Participation of GABAA-Mediated Inhibition in Ictallike Discharges in the Rat Entorhinal Cortex

Valeri Lopantsev and Massimo Avoli

Research Group on Cell Biology of Excitable Tissue, Montreal Neurological Institute; and Department of Neurology and Neurosurgery and Department of Physiology, McGill University, Montreal, Quebec H3A 2B4, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Lopantsev, Valeri and Massimo Avoli. Participation of GABAA-mediated inhibition in ictallike discharges in the rat entorhinal cortex. J. Neurophysiol. 79: 352-360, 1998. The spontaneous, synchronous activity induced by 4-aminopyridine (4AP, 50 µM) in the adult rat entorhinal cortex was analyzed with simultaneous field potential and intracellular recordings in an in vitro slice preparation. Four-AP induced isolated negative-going field potentials (interval of occurrence = 27.6 ± 9.9 (SD) s; n = 27 slices) that corresponded to intracellular long-lasting depolarizations (LLDs), and ictallike epileptiform discharges (interval of occurrence = 10.4 ± 5.7 min; n = 27 slices) that were initiated by the negative field potentials. LLDs recorded with K-acetate-filled microelectrodes triggered few action potentials of variable amplitude and had a duration of 1.7 ± 0.8 s (n = 26 neurons), a peak amplitude of 11.8 ± 5.0 mV (n = 26 neurons) and a reversal potential of -66.2 ± 3.9 mV (n = 17 neurons). The ictal discharges studied with K-acetate microelectrodes consisted of prolonged depolarizations (duration = 72.9 ± 44.3 s; peak amplitude = 29.2 ± 11.4 mV; n = 25 neurons) with action-potential firing during both the tonic and the clonic phase. These depolarizations had a reversal potential of -45.3 ± 3.8 mV (n = 4 neurons). Intracellular Cl- diffusion from KCl-filled microelectrodes made both LLDs and ictal depolarizations increase in amplitude (30.5 ± 8.2 mV, n = 8 and 41.8 ± 9.8 mV, n = 6 neurons, respectively). LLDs recorded with KCl and 2-(trimethyl-amino)N-(2,6-dimethylphenyl)-acetamide (QX-314) microelectrodesreached an amplitude of 36.3 ± 5.2 mV, lasted 12.5 ± 6.5 s, and had a reversal potential of -31.3 ± 2.5 mV (n = 4 neurons); under these recording procedures the ictal discharge amplitude was 41.5 ± 5.0 mV and the reversal potential -24.0 ± 7.0 mV (n = 4 neurons). The N-methyl-D-aspartate (NMDA) receptor antagonist 3,3-(2-carboxy-piperazine-4-yl)-pro-pyl-l-phosphonate (10 µM, n = 5 neurons) alone or concomitant with the nonNMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (10 µM, n = 4 neurons) abolished ictal discharges, without influencing LLDs. LLDs were blocked by the gamma -aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI, 10 µM, n = 6 neurons) or the µ-opioid receptor agonist (D-Ala2-N-Me-Phe, Gly-ol) enkephalin (DAGO, 10 µM, n = 2 neurons). Application of BMI (n = 4 neurons) or DAGO (n = 2 neurons) to control the medium abolished LLDs and ictal discharges but disclosed a novel type of epileptiform depolarization that lasted 3.5 ± 1.2 s and occurred every 5.2 ± 2.6 s (n = 6 neurons). Our data indicate that 4AP induces in the rat entorhinal cortex a synchronous, GABA-mediated potential that is instrumental in initiating NMDA-dependent, ictal discharges. Moreover we present evidence for an active role played by GABAA-mediated potentials in the maintenance and termination of these prolonged epileptiform events.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Inhibitory potentials mediated through the postsynaptic activation of gamma -aminobutyric acid (GABA) receptor subtypes control neuronal excitability in the neocortex, hippocampus, and other forebrain structures (see for review Kaila 1994; Krnjevic 1991). In several models of epilepsy the efficacy of GABA-mediated inhibition (in particular that caused by activation of the type A receptor) decreases shortly before the onset of, as well as during seizure activity, even when experimental procedures that do not antagonize the GABAA receptor are used to induce epileptiform discharges (Avoli et al. 1995; Ben-Ari et al. 1979; Korn et al. 1987; McCarren and Alger 1985; Mody et al. 1987; Whittington et al. 1995b). However GABA-mediated inhibition is present and even potentiated in other models (McLean et al. 1996; Traub et al. 1994, 1996), including the epileptiform discharges induced in the hippocampus by low doses of the K+-channel blocker 4-aminopyridine (4AP) (Perreault and Avoli 1991, 1992; Rutecki et al. 1987).

Recent evidence obtained in the adult rat entorhinal cortex indicates that ictallike (thereafter referred to as ictal) discharges induced by 4AP are preceded, and thus may be initiated, by synchronous, GABA-mediated potentials that are associated with transient increases in [K+]o (Avoli et al. 1996a). The entorhinal cortex provides the main inputs to the hippocampus proper (Bartesaghi 1994; Charpak et al. 1995; Empson and Heinemann 1995; Lopes da Silva et al. 1990; Witter 1993) and it may play a key role in the development of limbic seizures (Avoli et al. 1996a; Heinemann et al. 1993; Nagao et al. 1996; Paré et al. 1992). A dysfunction of the entorhinal cortex was also documented in patients with temporal lobe epilepsy (Rutecki et al. 1989; Spencer and Spencer 1994), where the surgical removal of this structure may be essential for achieving seizure control (Goldring et al. 1992).

Knowledge of the mechanisms responsible for the occurrence of epileptiform activity in the entorhinal cortex is therefore important for understanding the pathogenesis of temporal lobe seizures. In this study we used conventional field potential and intracellular recordings to analyze the synchronous activity induced by 4AP in the adult rat entorhinal cortex. The epileptogenic action of 4AP results primarily from an increase in transmitter release at both excitatory and inhibitory synapses (Perreault and Avoli 1991; Rutecki et al. 1987); hence, epileptiform activity in this in vitro model is the result of the potentiation of both types of synaptic transmission, rather than to the blockade of inhibition or the enhancement of a specific excitatory mechanism, as in other models of epileptiform discharge. Our data demonstrate the occurrence of synchronous, GABAA-mediated depolarizations that have properties similar to those seen in the hippocampus (cf., Perreault and Avoli 1991, 1992) and participate to the initiation of ictal discharges. Moreover GABAA-mediated, Cl--dependent potentials do operate during the ictal discharge and may contribute to its maintenance and termination. A preliminary account of these findings was published (Lopantsev and Avoli 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Preparation and maintenance of the slices

Adult, male Sprague-Dawley rats (200-300 g) were used. The procedures for preparing and maintaining slices of combined hippocampus-entorhinal cortex were previously described (Avoli et al. 1996a; Nagao et al. 1996). In brief horizontal slices (450-500 µm thick) were cut with a vibratome and were transferred to a tissue chamber where they were kept at 33.5 ± 0.5°C (SD) in an interface between humidified gas (95% O2-5% CO2) and oxygenated artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 124 NaCl, 2 KCl, 2 MgSO4, 2 CaCl2, 1.25 KH2PO4, 26 NaHCO3, and 10 glucose (pH = 7.4). 4AP (50 µM, Sigma), 3,3-(2-carboxy-pi-pe-razine-4-yl)-propyl-l-phosphonate (CPP, 10 µM, Tocris Cookson), 6-cyano-7-nitro-quinoxa-line-2,3-dione (CNQX, 10 µM, Tocris Cookson), (D-Ala2-N-Me-Phe, Gly-ol) enkephalin (DAGO, 10 µM, Sigma), and bicuculline methiodide (BMI, 10 µM, Sigma) were bath applied. The rate of perfusion (0.3-1 ml/min) was kept constant in each experiment.

Recording procedures

Field potential recordings were made with glass pipettes filled with ACSF (resistance = 1-5 MOmega ). Microelectrodes for intracellular recordings were filled with one of the following solutions: 1) 3 M K-acetate (resistance = 60-100 MOmega ); 2) 3 M K-acetate and 50 mM 2-(trimethyl-amino)-N-(2,6-dimethylphenyl)-acetamide (QX-314, Astra; resistance = 60-100 MOmega ); 3) 3 M KCl (resistance = 40-70 MOmega ); or 4) 3 M KCl and 50 mM QX-314 (resistance = 50-70 MOmega ). The distance between extracellular and intracellular microelectrodes ranged from 100 to 300 µm. Signals were fed to high-impedance amplifiers with internal bridge circuit (Axoclamp 2) that allowed intracellular current injection. The bridge was monitored carefully throughout the experiment and adjusted as necessary. Signals were displayed on oscilloscope and on a Gould WindoGraf recorder; they were also recorded on video cassette recorder for later analysis.

Database and analysis

The intracellular activity of 62 neurons was analyzed in the presence of 4AP in the lateral portion of the entorhinal cortex of 50 slices. The neuron depth ranged from 220 to 1,320 µm from the pia, which corresponds to layers II-VI (Köhler 1988). Their fundamental electrophysiological characteristics when recorded with K-acetate-filled microelectrodes were the following: 1) resting membrane potential (RMP) measured after withdrawal from the cell -75.1 ± 7.0 mV (n = 41); 2) action-potential amplitude calculated from the baseline = 96.8 ± 10.5 mV (n = 39); and 3) apparent input resistance, obtained from the maximum voltage response induced by hyperpolarizing current pulses (100-200 ms, -0.4 to -0.5 nA) = 39.9 ± 13.8 MOmega (n = 33). These cells generated regular spiking activity (27/31 cells) or bursts of action potentials (4/31 cells) during injection of depolarizing current pulses (0.1-0.7 nA; 100-200 ms). Bursting cells were located 480-730 µm from the pia, which corresponds to layers III-IV (Köhler 1988).

The amplitude of the intracellular potentials was measured from the RMP, unless otherwise specified. Quantitative results are expressed as means ± SD and n indicates the number of neurons analyzed, unless otherwise specified. Statistical analysis of the data obtained under control conditions and during any experimental manipulation was performed with paired or unpaired Student's t-tests. Data were considered significantly different if P < 0.05.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Spontaneous synchronous activity induced by 4AP

The spontaneous field potential activity induced by 4AP consisted of isolated, negative potentials (interval of occurrence = 27.6 ± 9.9 s, n = 27 slices) (Fig. 1A, *) and ictal discharges (duration = 86.5 ± 52.5 s; interval of occurrence = 10.4 ± 5.7 min, n = 27 (Fig. 1A, continuousline). Each ictal discharge was usually preceded (and thus it appeared to be initiated) by a negative potential (Fig. 1A, open circle ). The different types of spontaneous synchronous activity continued to occur for several hours during continuous application of 4AP. In eight slices the spontaneous synchronous activity induced by 4AP consisted of negative potentials only.


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FIG. 1. 4-Aminopyridine (4AP)-induced synchronous activity recorded in entorhinal cortex. A: simultaneous field potential (top) and intracellular (bottom, -85 mV) recordings at a depth of 870 µm illustrate long-lasting depolarizations (LLDs) (*) and one ictal discharge (------). B: expanded traces of recordings shown in Aa: an LLD is associated with action potentials of variable amplitude. Ab: an LLD similar to that illustrated in Ba occurs at onset of ictal discharge; rhythmic depolarizations (down-triangle) with action-potential discharge are generated during initial tonic phase of ictal discharge. C: clonic discharges are illustrated. Note that these depolarizations do not reach membrane level characteristic of tonic depolarizations.

Intracellular recordings with K-acetate microelectrodes (43 cells from 35 slices) showed that the negative field potentials corresponded to long-lasting depolarizations (LLDs) that lasted 1.7 ± 0.8 s (n = 26) and attained maximal amplitude of 11.8 ± 5.0 mV (n = 26) (Fig. 1Ba, down-triangle). Action potentials of variable amplitude (4-96 mV) occurred during the LLD, but small-amplitude, action potentials were most often seen at its onset. Steady membrane depolarization obtained by injecting positive DC current made the LLD reverse in polarity at -66.2 ± 3.9 mV (n = 17, not illustrated).

The ictal discharge recorded intracellularly with K-acetate microelectrodes consisted of a prolonged depolarization (overall duration = 72.9 ± 44.3 s, n = 25) with an initial tonic phase that reached values of 29.2 ± 11.4 mV (n = 25) and a late clonic phase (Fig. 1A). Rhythmic depolarizations (frequency = 10-12/s, amplitude measured from the sustained ictal depolarization level = 7-40 mV, duration = 20-100 ms) capped by single action potentials occurred during the tonic phase (Fig. 1Bb, Up-arrow ). The amplitude of the tonic depolarization decreased over time and clonic discharges appeared (Fig. 1Bc). Each clonic depolarization increased gradually in duration and amplitude as the membrane potential returned toward RMP; however, their amplitude never reached that of the tonic depolarizations. Both tonic and clonic discharges corresponded to negative field potential transients. The ictal depolarization had largest amplitudes in cells recorded 500-900 µm from the pia. The ictal discharge onset was associated with an LLD that developed into the sustained, tonic depolarization (Fig. 1Bb). The peak amplitude of these LLDs (16.9 ± 8.6 mV, n = 20) was significantly larger than the value of isolated LLDs recorded in the same neurons between ictal discharges (11.6 ± 6.2 mV, n = 20; Fig. 1Bb, Up-arrow ), but was smallerthan the peak amplitude of the ictal depolarization(Fig. 1Bb).

Changing the membrane potential with steady intracellular injection of hyperpolarizing or depolarizing current modified the amplitude of both ictal depolarizations and preceding LLDs without influencing their rate of occurrence. As shown in Fig. 2A (-55 mV sample) the initial LLD became hyperpolarizing at depolarized membrane potentials and thus the ictal depolarization originated from a hyperpolarization. Both LLDs and ictal discharges increased in amplitude during steady membrane hyperpolarization (Fig. 2B; -80 mV sample). The reversal potential of the sustained ictal depolarization was -45.3 ± 3.8 mV (n = 4, not illustrated). The input membrane resistance tested with brief hyperpolarizing current pulses (-0.4 to -0.7 nA, 30-50 ms) decreased by 83.5 ± 4.4% (n = 13) during the LLDs recorded between ictal discharges (Fig. 2Ba) and by 84.4 ± 5.3% (n = 6) during those preceding the ictal discharge (Fig. 2Bb). The input membrane resistance also diminished during the ictal discharge (Fig. 2Bb).


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FIG. 2. Characteristics of LLDs occurring at onset of ictal discharge. A: when membrane is depolarized by intracellular injection of steady positive current (-55 mV) amplitude of sustained ictal depolarization decreases while initial LLD becomes hyperpolarizing as compared with recording obtained at resting membrane potential (RMP, -70 mV). When membrane is hyperpolarized by intracellular injection of steady negative current (-80 mV) both LLD and ictal depolarization increase in amplitude as compared with RMP samples. Time occupied by this initial LLD is indicated by continuous line on top of -55 mV trace. B: changes in input membrane resistance during an isolated LLD (a) and during LLD occurring at onset of ictal discharge (b); input resistance was monitored by passing short pulses of negative current (-0.4 nA; 40 ms).

Ionic mechanisms and pharmacological properties of the long-lasting depolarizations

LLDs recorded with KCl microelectrodes increased in amplitude over time and reached a steady value of 30.5 ± 8.2 mV (n = 8) within 5-10 min (Fig. 3A). This recording procedure did not cause any significant change in the RMP. The amplitude of these LLDs was therefore nearly three times higher compared with that observed with K-acetate microelectrodes, although the duration (1.6 ± 0.5 s, n = 7) was not significantly different. LLDs recorded with K-acetate/QX-314 microelectrodes (Connors and Prince 1982) were not coupled to fast, Na+-dependent action potentials (Fig. 3B, -73 mV sample). These LLDs had amplitude of 16.2 ± 4.0 mV, duration of 2.0 ± 0.9 s, and reversal potential of -61.0 ± 1.4 mV (n = 5).


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FIG. 3. Ionic mechanisms underlying LLDs. A: progressive increase in LLD amplitude during intracellular recording with a KCl-filled microelectrode; action potentials are truncated. B: intracellular recording with aK-acetate and 2-(tri-methyl-amino)-N-(2,6-dimethylphenyl)-acetamide (QX-314)-filled microelectrode shows absence of fast action potentials both at RMP (-73 mV) and during intracellular injection of positive current (-55 mV). C: during intracellular recording with a KCl/QX-314-filled microelectrode fast action potentials are abolished; moreover increase in LLD amplitude is accompanied by a slowing of LLD repolarizing phase during which fast events occur. In A and C, time after penetration is indicated in minutes.

Fast, Na+-dependent action potentials did not occur during LLDs recorded with KCl/QX-314 microelectrodes. These LLDs had a peak amplitude of 36.3 ± 5.2 mV, a duration of 12.5 ± 6.5 s, and a reversal potential of -31.3 ± 2.5 mV (n = 4) (Fig. 3C). Moreover they were characterized by a slow repolarizing phase that was associated with the occurrence of fast, mainly depolarizing events that increased over time during the recording. This phenomenon was associated with a gradual slowdown of the LLD repolarization (Fig. 3C, 4 vs. 18 min samples). Some electrophysiological features of the LLDs recorded with microelectrodes containing different solutions are summarized in Table 1.

 
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TABLE 1. Electrophysiological features of the LLDs recorded in entorhinal cortex neurons with microelectrodes containing different solutions

Application of the NMDA receptor antagonist CPP (10 µM, n = 5) did not modify the shape, the amplitude, and the rate of occurrence of LLDs recorded with K-acetate microelectrodes (not illustrated but cf. Avoli et al. 1996a; Lopantsev and Avoli 1996). Application of the non-NMDA receptor antagonist CNQX (10 µM) either alone (n = 2) or concomitantly with CPP (n = 3) induced a nonsignificant decrease of the peak amplitudes of the LLDs (from 17.3 ± 8.2 mV to 14.0 ± 6.8 mV, n = 4) (not illustrated but cf. Avoli et al. 1996a; Lopantsev and Avoli 1996). Further application of the GABAA receptor antagonist BMI (10 µM, n = 3) or the µ-opioid receptor agonist DAGO (10 µM, n = 2) abolished the LLDs (not illustrated, cf. Avoli et al. 1996a).

Ionic mechanisms and pharmacological properties of the ictal discharges

When recorded with KCl microelectrodes, the LLDs preceding the ictal discharges had a peak amplitude of 35.2 ± 4.4 mV (n = 6), which was similar to that seen during the tonic phase of the ictal depolarization (41.8 ± 9.8 mV, n = 6; Fig. 4Aa). The latter value was significantly larger than what was observed during epileptiform depolarizations recorded with K-acetate microelectrodes in cells located at comparable depths (i.e., 31.4 ± 4.2 mV, n = 9; depth range = 670-930 µm). The clonic depolarizations recorded with KCl microelectrodes had values similar to those of the tonic depolarization (Fig. 4B, - - -).


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FIG. 4. Intracellular diffusion of Cl- from KCl-filled microelectrode increases amplitude of LLD preceding ictal discharge. Note in B that clonic depolarizations have amplitudes similar to those of tonic depolarization(- - - ). A and B: top traces are field potential and bottom traces are intracellular recordings.

Ictal discharges recorded with K-acetate/QX-314 microelectrodes were accompanied by a 30.8 ± 8.8 mV (n = 5) sustained depolarization that was not capped by fast action potentials (Fig. 5, -72 mV sample). Under these recording procedures, the tonic ictal depolarization was associated with rhythmic depolarizing events (frequency = 10-12/s, amplitude measured from the sustained depolarization level = 10-18 mV, duration = 70-100 ms) (Fig. 5, -72 mV, a), whereas clonic depolarizations were capped by phasic events (amplitude measured from the level of the underlying clonic depolarizations = 20-44 mV, duration = 40-65 ms) (Fig. 5, -72 mV, b). Steady depolarization of the membrane revealed a reversal potential of -41 ± 3.4 mV (n = 3) for the sustained ictal event and made both tonic and clonic potentials invert in polarity (Fig. 5, -22 mV). These inverted potentials had a complex waveform consisting of an initial negative and a subsequent positive component (Fig. 5, -22 mV,  and black-down-triangle , respectively, in a and b); they increased in duration during transition from tonic to clonic phase, while the reversal potential of the initial component became more negative (Fig. 5, -22 mV, - - -). The reversal potential of tonic events was -30.4 ± 5.1 mV and that of clonic events -47.9 ± 4.4 mV (n = 5).


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FIG. 5. Fast action potentials do not occur during ictal discharge recorded with K-acetate/QX-314-filled microelectrode at RMP (-72 mV). Note that clonic depolarizations have an amplitude that is lower that tonic depolarizations [a and b (- - -) obtained at RMP = -72 mV]. Ictal discharge is inverted at -22 mV; at this membrane potential level fast events occurring during both tonic and clonic discharges are also inverted in polarity and characterized by an initial, more negative () and subsequently more positive (black-down-triangle ) component (a and b). Note also shift of reversal potential in negative direction of clonic discharges during progression of ictal activity (- - -). a and b: expanded traces of tonic and clonic events at each respective membrane potential. Simultaneous field potential recording is only illustrated for -72 mV sample.

Ictal discharges recorded at RMP with KCl/QX-314 microelectrodes (Fig. 6, -74 mV) were associated with a 41.5 ± 5.0 mV sustained depolarization (n = 4). LLDs occurring at the ictal discharge onset had amplitudes similar to those seen during the tonic phase of the ictal events and merged with them. Moreover the amplitude of the clonic depolarizations reached the level of the tonic depolarization (Fig. 6, -74 mV, - - -). Rhythmic, short-lasting depolarizations (frequency = 10-12/s, amplitude = 10-20 mV, duration = 100-150 ms) occurred during the tonic phase (Fig. 6,-74 mV, a). During steady membrane depolarization, the sustained ictal depolarizations inverted in polarity at -24.0 ± 7.0 mV, while tonic and clonic potentials developed a negative polarity (Fig. 6, -16 mV). These potentials had an initial negative and subsequently a more positive component (Fig. 6, -16 mV, a,  and down-triangle, respectively) and were characterized by similar reversal values (i.e., -15.3 ± 3.5 mV for the tonic and -16.5 ± 2.4 mV for the clonic events, n = 4). Some electrophysiological features of the ictal discharges recorded with microelectrodes containing different solutions are summarized in Table 2.


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FIG. 6. Fast action potentials do not occur at RMP (-74 mV) during an ictal discharge recorded with a KCl/QX-314 microelectrode. Note that amplitude of clonic depolarizations has values similar to those of tonic depolarizations (- - -). At a membrane potential of -16 mV both tonic (a) and clonic (b) discharges invert in polarity and contain an initial, negative () and subsequent, more positive (down-triangle) component. Note absence of reversal potential shift in negative direction during progression of ictal discharge (- - -). a and b: expanded traces of tonic and clonic events at each respective membrane potential. Simultaneous field potential recording is shown only for -74 mV sample.

 
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TABLE 2. Electrophysiological characteristics of the ictal epileptiform potentials recorded in entorhinal cortex neurons with microelectrodes containing different solutions

As reported previously (Avoli et al. 1996a), ictal discharges were abolished by CPP (10 µM, n = 5) and/or CNQX (10 µM, n = 4) (not shown). Application of the GABAA receptor antagonist BMI (10 µM) modified the pattern of epileptiform activity in six entorhinal neurons recorded with K-acetate microelectrodes. As illustrated in Fig. 7A, an ictal event occurred 2 min after BMI onset, but it was shorter than the discharge recorded under control conditions. Later during steady application of BMI, short (duration = 3.5 ± 1.2 s, n = 6) epileptiform discharges occurred regularly at intervals of 5.2 ± 2.6 s (n = 6) (Fig. 7B). In contrast to the ictal discharges induced by 4AP only, these relatively brief epileptiform events were characterized by an initial positive field potential (Fig. 7C). The intracellular counterpart of the epileptiform discharges induced by 4AP and BMI consisted of a slow depolarizing envelope (amplitude = 24.5 ± 6.7 mV, n = 6) with rhythmic depolarizations capped by action potential bursts (Fig. 7C). These epileptiform events decreased in amplitude during positive current injection (Fig. 7D). This procedure, however, did not disclose any initial hyperpolarization as seen at the onset of ictal discharges induced by 4AP only. DAGO (10 µM; n = 2) also blocked LLDs and ictal discharges, while disclosing epileptiform events similar to those induced by BMI (not illustrated, but see Avoli et al. 1996a).


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FIG. 7. Effect induced by gamma -aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI, 10 µM) on 4AP-induced ictal activity. A and B: BMI abolished ictal discharges and LLDs while disclosing rhythmic short-lasting epileptiform discharges. C: expanded trace from B illustrating BMI-induced epileptiform discharges that at RMP are not preceded by LLD and consist of a stereotyped discharge of fast spikes riding over a slow membrane depolarization. D: a hyperpolarizing potential does not occur at onset of BMI-induced epileptiform discharge during steady membrane depolarization (sample -53 mV).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

GABA-mediated long-lasting depolarizations

Our findings indicate that low concentrations of 4AP induce in the entorhinal cortex synchronous, GABA-mediated potentials with electrophysiological and pharmacological properties that resemble those described in the hippocampus (Barbarosie et al. 1994; Perreault and Avoli 1991, 1992). These similarities include 1) an increase in Cl--conductance as the main mechanism responsible for the LLD, 2) the insensitivity to ionotropic excitatory amino acid receptor antagonists, and 3) the suppression exerted by the GABAA-receptor antagonist BMI and the µ-opioid receptor agonist DAGO, which prevents GABA release from inhibitory terminals (Capogna et al. 1993). These findings are also in line with those obtained in the entorhinal cortex with field potential and [K+]o recordings (Avoli et al. 1996a) and further indicate that LLDs represent GABAA-mediated postsynaptic responses to GABA released by inhibitory interneurons. These GABAA-mediated depolarizations may be contributed by several mechanisms such as [K+]o elevations, changes in Cl- driving force and a conductance increase to HCO-3 (Alger and Nicoll 1982; Kaila 1994; Staley et al. 1995; Wong and Watkins 1982). As reported in hippocampal neurons (Perreault and Avoli 1991, 1992), the entorhinal cortex LLDs triggered only occasional, action-potential firing in spite of their large amplitude. This phenomenon results presumably from the membrane shunt because of the large conductance increase that accompanies the LLD.

NMDA-mediated postsynaptic responses were documented in the entorhinal cortex (Jones 1987, 1994; Jones and Heinemann 1988). However LLDs are not influenced by the NMDA receptor antagonist CPP, which may be because of the masking effect exerted on NMDA potentials by GABAA receptor activation during the LLD. LLDs also occur during blockade of nonNMDA receptors, although this pharmacological procedure could cause a nonsignificant decrease of LLD amplitude. Recently we have shown participation of nonNMDA-dependent circuits in the activation of inhibitory interneurons in the entorhinal cortex (Lopantsev and Avoli 1996). The resistance of 4AP-induced, GABA-mediated potentials to excitatory amino acid receptor antagonists was described in several cortical structures (Michelson and Wong 1991; Muller and Misgeld 1990; Perreault and Avoli 1991, 1992).

Ictal discharges

4AP-induced ictal activity in the entorhinal cortex results from the powerful and synchronous activation of excitatory synaptic mechanisms as proposed in other models of interictal and ictal discharge (Gutnick et al. 1982; Johnston and Brown 1981; Nagao et al. 1996). This conclusion is based on the effects of ionotropic excitatory amino acid receptor antagonists, which blocked the ictal activity (cf. Avoli et al. 1996a) and the changes induced by intracellular injection of steady current, a procedure that modified the ictal depolarization amplitude as expected for a potential that is caused by synaptic currents. As documented in other models of entorhinal epileptiform discharge (Heinemann et al. 1993; Jones and Heinemann 1988; Nagao et al. 1996), our findings indicate that NMDA-mediated mechanisms play a primary role in the occurrence of the ictal depolarizations induced by 4AP. Moreover these prolonged epileptiform events have maximal amplitude in entorhinal cortex neurons located 500-900 µm from the pia. This evidence, along with the results obtained by using field-potential and [K+]o recordings (Avoli et al. 1996a), implies that middle and deep layer cells play a fundamental role in the generation of these ictal discharges. Inhibition may be weaker in these layers, thus allowing a greater expression of NMDA-dependent currents (Jones 1994, 1987; Jones and Heinemann 1988). Moreover, intrinsically bursting neurons were found at the depth of maximal ictal depolarization; bursting cells contribute to the generation of epileptiform discharges (Chagnac-Amitai and Connors 1989; Connors 1984; Miles and Wong 1983). However propagation of ictal activity to the hippocampus proper must involve superficial layer neurons, as these cells give rise to the perforant path projection (Charpak et al. 1995; Empson and Heinemann 1995; Heinemann et al. 1993; Jones 1994). Laminar differences in 4AP-induced epileptiform activity were reported in the neocortex (Barkai et al. 1995).

The reversal potential of the ictal depolarization recorded with K-acetate/QX-314 microelectrodes has values more negative than what was expected for an isolated EPSP or an epileptiform depolarization induced by GABAA-receptor antagonists (Gutnick et al. 1982; Johnston and Brown 1981). This evidence suggests that GABAA-mediated, Cl- conductances are present during the ictal activity induced by 4AP in the entorhinal cortex. Accordingly the ictal depolarization recorded with KCl or KCl/QX314 microelectrodes increased in amplitude and its reversal potential became more positive; both recording procedures lead to intracellular Cl- leakage, which makes the reversal potential of GABAA-mediated potentials more positive. Under these recording conditions clonic depolarizations increased in amplitude up to values comparable with those associated with the tonic depolarization. Similar findings were reported to occur during intracellular Cl- injection in neonatal hippocampal neurons (McLean et al. 1996).

When K-acetate/QX-314 microelectrodes were used the reversal potential of the tonic and clonic depolarizations associated with the ictal event became more negative as the discharge progressed from the tonic to the clonic phase; such a shift was not observed with KCl/QX-314-filled microelectrodes. Although these reversal potential values were eventually influenced by the [K+]o elevation associated with the ictal activity (Avoli et al. 1996a,b; McCarren and Alger 1985), these data indicate that a progressive enhancement of Cl--dependent inhibition occurs during the progression of the ictal discharge and may contribute to its termination.

The GABAA receptor antagonist BMI abolished both LLDs and ictal discharges, while disclosing a novel type of epileptiform activity that resembled interictal discharges induced by BMI in neocortex (Gutnick et al. 1982) and hippocampus (Schwartzkroin and Prince 1980). DAGO, which prevents GABA release from inhibitory interneurons through the activation of presynaptic µ-opioid receptors (Capogna et al. 1993) had similar effects. Hence, in the entorhinal cortex Cl--dependent GABAA-mediated inhibition participates in the maintenance of the 4AP-induced ictal activity and to contribute to its termination (in addition to playing a significant role in the initiation process, see below).

Higashima et al. (1996) have shown that activation of GABAergic mechanisms is necessary for the generation of afterdischarges recorded in hippocampal slices after electrical stimuli. Experimental and computational data obtained by Traub et al. (1996) also suggest a role played by GABAA-mediated depolarizing conductances in the epileptiform synchronization that occurs in some models of epileptiform discharge (in particular that induced by 4AP application). GABAergic inhibitory networks can also synchronize principal cells in the neocortex and hippocampus (Cobb et al. 1995; Whittington et al. 1995a).

Role of the LLDs in initiation of ictal discharge

The synchronous, GABAA-mediated potential induced by 4AP represents a mechanism capable of initiating prolonged epileptiform discharges in the entorhinal cortex (cf. Avoli et al. 1996a). This conclusion rests on the finding that the ictal events were usually preceded and thus they appeared to be initiated by LLDs, as well as by the ability of pharmacological procedures that interfere with the occurrence of LLDs (i.e., application of BMI or DAGO) to abolish the ictal discharge. Moreover the amplitude of the LLD occurring at the onset of the ictal discharge had values that were larger that those seen with isolated LLDs. In the neonatal rat hippocampus a GABAB receptor antagonist potentiates giant GABAergic potentials leading to the occurrence of ictallike epileptiform discharges (McLean et al. 1996).

By employing [K+]o recordings we have shown that the larger amplitude of the LLD recorded in the entorhinal cortex at the onset of the ictal discharge corresponds to a transient [K+]o increase that is greater than that seen in coincidence with the isolated LLDs (Avoli et al. 1996a). A similar mechanism occurs in isolated hippocampal slices obtained from young rats at the outset of ictal discharges induced by 4AP (Avoli et al. 1993, 1996b). It is well known that the appearance of epileptiform activity is facilitated by high K+ (Korn et al. 1987; Traynelis and Dingledine 1988). The increases in [K+]o during the GABA-mediated potential can induce a positive shift of GABA-mediated postsynaptic inhibition (Korn et al. 1987; McCarren and Alger 1985), depolarize neurons, and thus disinhibit excitatory postsynaptic interactions. All these processes can lead to the appearance of ictal epileptiform activity induced by 4AP in the entorhinal cortex. Therefore GABAA-mediated LLDs may serve as a powerful implement for the initiation and the synchronization of neuronal activity in the entorhinal cortex. This conclusion is in line with recent findings obtained in epileptic human temporal lobes, where inhibitory neuronal interactions are increased in regions of seizure initiation (Colder et al. 1996).

    ACKNOWLEDGEMENTS

  We thank Siobhán McCann for secretarial assistance.

  This study was supported by Medical Research Council of Canada Grant MT-8109, the Savoy Foundation, Hospital for Sick Children Foundation Grant XG-93-056, and the Quebec Heart and Stroke Foundation.

    FOOTNOTES

  Address for reprint requests: M. Avoli, 3801 University, Montreal, Quebec H3A 2B4, Canada.

  Received 16 December 1996; accepted in final form 9 September 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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