- 8.01 mV, n = 5) that were interrupted by ectopic spiking. Our findings demonstrate that, in an acute seizure model developed in the adult guinea pig brain, hypersynchronous GABA(A)-receptor-mediated interictal events are generated from independent sources and propagate within limbic cortices in the absence of excitatory synaptic transmission. As spared or enhanced inhibition was reported in models of epilepsy, our data may support a role of GABA-mediated signaling in ictogenesis and epileptogenesis.

Fig. 1

Stimulation of the LOT induces the sequential activation of the PC, l-EC and subfield CA1 of the hippocampus under control conditions. Note that perfusion with 4AP induces further re-entrant propagation of LOT-evoked activity from the hippocampus to the m-EC. The asterisk indicates the delayed slow potential induced by 4AP in the PC. The scheme of the position of the electrode is illustrated in the upper panel.

Fig. 2

(A) Paired-pulse (Pp) stimulation with different ISIs induces suppression of disynaptic potentials in the conditioned PC response. The disynaptic component (arrows) recovers at an ISI of 70 ms under control conditions. During perfusion with 4AP the paired-pulse depression of disynaptic potential did not recover at an ISI of 70 ms. The asterisk marks the delayed slow potential induced by 4AP in the PC. (B) Ratio between the amplitudes of the conditioned (second) and the conditioning (first) disynaptic components at ISIs of 20 and 70–80 ms in control solution (gray columns) and during 4AP application (black columns). (C) PC response to single stimulation (a) and to paired-pulse stimulation at 20 ms ISI (b), and digitally subtracted trace (b–a). The CSD contour plot of the b–a laminar profile shows that only the monosynaptic components located in the most superficial cortex remain after the conditioned stimulus. Isocurrent lines at 0.14 mV / mm². (D) Field potentials simultaneously recorded in the PC, l-EC, CA1 and m-EC after double-pulse stimulation of the LOT in control condition and during 4AP perfusion. 4AP blocks the propagation of activity evoked by the second stimulus of the pair.

Fig. 3

Epileptiform ictal discharges recorded during 4AP perfusion. Simultaneous extracellular recordings were obtained from the PC, l-EC, CA1 region of the hippocampus and m-EC. The ictal discharge originates from the l-EC and propagates to the hippocampus and m-EC. The scheme of the position of the electrodes is illustrated in the upper panel.

Fig. 4

(A) Modification of the LOT-evoked responses in the PC, l-EC, hippocampus (CA1) and m-EC during application of 4AP alone or co-perfusion with glutamatergic and GABA_A receptor antagonists. Note that glutamatergic receptor antagonism [DNQX (50 μM) and AP5 (100 μM)] abolishes the evoked response. Additional perfusion of bicuculline (BIC) (50 μM) does not change the response. (B) Arterial perfusion of the glutamatergic receptor blockers DNQX (50 μM) and AP5 (100 μM) abolishes the field potentials induced by LOT stimulation. The subsequent addition of 4AP to the perfusate has no effect on the evoked potentials. Partial recovery is observed upon washout.

Fig. 5

(A) Epileptiform activity induced by 4AP application in the PC, l-EC, CA1 and m-EC of the isolated brain preparation is abolished by DNQX and AP5. The application of these glutamatergic receptor antagonists reveals potentials in the EC / hippocampal region that are abolished by further administration of the GABA_A receptor blocker bicuculline (BIC) (50 μM). (B) Spontaneous potentials recorded in the PC, CA3 and m-EC and in the CA1 of the contralateral hemisphere during 4AP, CNQX (50 μM) and AP5 (100 μM) perfusion. On the left, traces are presented with a slow time scale to highlight the periodicity of these field potentials. An expansion of the trace outlined by the dotted line is shown on the right. Note that type 1 potentials do not propagate outside the hippocampus, whereas type 2 activity propagates to CA3, m-EC and the contralateral CA1. (C) Examples of type 2 potentials that originate from and propagate to different structures.

Fig. 6

(A) Distribution histogram of the site of initiation of type 2 potential events in the PC, l-EC, hippocampus and m-EC. The hippocampus was subdivided into the DG / CA1 region and DG hilus (DGh) / CA3 region on the basis of the reconstructed position of the recording electrode. The analysis includes 528 events from nine experiments. (B) Distribution histogram of the onset delays of type 2 potentials originating in the m-EC and propagating to different portions of the hippocampus (DG / CA1 and DGh / CA3, respectively). (C) Distribution histogram of the onset delays of the type 2 potential originating in the m-EC and propagating to the contralateral m-EC.

Fig. 7

(A) Type 1 and type 2 potentials recorded from the same position with a recording silicon probe during one experiment. (a) The calculated position of the recording contacts is superimposed on the histological reconstruction of the track of the 16-channel silicon probe (100 μm separation between recording sites). Calibration bar: 500 μm. (b and c) Laminar profiles were recorded with the same probe position. (b) Field potential profile of type 1 potential recorded in the hilus of the DG as shown by the CSD contour plot (isocurrent lines at 0.01 mV / mm²). No activity was recorded with a glass electrode positioned in the m-EC. (c) Type 2 potential recorded in the hippocampus; the simultaneous activity in the m-EC is also illustrated. Corresponding CSD contour plot shows a local generation in CA3 (isocurrent lines at 0.05 mV / mm²). (B) Type 2 potential recorded in the m-EC. On the left is shown the reconstruction of the position of the multichannel probe (100 μm between recording sites) on the histological section (calibration bar: 500 μm). Field potential profile of the type 2 potential recorded in the m-EC and the trace simultaneously recorded in the hippocampus showing the propagation of the potential (represented in the middle). On the right, the relative CSD contour plot (isocurrent lines at 0.02 mV / mm²) shows an early sink in layer II–III (500–700 μm depth) followed by an associative sink in the superficial layers (0–200 μm depth).

Fig. 8

Intracellular recordings performed in the m-EC during GABA_A-receptor-mediated spontaneous interictal potentials obtained with the simultaneous perfusion of DNQX, AP5 and 4AP. (A) Intracellular (upper traces) and extracellular activities recorded in the m-EC ipsilateral and contralateral to the intracellular recording site and in ipsilateral CA1 during a type 2 potential that initiated in the m-EC (a) and during a GABAergic event that is propagated from the m-EC contralateral to the intracellular recording site (b). The two events were recorded from the same m-EC neuron located in layers II–III. The cell was depolarized to highlight the IPSP that correlates to the GABAergic potential. (B) Superimposed intracellular and extracellular activities recorded in the m-EC ipsilateral and contralateral to the intracellular recording side and in the ipsilateral CA1 during a type 2 GABAergic potential that initiates in the m-EC. The intracellular correlates of two identical GABAergic potentials were recorded at different membrane polarizations during the injection of steady intracellular currents (resting membrane potential, −69 mV) to illustrate the IPSP reversal. Note that action potential firing interrupted the IPSP.