INTRODUCTION

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Epilepsy is characterized by the periodic and unpredictable occurrence of seizures. Clinical electroencephalographic observations show that epileptiform activity is composed of interictal discharges, ictal events (seizures), and periods of postictal depression. Although the distinction between an interictal discharge and an ictal event can be debated, in general, the interictal discharges are characterized by a brief burst of synchronized paroxysmal activity lasting <500 ms (usually 50-100 ms). Ictal events have an overall duration of seconds to tens of seconds. Efforts to understand the mechanisms underlying epileptiform activity have focused primarily on in vitro models of interictal discharges. Although physiological abnormalities may be identified in the interictal state, an understanding of the mechanisms underlying the longer ictal events is also important.

A major obstacle in studying seizure (ictal) activity has been the lack of an in vitro model that closely mimics the in vivo situation. Ictal-like discharges, which can last a few seconds up to tens of seconds, were first described in the CA1 region (Jefferys and Haas 1982; Taylor and Dudek 1982) and were later observed in the dentate gyrus (Schweitzer et al. 1992) when perfusing the slices with no added calcium and high potassium solutions. Although extracellular calcium levels have been recorded to fall as low as 0.5 mM after many seconds of seizure activity (Pumain et al. 1985), the role of low calcium levels in the onset of the synchronized seizure activity is not known. The goal of this study was to determine whether the ictal-like discharges could occur in normal calcium levels and, if so, what conditions were required for them to appear.

	METHODS

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Hippocampal slices were prepared by conventional methods from Sprague-Dawley rats (100-180 g, both sexes). After anesthetizing the rats (ketamine 25 mg/kg, xylazine 5 mg/kg, acepromazine 0.8 mg/kg ip), the brains were removed. Transverse slices (400-500 µm) through the hippocampus were cut with a Vibratome (Technical Products International). Slices were placed in an interface-type chamber and continuously perfused with artificial cerebrospinal fluid (ACSF) at 32°C under a stream of humidified 95% O₂-5% CO₂. Composition of the ACSF was (in mM) 127 NaCl, 3.5 KCl, 1.5 MgCl, 1.1 KH₂PO₄, 26 NaHCO₃, 2 CaCl₂, and 10 glucose. All solutions were bubbled constantly with 95% O₂-5% CO₂. Slices were allowed to equilibrate for 1 h before electrophysiological recording was begun.

Recording electrodes were made of microfilament capillary thin-walled glass (A-M Systems, 0.9 mm ID, 1.2 mm OD) pulled on a micropipette puller (P-87, Sutter Instruments). Electrodes were filled with 2 M NaCl and had impedances between 4 and 10 M. Recording electrodes were placed in the cell body layer of CA1, CA3, and the dorsal dentate gyrus. Synaptic transmission was blocked in some experiments by the addition of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM), D,L-2-amino-5-phosphonopentanoic acid (APV, 100 µM), and bicuculline (20 µM) or perfusion with Cd²⁺ (200 µM).

Cesium chloride (Cs⁺), 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA), and cadmium chloride (Cd²⁺) were purchased from Sigma Chemical (St Louis, MO). CNQX, DL-APV, and bicuculline methiodide were purchased from Tocris (Ballwin, MO). All chemicals were dissolved directly into the perfusing solution. When cadmium was added to the perfusing solution, the potassium phosphate was omitted and replaced with equimolar amounts of potassium chloride.

	RESULTS

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The first experiments confirmed that perfusion with cadmium (Cd²⁺, 200 µM, n = 6) or neurotransmitter blockers (CNQX, 20 µM; APV, 100 µM; and bicuculline, 20 µM, n = 6) blocked synaptically evoked responses, and ictal-like discharges did not appear in normal ACSF in any region of the hippocampus with at least 2 h of perfusion.

Next, neuronal excitability was increased using elevated extracellular potassium (Fig. 1, Table 1). Bath application of 12 mM potassium resulted in spontaneous interictal discharges in CA1 and CA3 within 30 min (n = 33). The dentate gyrus remained silent (Fig. 1, A and C). In eight slices, the high potassium perfusion was continued for up to 3 h. In these slices, the interictal activity continued, and ictal-like discharges were never recorded in any hippocampal region. In the remaining 25 slices, synaptic transmission was blocked by the addition of either cadmium (200 µM, n = 13) or neurotransmitter blockers (CNQX, APV, and bicuculline, n = 12), and the slices were monitored for the presence of ictal-like discharges for at least 2 h. Within 20 min after switching to the perfusing solution containing blockers of synaptic transmission, interictal activity stopped. After prolonged perfusion (up to 40 min), ictal-like discharges developed (Fig. 1, B and D). In the presence of cadmium, ictal-like discharges were seen in CA1 in 40% of the slices and in the dentate gyrus in 30% of the slices. In the presence of neurotransmitter receptor blockers, ictal-like discharges developed in 33% of slices in CA1 and the dentate gyrus after 1 h of perfusion (Fig. 1D). No spontaneous ictal-like discharges were recorded in the CA3 region. Lower concentrations of potassium (8-10 mM) would induce interictal activity in CA1 and CA3 in all slices and ictal-like activity in CA1 and CA3 in about one-third of the slices, but the longer ictal-like discharges did not appear in the dentate gyrus, even after blocking synaptic transmission.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effect of blocking synaptic transmission on epileptiform activity induced by high potassium. A: simultaneous chart recordings of extracellular discharges in the CA1 (top), CA3 (middle), and the dentate gyrus (DG, bottom) after perfusion for 1.5 h with 12 mM potassium. Spontaneous interictal discharges are present in both CA1 and CA3 regions. The small upward spikes in the dentate gyrus, here and in subsequent figures, are far-field recording of the activity in CA3. The inset in A is a single evoked response recorded in CA1 after 15 min of perfusion in the high potassium solution. B: same slice as in A. After 50 min of perfusion with cadmium (Cd2+), spontaneous ictal-like discharges is present in CA1 and the dentate gyrus. The inset in B is a single evoked response recorded in CA1 just after the interictal activity had stopped. The same stimulus intensity was used for the responses in the insets in A and B. Two minutes later, no response was recorded in response to stimulation. C: spontaneous epileptiform activity after perfusion for 60 min in 12 mM potassium. D: same slice as in C, addition of bicuculline, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and D,L-2-amino-5-phosphonopentanoic acid (APV) blocked the interictal discharges in all regions and ictal-like discharges appeared 1 h later. In A, the interictal activity is shown on the same time scale as the longer ictal-like activity. Elsewhere the interictal activity is shown on a faster time scale to show the regularity of the discharges.

To determine whether these results are specific for high potassium, in additional experiments excitability was increased by perfusion with the potassium channel blockers, cesium, TEA, or 4-AP (Table 1). Addition of cesium (5 mM, n = 32, Fig. 2, A and C), TEA (5 mM, n = 19, Fig. 3A), or 4-AP (1 mM, n = 12) induced spontaneous interictal discharges that were synchronized in the CA3 and CA1 regions. The dentate gyrus was silent. Lower concentrations of 4-AP (100 and 500 µM) would produce interictal activity, but no ictal-like activity appeared after blocking synaptic transmission. Addition of cadmium (200 µM), to block synaptic transmission, blocked the interictal activity in both CA1 and CA3 in all slices tested (n = 20 in cesium, n = 9 in TEA, n = 6 in 4-AP). Spontaneous ictal-like discharges appeared after 40-60 min of perfusion with cadmium in CA3 (40% of slices in cesium), CA1 (20% of slices in cesium, 30% in TEA, 25% in 4-AP), and the dentate gyrus (55% of slices in cesium, 20% in TEA, 12% in 4-AP; Figs. 2B and 3B). Addition of neurotransmitter receptor blockers (CNQX, APV, and bicuculline) also blocked the interictal activity in all slices (n = 12 in cesium, n = 10 in TEA, n = 6 in 4-AP). Spontaneous ictal-like discharges appeared after 40-60 min of perfusion with neurotransmitter receptor blockers in CA3 (33% of slices in cesium), CA1 (20% of slices in cesium, 40% in TEA, 25% in 4-AP), and the dentate gyrus (50% of slices in cesium, 20% in TEA, 17% in 4-AP; Figs. 2D and 3C).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Effect of blocking synaptic transmission on spontaneous epileptiform activity induced by 5 mM cesium (Cs+). A: simultaneous chart recordings of extracellular discharges in CA1 (top), CA3 (middle), and the dentate gyrus (DG, bottom) induced by perfusion for 1 h with 5 mM cesium. B: same slice as in A. After 40 min of perfusion with cadmium (Cd2+), spontaneous ictal-like discharges developed in CA3 and the dentate gyrus. C: spontaneous epileptiform activity after perfusion for 1 h with 5 mM cesium. D: same slice as in C, addition of bicuculline, CNQX, and APV blocked the interictal discharges and ictal-like discharges appeared after 50 min. The small-amplitude activity in CA1 is a far-field recording of the larger amplitude activity in CA3.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Effect of blocking synaptic transmission on epileptiform activity induced by TEA. A: chart recordings of extracellular discharges in CA1 and the dentate gyrus 40 min after beginning perfusion with TEA 5 mM. B: addition of cadmium (0.2 mM) blocked the interictal discharges, and ictal-like discharges appeared in CA1 and the dentate gyrus in some slices. C: in the presence of bicuculline, CNQX, and APV, spontaneous interictal discharges were blocked and ictal-like discharges appeared. Recordings in CA1 in A and C are from the same slices. Recordings in the dentate gyrus in A and B are from the same slice.

	DISCUSSION

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In this study, epileptiform activity was induced by perfusing hippocampal slices with high potassium or potassium channel blockers until spontaneous interictal discharges developed in both CA1 and CA3 (in normal levels of calcium). Synaptic transmission was then blocked by the addition of neurotransmitter receptor blockers (CNQX, APV, and bicuculline) or the calcium channel blocker cadmium, resulting in complete blockade of the interictal discharges and the appearance of ictal-like discharges in some slices. These results demonstrate that ictal-like discharges can develop in normal levels of extracellular calcium, which is in agreement with previous studies (Patrylo et al. 1994). But the present experiments further define the conditions required for the appearance of the ictal-like events. The results suggest that before ictal-like events can occur in the hippocampus there must be an increase in neuronal excitability, which in these experiments was achieved by increasing the extracellular potassium or adding potassium-channel blockers, but that this increase in neuronal excitability is not sufficient. The present experiments suggest that, in addition, there must be either blockade of synaptic transmission or elimination of interictal discharges.

Is it a reduction in synaptic transmission that allows the appearance of the ictal-like bursts, or do frequent interictal discharges suppress the ictal-like activity? There is evidence in support of each of these possibilities. The long duration bursts were first observed in zero-added calcium conditions (Jefferys and Haas 1982; Schweitzer et al. 1992; Taylor and Dudek 1982), in which there is no synaptic transmission, suggesting that the absence of synaptic transmission is critical. It is possible that in the presence of synaptic transmission that there is sufficient GABA release or activation of inhibitory synapses to terminate bursting. Reduction, or elimination, of synaptic transmission may allow longer duration discharges to appear. However, in bicuculline alone, which would block GABA_A receptors, longer duration discharges have not been observed (unpublished observations). Although there is some controversy about the interplay between interictal discharges and ictal events, there is some evidence that frequent short discharges will suppress longer duration discharges (Wilson and Bragdon 1993; Xiong and Stringer 1999). In the CA3 region of the hippocampus in zero-added magnesium, it has been shown that constant interictal-like activity can suppress the longer seizure-like events (Anderson et al. 1986; Swartzwelder et al. 1987). In CA1 in high potassium, cadmium has been shown to block the interictal activity, but have no effect on the longer ictal-like events (Jensen and Yaari 1988), suggesting different mechanisms for the two different types of epileptiform discharges. Finally, in the dentate gyrus, frequent short bursts appear to block the longer duration bursts (Xiong and Stringer 1999).

When the long-duration bursts were first observed in zero-added calcium, in the absence of synaptic transmission, it was postulated that nonsynaptic mechanisms were sufficient to synchronize neurons (Jefferys and Haas 1982; Jensen and Yaari 1988; Taylor and Dudek 1982). Nonsynaptic mechanisms that may result in synchronization of neuronal activity have been extensively investigated (see reviews by Dudek et al. 1998; Jefferys 1995). It is generally accepted that nonsynaptic mechanisms are sufficient to synchronize neuronal activity (at least in some conditions), but whether these mechanisms operate to synchronize neurons in the presence of synaptic activity is not known. If one assumes that these long-duration bursts are only synchronized by nonsynaptic mechanisms, then the fact that they can be generated in normal levels of extracellular calcium suggests that nonsynaptic mechanisms may operate in the presence of synaptic transmission.