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Vol. 303, Issue 3, 1102-1113, December 2002


Epileptiform Synchronization and GABAB Receptor Antagonism in the Juvenile Rat Hippocampus

Rita Motalli, Margherita D'Antuono, Jacques Louvel, Irene Kurcewicz, Giovanna D'Arcangelo, Virginia Tancredi, Mario Manfredi, René Pumain and Massimo Avoli

Montreal Neurological Institute and Departments of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada (R.M., M.A.); Istituto di Ricovero e Cura a Carattere Scientifico Neuromed, Pozzilli (Isernia), Italy (M.D., M.M., M.A.); Centre Paul Broca and Institut National de la Santé et de la Recherche Médicale U109, Paris, France (J.L., I.K., R.P.); and Dipartimento di Neuroscienze, Università degli Studi di Roma "Tor Vergata", Roma, Italy (G.D., V.T.)

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The GABAB receptor agonist baclofen enhances the epileptiform activity induced by 4-aminopyridine (4AP) in juvenile rat hippocampal slices. In this study, we used a similar experimental approach (i.e., field potential, intracellular, and [K+]o recordings in the CA3 area of slices obtained from 15-23-day-old rats) to establish whether antagonizing GABAB receptors could exert opposite (presumably anticonvulsant) effects. Bath application of 4AP (50 µM) induced spontaneous interictal and ictal discharges along with synchronous GABA receptor-mediated potentials. All types of 4AP-induced synchronous activity occurred more frequently during application of the GABAB receptor antagonist p3-amino-propyl,p-diethoxymethylphosphonic acid (CGP 35348) (0.1-1 mM; EC50 = 0.25 mM). Moreover, CGP 35348 augmented the frequency and, to a lesser extent, the duration of the asynchronous synaptic activity recorded intracellularly from CA3 pyramids (n = 19). In medium containing 4AP + ionotropic glutamatergic antagonists (which abolished interictal and ictal activity), CGP 35348 prolonged both GABA-receptor-mediated field potentials and the accompanying intracellular long-lasting depolarizations without modifying their rate (n = 12). The transient elevations in [K+]o associated with GABA receptor-mediated potentials in 4AP-containing medium (n = 7 slices) became larger during CGP 35348 application. Similar findings were obtained when CGP 35348 was applied to medium containing 4AP + ionotropic glutamatergic antagonists (n = 6). Thus, the effect of CGP 35348 on 4AP-induced epileptiform activity is not anticonvulsant and to some extent similar to what was reported in this model during GABAB receptor activation. We propose that the facilitation of ictal activity by CGP 35348 is mainly caused by the blockade of presynaptic GABAB receptor, leading to an increase in GABA release and subsequent larger [K+]o elevations.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Seizure disorders are often associated with a decreased efficacy of GABA receptor-mediated inhibition that is mainly mediated by two receptor subtypes (termed A and B) located pre- and postsynaptically on both interneurons and principal cells (Avoli, 2000; Olsen and Avoli, 1997). The contribution of GABAB receptor-mediated mechanisms to seizure generation remains unclear. For instance, GABAB receptor antagonists can prolong epileptiform discharges in cortical networks (McCormick, 1989; Scanziani et al., 1991, 1994; Sutor and Luhmann, 1998), but in other studies, GABAB receptor activation exerts proconvulsant effects. This has been documented both in clinical practice (Rush and Gibberd, 1990; Kofler et al., 1994) and in experimental models (Lewis et al., 1989; Mott et al., 1989; Watts and Jefferys, 1993; Motalli et al., 1999).

Recently, we have reported that baclofen promotes seizure-like discharges in hippocampal slices obtained from juvenile rats during application of low doses of the convulsant drug 4-aminopyridine (4AP) (but see also Watts and Jefferys, 1993; Motalli et al., 1999). In this in vitro model, interictal and ictal discharges occur along with synchronous GABA receptor-mediated potentials (Avoli et al., 1996b). Notably, the proconvulsant action of baclofen resulted from activity-dependent changes in hippocampal network excitability along with weakening of asynchronous GABA receptor-mediated potentials leading to disinhibition (Motalli et al., 1999). This evidence suggested that in this in vitro model of epileptiform discharge the antagonism of GABAB receptors might exert an anticonvulsant action.

In the present study, we have tested the validity of this hypothesis by using the GABAB receptor antagonist CGP 35348 (Olpe et al., 1990). To this end, we used field potential, intracellular, and [K+]o recordings in isolated hippocampal slices obtained from 15- to 23-day-old rats to investigate whether and how CGP 35348 influences the epileptiform activities induced by 4AP. Herein, we report that CGP 35348 increases the rate of occurrence of all types of spontaneous activity recorded in the CA3 subfield and induces ictal discharges whenever these events were not seen under control conditions (i.e., during application of 4AP-containing medium). Moreover, our findings suggest that the increased occurrence of ictal discharges during CGP 35348 application may be caused by blockade of presynaptic GABAB receptors, thus leading to a greater release of GABA from inhibitory terminals and consequent increase of the transient [K+]o elevations seen during interneuron synchronization.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Sprague-Dawley rats (15-23 days old) were anesthetized with halothane and decapitated. The brain was quickly removed and placed in cold, oxygenated artificial cerebrospinal fluid (ACSF) composed of 124 mM NaCl, 2 mM KCl, 1.25 mM KH2PO4, 2 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose. Isolated 500-µm-thick hippocampal slices were cut with a vibratome and transferred to a tissue chamber where they lay at an interface between oxygenated ACSF and humidified atmosphere (95% O2/5% CO2) at 33-35°C (pH = 7.4). 4AP (50 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM), 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP; 10 µM), and CGP 35348 (0.1-1 mM) were bath applied. All chemicals were acquired from Sigma-Aldrich (St. Louis, MO) with the exception of CNQX and CPP (obtained from Tocris Cookson, Inc., Ballwin, MO) and CGP 35348 (a generous gift from Novartis Ltd, Basel, Switzerland).

Field potential recordings were made in the CA3 stratum radiatum with ACSF-filled electrodes or through the reference channel of the ion-selective electrode. Measurements of free [K+]o were obtained from the same region with a double-barreled ion-selective microelectrode (tip diameter = 2-6 µm) filled with the valinomycin-based ion exchanger (product number 60398, Fluka, Buchs, Switzerland) (Heinemann et al., 1977; Motalli et al., 1999). The reference channel was back-filled with 150 mM NaCl and the ion selective channel with 100 mM KCl. In calibration solutions containing 124 mM NaCl, the electrodes considered suitable for recording showed a potential change of approximately 57 mV for a 10-fold increase in [K+].

Sharp-electrode intracellular recordings were performed from CA3 pyramids with pipettes that were filled with one of the following solutions: 1) 3 M potassium acetate (tip resistance = 70-120 MOmega ), 2) 3 M KCl (tip resistance = 60-90 MOmega ), or 3) 3 M KCl + 50 mM 2-(trimethyl-amino)-N-(2-6-dimethyl-phenyl)-acetamide (QX-314; a kind gift from Astra, Toronto, ON) (tip resistance = 60-100 MOmega ). Signals were fed to a high-impedance amplifier with an internal bridge circuit for passing intracellular current (Axoclamp2A; Axon Instruments, Inc., Foster City, CA). The bridge balance was routinely checked. Signals were displayed on an oscilloscope and/or on a Gould recorder and recorded on tape for later analysis.

Our study is based on the use of over 70 slices that were analyzed with field potential, intracellular, and/or [K+]o recordings. The electrophysiological properties of CA3 pyramids recorded with either potassium acetate- or KCl-filled electrodes were similar to those reported in previous studies (Motalli et al., 1999), including the ability to fire regular spiking with adaptation in response to intracellular injection of depolarizing current pulses. Some basic electrophysiological characteristics observed in a representative group of cells (n = 18) that were analyzed in 4AP-containing ACSF with potassium acetate-filled electrodes were as follows: 1) RMP obtained after electrode withdrawal = -65.3 ± 4.2 mV (mean ± S.E.M.), 2) action potential amplitude calculated from the baseline = 92.3 ± 9.4 mV; and 3) apparent input resistance measured from the maximum voltage response induced by small (<0.5 nA) hyperpolarizing current pulses = 24.7 ± 6.8 MOmega . Measurements throughout the text are expressed as mean ± S.D., and n indicates the number of slices studied under each specific protocol. Dose-response curves were fitted with the four-parameter logistic equation using the Prism software package (GraphPad Software, Inc., San Diego, CA). Statistical comparisons were made with either the paired or the unpaired Student's t test. Differences were considered significant if p < 0.05.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

CGP 35348 effects on the Synchronous Field potential Activity Induced by 4-Aminopyridine. In line with previous studies (Avoli et al., 1996b; Motalli et al., 1999), field potential recordings made in the CA3 stratum radiatum during application of medium containing 4AP (50 µM) revealed three types of spontaneous synchronous activity. These events consisted of: 1) brief (duration = 0.25-0.40 s) positive-going potentials that occurred at 0.18 to 0.59 Hz and resembled interictal discharges (arrows in Fig. 1A; control); 2) prolonged series (duration = 7-18 s) of positive-going events that were reminiscent of electrographic ictal discharges and occurred at 0.002 to 0.008 Hz (continuous line in Fig. 1A; control); and 3) negative-going field potentials occurring at 0.0065 to 0.015 Hz (asterisks in Fig. 1A; control).


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  		Fig. 1.   Effects induced by CGP 35348 on 4AP-induced synchronous activity analyzed with field potential recordings obtained from the CA3 stratum radiatum. A, typical electrographic pattern consisting of brief interictal (arrows) and ictal (continuous lines) discharges, the latter being initiated by negative-going GABA receptor-mediated events (asterisks). Note that application of CGP 35348 (1 mM) increases the rate of occurrence of all activities. B-D, quantitative summary of the effects induced by CGP 35348 (1 mM) on the rate of occurrence of the three types of 4AP-induced synchronous activity analyzed in 12 slices. In this and the following figures, results are expressed as mean ± S.D.; single and double asterisks indicate significant differences with p < 0.05 and 0.02, respectively. E, experiment in which only interictal activity and GABA receptor-mediated potentials were recorded under control conditions (i.e., medium containing 4AP). In this case, CGP 35348 (1 mM) induces the appearance of ictal discharges that are preceded by a GABA receptor-mediated, negative-going potential.

We have demonstrated (Avoli et al., 1996b; Motalli et al., 1999) that: 1) 4AP-induced interictal and ictal discharges are abolished by the non-N-methyl-D-aspartate receptor antagonist CNQX, whereas 2) the negative-going potentials are blocked by GABAA receptor antagonists or following activation of µ-opioid receptor, a procedure that leads to inhibition of GABA released from interneuron terminals (Capogna et al., 1993). Hence, these events represent synchronous GABA receptor-mediated potentials caused by interneurons firing leading to GABA release and to consequent activation of postsynaptic GABA receptors (Perreault and Avoli, 1992). Interictal and GABA receptor-mediated potentials were seen in all hippocampal slices analyzed during 4AP application (n = 46), whereas ictal discharges occurred in 32 of these experiments. As illustrated in Fig. 1A (but also see Figs. 2 and 7), each ictal discharge was always preceded, and thus, it appeared to be initiated by a GABA receptor-mediated potential (Avoli et al., 1996b; Motalli et al., 1999).


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  		Fig. 2.   Effects induced by increasing concentrations of CGP 35348 on the synchronous field potential activity elicited by 4AP in the CA3 subfield. A, samples obtained under control conditions by recording the field potential in the stratum radiatum during application of three concentrations of CGP 35348, as indicated on the left of each trace, and after washout of the drug. Note the shortening of the interval between ictal discharges. B, dose-response curve of the decrease in ictal discharge interval induced by different concentrations of CGP 35348 in four experiments. This dose-response curve reveals an EC50 of 0.25 mM.

Application of CGP 35348 (0.5 or 1 mM in 21 and 7 experiments, respectively) to hippocampal slices generating interictal and ictal discharges along with GABA receptor-mediated potentials under control conditions (i.e., 4AP-containing medium) increased the rate of occurrence of all activities (Fig. 1A). The effects induced by these two concentrations of CGP 35348 were not different and therefore the results were pooled together (Fig. 1, B-D). In addition, there was a small increase in the amplitude of the GABA receptor-mediated potentials and a decrease in ictal discharge duration during CGP 35348 application. These changes, however, were not significant in either case. As illustrated in the experiment illustrated in Fig. 1E, CGP 35348 (0.5 or 1 mM in 11 and 4 slices, respectively) also uncovered ictal discharges when only interictal activity and GABA receptor-mediated potentials were recorded under control conditions. Furthermore, in these experiments, the ictal discharges disclosed by the application of CGP 35348 were preceded by synchronous GABA receptor-mediated potentials.

We also analyzed the dose-response of the changes induced by CGP 35348 on the rate of occurrence of 4AP-induced ictal discharges (Fig. 2A). Data obtained in four slices in which increasing doses (0.1-1 mM) of CGP 35348 were sequentially applied indicated an EC50 = 0.25 mM (Fig. 2B). As illustrated in Fig. 2A, the effects induced by CGP 35348 were fully reversible upon wash-out of CGP 35348 with control medium.

Intracellular Recordings under Control Conditions and during CGP 35348 Application. In line with what has been reported in previous studies (Motalli et al., 1999), the field potential ictal discharges, when recorded intracellularly with potassium acetate-filled electrodes from CA3 pyramids, corresponded to prolonged depolarizations (duration = 7.3-21.5 s) that were capped by sustained action potential firing (n = 11 cells) (Fig. 3A; control). By contrast, the interictal events, which occurred in all experiments (n = 18 cells), were associated with brief intracellular depolarizations triggering action potential bursts (duration = 1.2-2.0 s; Fig. 3, A and B, arrows). As shown in Fig. 3B (asterisk), GABA receptor-mediated potentials were mirrored by long-lasting depolarizations (LLDs; duration = 0.9-3.7 s) that triggered a minimal amount of action potential firing, even though their amplitude often exceeded that seen during the interictal discharge depolarization.


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  		Fig. 3.   Effects induced by CGP 35348 on the 4AP-induced activity recorded with field potential and intracellular recordings from CA3 pyramidal cells; intracellular electrodes in the experiments shown in A and B contained a potassium acetate solution. A, in this hippocampal slice, both ictal discharges (corresponding to prolonged depolarizations of CA3 pyramids with sustained action potential firing), and interictal events (associated with brief action potential bursts, arrows) occurred. Application of CGP 35348 (1 mM) increased the rate of occurrence of the interictal events and reduced the duration of the ictal depolarization that continued to be initiated by a long-lasting depolarization (curved arrows). B, in a different experiment in which only interictal discharges (arrows) and GABA receptor-mediated potentials associated with LLDs (asterisk) were recorded in control, CGP 35348 (1 mM) application made ictal discharges appear.

Spontaneous asynchronous postsynaptic potentials (PSPs) were also recorded from CA3 pyramids between the different types of synchronous activity (Perreault and Avoli, 1992; Motalli et al., 1999). Recordings with potassium acetate-filled electrodes revealed that at RMP, these PSPs consisted of hyperpolarizing and depolarizing events (Fig. 4A; control; open and filled circle, respectively). Membrane hyperpolarization at values more negative than RMP did not influence the rate of occurrence of these PSPs but made them become all depolarizing while increasing their amplitude (not illustrated).


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  		Fig. 4.   Effects induced by CGP 35348 on the interictal discharges and the asynchronous PSPs recorded with potassium acetate-filled electrodes from CA3 pyramidal cells. A, bath application of CGP 35348 (1 mM) increased the rate of occurrence and decreased the duration of the 4AP-induced interictal discharges; in addition, this GABAB receptor antagonist increased the rate of occurrence of both hyperpolarizing and depolarizing PSPs (identified in the control panel with open and filled circles, respectively). B and C, cumulative plots of the effects induced by CGP 35348 (1 mM) on the rate of occurrence and the amplitude of hyperpolarizing and depolarizing PSPs recorded from five CA3 pyramidal cells in the presence of 4AP. D, superimposed intracellular traces of depolarizing and hyperpolarizing PSPs recorded in the same experiment shown in A under control conditions and during application of CGP 35348; traces were aligned in both cases by using the initial component of the depolarizing or hyperpolarizing deflection. Note that both types of PSPs increase in duration during application of CGP 35348.

We confirmed in these intracellular studies that CGP 35348 (1 mM) application to slices generating both interictal and ictal discharges under control conditions caused an increase in the occurrence of all spontaneous activities (Fig. 3A; +CGP 35348; n = 7), whereas it produced ictal discharges in those experiments in which only interictal discharges and GABA receptor-mediated potential occurred in medium containing 4AP (Fig. 3B; +CGP 35348; n = 5). CGP 35348 also increased the rate of occurrence and caused a small, not significant, decrease in the amplitude of the asynchronous PSPs (Fig. 4A). These effects are quantified in the histograms of Fig. 4, B and C. Moreover, this GABAB receptor antagonist caused a 30 to 47% increase of the duration of both depolarizing and hyperpolarizing asynchronous PSPs (Fig. 4D). No significant change in either RMP or input resistance was seen in CA3 pyramids treated with CGP 35348 (n = 10; not illustrated).

Effects of CGP 35348 on the Intracellular Activity Recorded in Medium Containing 4AP and Glutamatergic Antagonists. Next, we established the effects induced by CGP 35348 (1 mM) on the LLDs and on the asynchronous GABA receptor-mediated PSPs recorded intracellularly from CA3 pyramids (n = 5) with KCl-filled electrodes during application of the glutamatergic receptor antagonists CNQX and CPP. Under these conditions, interictal and ictal epileptiform discharges were abolished while negative-going field potentials associated with intracellular LLDs and synchronous, presumptive GABAA receptor-mediated, depolarizing PSPs continued to occur (Fig. 5A, control). In two of these five neurons, we could also record presumptive GABAB receptor-mediated hyperpolarizing PSPs that were not associated with any field potential activity (Fig. 5C, filed circles).


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  		Fig. 5.   Effects induced by CGP 35348 (1 mM) on the activity generated in the CA3 area during application of 4AP and excitatory amino acid receptor antagonists. Field potential and intracellular (KCl-filled electrode) recordings were obtained from three different slices. A and B, GABA receptor-mediated potentials and simultaneously recorded LLDs are generated under control conditions either spontaneously (A) or following electrical stimuli delivered in the dentate hilus (B); triangles in B indicate the time at which stimulation occurred. Note that in both experiments the LLD is followed by a long-lasting hyperpolarization (indicated by filled circles). Application of CGP 35348 markedly reduces the long-lasting hyperpolarization and causes a prolongation of the LLD. This effect is mirrored in the field potential recording by an increase in duration of the negative-going event. Note also that in the experiment shown in A, the LLD recorded in the presence of CGP 35348 is associated with an increased amount of action potential firing. C, CGP 35348 abolished the spontaneous, long-lasting hyperpolarizations recorded during application of 4AP and glutamatergic receptor antagonists. Note in the CGP 35348 sample that the membrane potential was depolarized by 5 mV to further disclose the occurrence of hyperpolarizing potentials. This procedure, however, only caused an increase of subthreshold membrane oscillations at 10 to 12 Hz (arrow-heads).

Addition of CGP 35348 to medium containing 4AP + CNQX + CPP did not influence the RMP or the input resistance of CA3 pyramidal cells but caused in all cases an increase in the duration of the spontaneous GABA receptor-mediated synchronous field potentials and of the associated LLDs (Fig. 5A). In contrast, this GABAB receptor antagonist did not modify their rate of occurrence (not illustrated but see Fig. 8A). Moreover, CGP 35348 decreased the post-LLD long-lasting hyperpolarization, thus indicating that this potential represented a GABAB receptor-mediated hyperpolarizing current (Fig. 5A). Similar results were obtained by studying the intracellular responses induced by electrical stimuli delivered in the dentate hilus or in the CA1 stratum radiatum (Fig. 5B). These CGP 35348-induced effects were not accompanied by any appreciable change in the rate of occurrence of spontaneous, asynchronous depolarizing PSPs. Moreover, CGP 35348 abolished the long-lasting hyperpolarizations that occurred spontaneously in two cells (Fig. 5C).


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  		Fig. 6.   Effects induced by CGP 35348 on the intracellular activity recorded with KCl + QX-314 filled electrodes from CA3 pyramidal cells bathed in 4AP and excitatory amino acid receptor antagonists. A, in these type of experiments, the spontaneously occurring LLD can trigger small amplitude regenerative events, and it is not followed by any long-lasting hyperpolarization. Note also that the apparent input membrane resistance decreases markedly during the LLD. Application of CGP 35348 causes a prolongation of the LLD. B, quantitative summary of the effects induced by CGP 35348 on the half-width duration of the LLD in four neurons recorded with KCl + QX-314-filled electrodes. C-F, spontaneous PSPs recorded intracellularly with KCl + QX-314-filled electrodes during application of 4AP and excitatory amino acid receptor antagonists and following bath addition of CGP 35348. Raw data in C and amplitude histograms in D were obtained from an experiment in which the CA3 pyramidal cell did not change its ability to generate PSPs. Raw data in E and amplitude histograms in F were from a neuron that produced an increased amount of PSPs larger than 10 mV during CGP 35348 application.


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  		Fig. 7.   Effects induced by CGP 35348 on the synchronous activity analyzed with simultaneous field potential and [K+]o recordings from the CA3 stratum radiatum during application of 4AP. A, under control conditions, most of the GABA receptor-mediated potentials do not initiate the ictal discharge; note that in these cases the increases in [K+]o attain peak values that are smaller than those seen with GABA receptor-mediated potentials that are followed by an ictal event. Application of CGP 35348 causes an increase in occurrence of all synchronous activities and also makes most of the negative-going field potentials trigger ictal activity. B, quantitative summary of peak values of the increases in [K+]o associated with the negative-going GABA receptor mediated potentials recorded under control conditions and during CGP 35348 application. Data were segregated in two groups according to whether ictal discharge was present or absent after the GABA receptor-mediated potential.


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  		Fig. 8.   Effects induced by CGP 35348 on the GABA receptor-mediated potentials occurring during application of 4AP and glutamatergic receptor antagonists; simultaneous field potential and [K+]o recordings were obtained from the CA3 stratum radiatum. A, simultaneous field potential and [K+]o recordings in control conditions indicate that the negative-going field potentials are associated to increases in [K+]o even though glutamatergic transmission was pharmacologically blocked. Application of CGP 35348 (1 mM) caused an increase in the amplitude of the elevations in [K+]o without modifying the rate of occurrence of the GABA receptor-mediated synchronous events. Note also that a clear prolongation of the GABA receptor-mediated synchronous events is clearly appreciable in the field potential recordings. B, quantitative summary of the peak values of the increases in [K+]o associated with the negative-going GABA receptor-mediated potentials recorded under control conditions and during CGP 35348 application. C, semilog plot of the time decay of the increases in [K+]o occurring during the negative-going GABA receptor-mediated field potential under control conditions and after bath application of CGP 35348. D, column charts of the duration of the increases in [K+]o obtained under control in the presence of CGP 35348 and during wash. Time values were measured at 50 and 20% of the maximal increases recorded in four slices.

These results demonstrate that GABAB receptor blockade leads to an increase in the duration of the synchronous GABA receptor-mediated potentials and of the associated LLDs. This evidence, however, does not denote whether the changes in LLD resulted from an augmented release of GABA from presynaptic terminals (following presynaptic GABAB receptor blockade) or from postsynaptic GABAB receptor antagonism leading to a reduction of the post-LLD hyperpolarization. Such an effect may produce an increase in LLD duration. Therefore, we analyzed the effects induced by CGP 35348 (1 mM) on the intracellular activity recorded with KCl-QX-314-filled electrodes from CA3 pyramids during application of medium containing 4AP and excitatory amino acid receptor antagonists. Intracellular diffusion of QX-314 leads to a blockade of the GABAB receptor-activated K+ conductance along with voltage-gated Na+ channels and other K+ conductances (Nathan et al., 1990). Application of CGP 35348 (1 mM) caused in all cases (n = 6) an increase in the duration and amplitude of the LLDs (Fig. 6, A and B).

In the experiments performed with KCl-QX-314-filled electrodes, we also analyzed the effects induced by CGP 35348 (1 mM) on the spontaneous depolarizing PSPs, which presumably represented GABAA receptor-mediated events. It should be emphasized that this type of analysis was more accurate than with KCl-filled electrodes as intracellular diffusion of QX-314 abolished the subthreshold oscillations generated by CA3 pyramidal cells (Fig. 5C, arrow-heads; Psarropoulou and Avoli, 1995). In four of seven neurons, CGP 35348 did not appear to influence the rate of occurrence of these PSPs (Fig. 6, C and D), whereas in three cells, it caused an increase in their rate. This effect was clearly identified when measuring the frequency of occurrence of the events that attained amplitudes larger than 10 mV (Fig. 6, E and F).

[K+]o and Effects Induced by CGP 35348 on the 4-Aminopyridine-Induced Synchronous Activity. The effects exerted by CGP 35348 on the 4AP-induced synchronous activity were also analyzed in nine experiments by employing simultaneous field potential and [K+]o recordings in the CA3 stratum radiatum. As illustrated in Fig. 7A (control), [K+]o transiently increased during each GABA receptor-mediated field potential (from a resting value of approximately 3.5 up to 7.5 mM) and remained elevated during the successive ictal discharge whenever this type of activity was present. Moreover, as reported by us in a previous study (Avoli et al., 1996b), the elevations in [K+]o associated with the isolated GABA receptor-mediated events were smaller than those seen during the GABA receptor-mediated potentials preceding the ictal discharges (Fig. 7, A and B; control). In line with what observed while studying the field potential and the intracellular activities, application of CGP 35348 increased the rate of occurrence of all synchronous activities, an effect that was accompanied by an increase of the [K+]o elevations associated with the GABA receptor-mediated potentials, both when they were followed by ictal discharges and when they occurred in isolation (Fig. 7, A and B; CGP 35348). The effects induced by CGP 35348 were not accompanied by any change in [K+]o baseline.

Next, we analyzed the effect of CGP 35348 (0.5-1 mM) on the pharmacologically isolated synchronous GABA receptor-mediated potentials that occur during concomitant application of 4AP + CNQX + CPP (n = 7). As reported in previous studies (Avoli et al., 1996b; Motalli et al., 1999), these synchronous events continued to be associated with transient elevations in [K+]o in spite of blockade of glutamatergic transmission. In these experiments as well, CGP 35348 application significantly increased the half-amplitude duration of the GABA receptor-mediated field potentials from 0.76 ± 0.089 to 1.34 ± 0.51 s without changing the rate of occurrence of these events (n = 7) (Fig. 8A). These effects were characterized by an increase in the peak values of the [K+]o elevations that were associated with the GABA receptor-mediated events (Fig. 8, A and B). Interestingly, CGP 35348 increased the half-amplitude duration of these [K+]o elevations to a lesser extent than their overall duration. Such an effect can be appreciated in the plot of the decay time of the [K+]o elevations shown in Fig. 8C as well as in the column histograms of Fig. 8D in which the duration of the increases in [K+]o were calculated at 50 and 20% of the peak amplitude.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The main conclusions of this study are that antagonizing GABAB receptors in the juvenile rat hippocampus: 1) increases the occurrence of all types of synchronous activity elicited by 4AP, and 2) facilitates the initiation of ictal discharges by synchronous GABA receptor-mediated potentials. Since the rate of the GABA receptor-mediated events recorded during blockade of ionotropic glutamatergic transmission was not influenced by CGP 35348, we believe that the increased occurrence of synchronous activity during GABAB receptor antagonism reflects a heightened network excitability that is mediated through glutamatergic mechanisms. Moreover, our findings indicate that the disclosure of ictal discharges by CGP 35348 may rest on the ability of this GABAB receptor antagonist to cause larger increases in [K+]o during the GABA receptor-mediated synchronous potentials. In line with this view, application of CGP 35348 during blockade of glutamatergic transmission caused larger elevations in [K+]o, thus suggesting that this pharmacological treatment leads to increased release of GABA from interneuron terminals through an action exerted on presynaptic GABAB autoreceptors.

GABAB Receptor Antagonism and Proconvulsant Effects. Clinical and experimental evidence indicates that the GABAB receptor agonist baclofen can exert proconvulsant effects (Lewis et al., 1989; Mott et al., 1989; Rush and Gibberd, 1990; Watts and Jefferys, 1993; Kofler et al., 1994; Motalli et al., 1999). Such an action has also been documented with the epileptiform discharges induced by bath application of 4AP to hippocampal slices (Watts and Jefferys, 1993; Motalli et al., 1999), leading us to hypothesize that GABAB receptor antagonists could exert anticonvulsant actions in this in vitro model of epileptiform discharge. Previous studies have indeed shown that CGP 35348 has anticonvulsant actions in rodent models of absence seizures (Liu et al., 1992; Bowery and Enna, 2000).

Contrary to our expectations, we have found that CGP 35348 makes CA3 networks generate interictal and ictal discharges that are more frequent than under control conditions. Moreover, when only interictal activity and GABA receptor-mediated potentials occurred in the presence of 4AP, GABAB receptor antagonism leads to the appearance of ictal discharges. The proconvulsant effects of CGP 35348 were, however, different from those seen with baclofen under similar experimental conditions (Motalli et al., 1999). Thus, although GABAB receptor activation abolishes interictal discharges and prolongs ictal events through activity-dependent changes in excitability (Motalli et al., 1999), we have found that CGP 35348 increases the frequency of 4AP-induced interictal and ictal discharges, the latter displaying shorter duration than under control conditions. Dose-response analysis of the CGP 35348-induced changes has revealed an EC50 of 0.25 mM.

The physiological role of GABAB receptors in regulating cortical neuron excitability has been studied in detail over the last few years (Davies et al., 1991; Bowery, 1993; Yamada et al., 1999; Bowery and Enna, 2000). Previous in vitro work has shown that antagonizing GABAB receptor is not sufficient per se to cause epileptiform synchronization (McCormick, 1989; Sutor and Luhmann, 1998), but it can potentiate epileptiform responses induced by GABAA receptor antagonists (McCormick, 1989; Scanziani et al., 1991, 1994; Karlsson et al., 1992; Sutor and Luhmann, 1998). Overall, these data suggested that weakening or abolishing GABAA receptor-mediated inhibition should be a sine qua non condition for expressing the proepileptogenic effects of CGP 35348. We have demonstrated here that similar effects can be obtained by blocking GABAB receptors in the 4AP model in which GABAA receptor inhibition is potentiated as the result of an increased release of GABA from interneuron terminals (Rutecki et al., 1987; Perreault and Avoli, 1991,1992). Hence, our data indicate that the capacity of GABAB receptor-mediated mechanisms to modulate epileptiform synchronization in cortical networks maintained in vitro does not depend on GABAA receptor antagonism but rather on the ability of ambient GABA to activate type B receptors located pre- and postsynaptically. This condition can be achieved by increasing neuronal excitability, and thus GABA release, either by blocking the GABAA receptor function (McCormick, 1989; Scanziani et al., 1991, 1994; Sutor and Luhmann, 1998) or by applying drugs capable of augmenting transmitter release (e.g., 4AP).

Such a conclusion is further supported by the ability of CGP 35348 to increase the rate of occurrence and the duration of asynchronous PSPs recorded in the presence of 4AP. These action potential-dependent synaptic events are not usually recorded with sharp electrodes from CA3 pyramidal cells under control conditions but are readily induced by bath application of 4AP (Perreault and Avoli, 1992; Motalli et al., 1999). Activation of presynaptic GABAB receptors by ambient GABA has been reported in thalamic cells (Le Feuvre et al., 1997).

The effects of CGP 35348 in juvenile hippocampal slices confirms that both presynaptic and postsynaptic GABAB receptors are functional at this age (Gaiarsa et al., 1995; McLean et al., 1996). In this study, we have shown that this GABAB receptor antagonist can decrease and eventually abolish the long-lasting hyperpolarization that follows the 4AP-induced LLD in intracellular recordings performed with KCl-filled electrodes. Under these recording conditions, GABAA receptor-mediated potentials became depolarizing due to intracellular leakage of Cl- from the recording electrode, and thus hyperpolarizing potentials should represent events caused by an increase in K+ conductance (Gähwiler and Brown, 1985). Indeed, several studies have documented that cortical neurons (including hippocampal pyramids) generate a postsynaptic, K+-dependent hyperpolarization that is abolished by GABAB receptor antagonists (Newberry and Nicoll, 1985; Dutar and Nicoll, 1988; McCormick, 1989).

We have also demonstrated that CGP 35348 increases the duration of the LLD analyzed intracellularly with QX-314-filled electrodes. This lidocaine derivative blocked the post-LLD hyperpolarization as expected for a GABAB receptor-mediated potential associated with an increase in K+ conductance (Nathan et al., 1990; Andrade, 1991). The prolongation induced by CGP 35348 under these experimental conditions should reflect an increase in transmitter release that is caused by interneuron firing leading to the generation of synchronous GABA receptor-mediated potentials (Benardo, 1997). Hence, we are inclined to interpret the LLD prolongation as caused by blockade of presynaptic GABAB autoreceptors. This conclusion, however, awaits confirmation by using selective antagonists for pre- and postsynaptic GABAB receptors (Yamada et al., 1999). McLean et al. (1996) have also reported an increase in the duration of 4AP-induced GABA receptor-mediated potentials recorded from CA3 pyramids in hippocampal slices obtained between postnatal day 3 and 7 during CGP 35348 application.

GABA-Mediated Synchronous Potentials and Ictal Discharge Initiation. We have found that CGP 35348 modulates the size of the GABA receptor-dependent increases in [K+]o both in medium containing 4AP and during concomitant application of 4AP and glutamatergic receptor antagonists. Previous studies from our laboratories have demonstrated that these synchronous GABA receptor-mediated potentials can initiate ictal activity in slices of the juvenile hippocampus (Avoli et al., 1996b; Motalli et al., 1999) and of the adult entorhinal cortex (Avoli et al., 1996a). In keeping with this view, we have confirmed here that these events precede and thus appear to trigger ictal discharges. GABA receptor-mediated synchronization also facilitates epileptiform activity in the Mg2+-free in vitro model (Köhling et al., 2000).

The ability of GABAA receptor-mediated mechanisms to entrain cortical networks into seizure-like activity relies on the transient elevations in [K+]o that accompany the GABA receptor-mediated potentials and are contributed by outward transport of K+ with a Cl-/HCO3- anion shift, Na+-dependent GABA uptake, and glial depolarization (reviewed in Avoli, 2000). Indeed, elevating [K+]o causes seizure activity both in in vivo and in vitro preparations by producing a positive shift of GABA-mediated postsynaptic inhibition, by depolarizing neurons and disinhibiting excitatory postsynaptic interaction (Zuckermann and Glaser, 1968; Traynelis and Dingledine, 1988; McBain et al., 1993). Hence, the ability of CGP 35348 to increase the amplitude of the GABA receptor-mediated elevations in [K+]o generated by CA3 networks in the juvenile rat hippocampus, strongly suggests that this phenomenon represents a mechanism by which GABAB receptor antagonism can lead to potentiation or disclosure of epileptiform ictal discharges in the 4AP model. In particular, we propose that blockade of presynaptic GABAB autoreceptors should cause an increase in the release of GABA during the synchronous firing of interneurons, thus leading to greater increases in [K+]o that implement pyramidal cells epileptiform synchronization both spatially and temporally.

This conclusion is further supported by the data obtained by applying CGP 35348 to medium containing 4AP and glutamatergic receptor antagonists. GABA receptor-mediated synchronous potentials generated by CA3 networks under these experimental conditions were associated with [K+]o elevations of larger amplitude. Similar findings have been recently reported in human neocortical networks (Louvel et al., 2001). Interestingly, in our experiments the GABA receptor-mediated synchronous potentials increased in frequency during application of CGP 35348 to hippocampal slices perfused with control, 4AP-containing medium. On the contrary, the rate of occurrence did not change when CGP 35348 was applied during blockade of glutamatergic transmission (Louvel et al., 2001). This evidence further support the contribution of glutamatergic mechanisms in modulating interneuron excitability and thus GABA release (McBain and Fisahn, 2001).

    Acknowledgments

We thank D. Wan-Chow-Wah for participating in some preliminary experiments, Dr. K. Babinski for helping with data analysis, and Dr. K. Krnjevic for constructive criticism on an early draft of this article.

    Footnotes

Accepted for publication August 9, 2002.

Received for publication June 25, 2002.

This study was supported by the Canadian Institutes of Health Research (Grant MT-8109) and the Savoy Foundation. MD is recipient of a Fragile X Foundation of Canada.

DOI: 10.1124/jpet.102.040782

Address correspondence to: Dr. M. Avoli, Montreal Neurological Institute, 3801 University St., Montreal, Quebec, H3A 2B4, Canada. E-mail: massimo.avoli{at}mcgill.ca

    Abbreviations

4AP, 4-aminopyridine; CGP 35348, p3-amino-propyl,p-diethoxymethylphosphonic acid; ACSF, artificial cerebrospinal fluid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CPP, 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate; QX-314, 2-(trimethyl-amino)-N-(2-6-dimethyl-phenyl)-acetamide; LLD, long-lasting depolarization; PSPs, asynchronous postsynaptic potentials; RMP, resting membrane potential.

    References
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Abstract
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Materials and Methods
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