Independent epileptiform discharge patterns in the olfactory and limbic areas of the in vitro isolated Guinea pig brain during 4-aminopyridine treatment

Abstract

In vitro studies performed on brain slices demonstrate that the potassium channel blocker 4-aminopyridine (4AP, 50 microM) discloses electrographic seizure activity and interictal discharges. These epileptiform patterns have been further analyzed here in a isolated whole guinea pig brain in vitro by using field potential recordings in olfactory and limbic structures. In 8 of 13 experiments runs of fast oscillatory activity (fast runs, FRs) in the piriform cortex (PC) propagated to the lateral entorhinal cortex (EC), hippocampus and occasionally to the medial EC. Early and late FRs were asynchronous in the hemispheres showed different duration [1.78 +/- 0.51 and 27.95 +/- 4.55 (SD) s, respectively], frequency of occurrence (1.82 +/- 0.49 and 34.16 +/- 6.03 s) and frequency content (20-40 vs. 40-60 Hz). Preictal spikes independent from the FRs appeared in the hippocampus/EC and developed into ictal-like discharges that did not propagate to the PC. Ictal-like activity consisted of fast activity with onset either in the hippocampus (n = 6) or in the mEC (n = 2), followed by irregular spiking and sequences of diffusely synchronous bursts. Perfusion of the N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid (100 microM) did not prevent FRs, increased the duration of limbic ictal-like discharges and favored their propagation to olfactory structures. The AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (50 microM) blocked ictal-like events and reduced FRs. In conclusion, 4AP-induced epileptiform activities are asynchronous and independent in olfactory and hippocampal-entorhinal regions. Epileptiform discharges in the isolated guinea pig brain show different pharmacological properties compared with rodent in vitro slices.

FIG. 1

A: scheme of the isolated guinea pig brain maintained in vitro with the position of the stimulating electrode on the lateral olfactory tract (LOT) and the recording electrodes positioned in piriform cortex (PC), lateral entorhinal cortex (l-EC), CA1 area of the hippocampus, medial entorhinal cortex (m-EC), and contralateral medial entorhinal cortex (m-EC contr). LOT stimulation was performed to test the viability of the brain and the evoked responses in the different structures are shown (right). B: arterial perfusion with 4-aminopyridine (4AP, 50 μM) induces a highly reproducible pattern. Spontaneous activity begins with early fast runs (FRs) originating in the PC (PC trace, *), that are followed by late FR (PC trace, **). Note that differently from the early FRs, late FRs propagate to both l-EC and hippocampus CA1. Later on interictal spikes appear in m-EC (m-EC trace, ●) and propagate bilaterally before the seizure onset. Note also that the seizure propagates to the contralateral hemisphere but spares the PC that continues to exhibit FR activity (***). The period of application of 4AP is indicated by the line above the electrophysiological traces.

FIG. 2

A: early (*) and late (**) FRs induced by 4AP recorded in PC, l-EC, CA1, and m-EC. In both cases, the fast activity propagates from the PC to the m-EC. B: expansions of early FRs and late FRs in all the recorded structures and relative joint time-frequency analysis. Note that early FRs are mainly characterized by frequency around 20 Hz, whereas in the late FRs, peak frequency was higher than 30 Hz. With the joint time-frequency analysis it is possible to follow the spread of FR from PC to m-EC. C: average of 6 early and late FRs recorded in PC.

FIG. 3

Indipendent FRs recorded in PC and contralateral PC.

FIG. 4

Correlation analysis of the early FRs. A: field potential activity recorded in the PC before the onset of FRs (control) and during early FRs induced by 4AP. B: representation of the correlation coefficient (r²) of PC vs. l-EC, PC vs. CA1, l-EC vs. CA1, and CA1 vs. m-EC were evaluated during a period of 30 s in control condition and during the early FRs. Note the increase of r² with the appearance of FRs. C: histogram representation of r² during control (black), early-FRs (dark gray), late-FRs before seizure (gray), and FRs after seizure (white). Note that r² increases in all the possible pairs of structures from the control phase to the l-FRs recorded before seizure onset in 4 different experiments.

FIG. 5

A: late FRs were followed by interictal spikes (◀). We compared these spike (a, ◀) with the preictal spikes (b, ●). B: expansions of the 2 sections shown in A. Note that the interictal spikes that follow the late FRs originate in PC and propagated to l-EC, CA1, and m-EC. In contrast, the preictal spikes originate in m-EC and propagated to CA1 and l-EC, without invading the PC. C: further expansion of spikes shown in B.

FIG. 6

4AP-induced epileptiform activity in the in vitro isolated guinea pig brain. A: the entire sequence of epileptiform activity recorded simultaneously in olfactory and limbic structures is shown. B: expansion of sections as indicated in A. Note in a that preictal spikes are recorded in the contralateral hemisphere (CA1-c and m-EC-c). Some of these interictal events (●) resemble the GABA-mediated potentials reported in a previous work during co-perfusion of 4AP and glutamate receptor antagonists. Note that after the preictal GABA-mediated spikes (●) and complex spikes (● ●) fast onset activity is recorded but not in the olfactory areas. Note also in b that irregular spiking appears later on, whereas in c, hypersynchronous burst activity originates in the hippocampus with components that propagate to the EC. Note also in c that FRs appear in the PC during the burst activity (***).

FIG. 7

A: field activity occurring during arterial perfusion with 4AP. B: co-perfusion of 4AP and 2-amino-5-phosphonopentanoic acid (AP5) induces a prolongation of the seizure activity that spreads to PC. C: co-perfusion of 4AP and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocks seizure-like activity while interictal spikes (●) and low-amplitude FRs continue to occur. In A–C, examples of interictal spikes and FRs are expanded in a and b. **, late FRs; ***, FRs during and after seizure; ●, interictal spikes).

FIG. 8

A: seizures onset and duration recorded in CA1. Co-perfusion of 4AP and AP5 100 μM induced an increase statistically significant both in time onset and duration of ictal events, whereas 4AP+CNQX 50 μM completely abolished seizure occurrence. B: duration and amplitude of the slow wave of the interictal spikes recorded in CA1. Co-perfusion of 4AP and AP5 and CNQX induced, respectively, a decrease and an increase statistically significant of the duration of the preictal spike slow wave compared with 4AP alone. The amplitude of the peak of the interictal slow wave was increased during co-perfusion with 4AP+AP5 and 4AP+CNQX compared with 4AP alone (P < 0.05). C: duration and amplitude of late FRs recorded in PC. Co-perfusion of 4AP and AP5 induced, respectively, an increase and a decrease statistically significant of both duration and amplitude of FRs. Otherwise 4AP+CNQX induced a decrease in the duration of FRs and a statistically significant decreased in amplitude of FRs.