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. 2001 May 1;532(Pt 3):713–730. doi: 10.1111/j.1469-7793.2001.0713e.x

Seizure-like activity in the disinhibited CA1 minislice of adult guinea-pigs

Sergei Karnup 1, Armin Stelzer 1
PMCID: PMC2278566  PMID: 11313441

Abstract

  1. Spontaneous activity was monitored during pharmacological blockade of GABAA receptor function in the CA1 minislice (CA3 was cut off). Synaptic inhibition was blocked by competitive GABAA antagonists bicuculline-methiodide (Bic) or GABAZINE (GBZ) and the chloride channel blocker picrotoxin (PTX). Extra- and intracellular recordings using sharp electrodes were carried out in stratum radiatum and pyramidale.

  2. At low antagonist concentrations (Bic, GBZ: 1-10 μm; PTX: < 100 μm), synchronized bursts (< 500 ms in duration, interictal activity) were seen as described previously. However, in the presence of high concentrations (Bic, GBZ: 50-100 μm; PTX: 100-200 μm), seizure-like, ictal events (duration 4-17 s) were observed in 67 of 88 slices. No other experimental measures to increase excitability were applied: cation concentrations ([Ca2+]o= 2 mm, [Mg2+]o= 1.7 mm, [K+]o= 3 mm) and recording temperature (30-32 °C) were standard and GABAB-mediated inhibition was intact.

  3. In whole-slice recordings prominent interictal activity, but fewer ictal events were observed. A reduced ictal activity was also observed when interictal-like responses were evoked by afferent stimulation.

  4. Ictal activity was reversibly blocked by antagonists of excitatory transmission, CNQX (40 μm) or d-AP5 (50 μm).

  5. Disinhibition-induced ictal development did not rely on group I mGluR activation as it was not prevented in the presence of group I mGluR antagonists (AIDA or 4CPG).

  6. (RS)-3,5-DHPG prevented the induction and reversed the tertiary component of the ictal event through a group I mGluR-independent mechanism.

Two main forms of epileptiform neuronal activity are recorded electrographically: short (< 500 ms) interictal bursts and seizure-like ictal events, lasting seconds or minutes. Although long considered a hallmark of epileptiform activity (Alger, 1984), notably as possible precursors of ictal events, interictal events have no behavioural correlate in situ and may even represent physiological activity (Schneiderman, 1986; Schwartzkroin & Haglund, 1986). In contrast, ictal events may result in severe neurological dysfunction and brain damage (Lynch et al. 1996; Meldrum, 1997).

The pharmacological blockade of synaptic inhibition is one of the most frequently used models for studying mechanisms of epilepsy. The application of antagonists of GABAA receptor-mediated inhibition in the hippocampal slice preparation was shown to result in synchronized short bursts (Schwartzkroin & Prince, 1978) or intermediate events which contain afterdischarges (Wong et al. 1986). Studies in the CA3 subfield of PTX-treated hippocampal slices have shown that synchronized bursting occurred when latent recurrent excitatory connections became functional (Miles & Wong, 1986, 1987). However, even in CA3, which is widely believed to contain a higher connectivity of recurrent excitatory synapses (than CA1), disinhibition led to ictal events only under special conditions: in immature CA3 slices (Swann & Brady, 1984) or in ventral, but not dorsal, CA3 slices in the presence of elevated [K+]o (Traub et al. 1996; Borck & Jefferys, 1999). A prolongation of afterdischarges was observed in CA1 when activators of group I metabotropic glutamate receptors (mGluRs) were added to PTX (Merlin & Wong, 1997).

Two factors may preclude disinhibition-induced ictal activity in the slice. First, the neuronal population of the slice may be too small to generate ictal activity during disinhibition. This was suggested by a recent study which showed ictal-like events during disinhibition in the whole in vitro hippocampus, but not in the slice (Khalilov et al. 1997). Second, in contrast to other epileptogenic conditions shown to generate ictal-like activity in the slice, e.g. elevation of [K+]o (Traynelis & Dingledine, 1988; Jensen & Yaari, 1988) or electrical stimulation (Swartzwelder et al. 1987), a removal of synaptic inhibition alone may not suffice to implement the mechanisms underlying ictal activity, i.e. presynaptic increases of excitability (Traub et al. 1996), elimination of the burst afterhyperpolarization (AHP) (Spencer & Kandel, 1969; Alger, 1984) and the development of a sustained afterdepolarization (ADP). Additional actions such as the activation of mGluRs (Wong et al. 1999) may be necessary. Here we show, however, that seizure-like activity can develop in the CA1 minislice of the guinea-pig hippocampus solely through a pharmacological blockade of GABAA receptor function.

METHODS

Slice preparation

Transverse hippocampal slices were obtained from adult guinea-pigs (Hartley, from Harlan Sprague Dawley, Inc., Indianapolis, IN, USA; 150-200 g). Guinea-pigs were anaesthetized by inhalation of halothane before decapitation with an animal guillotine (in conformation with the guidelines of the Institutional Animal Care and Use Committee (protocol 9808069)). After removal of the brain and isolation of the hippocampus, slices of 450 μm thickness were cut on a Vibrotome. CA1 ‘mini’ slices were obtained by cutting off CA2/3 and the subiculum under microscopic control. Slices were superfused in an interface recording chamber (Fine Science Tools, Belmont, CA, USA) with a solution saturated with 95 % O2-5 % CO2 (temperature 30-32 oC) of the following composition (mm): NaCl 118, KCl 3, NaHCO3 25, NaH2PO4 1.2, MgCl2 1.7, CaCl2 2.0 and d-glucose 11.

Recordings

Recording electrodes (World Precision Instruments, Inc., Sarasota, FL, USA) were pulled by a Brown-Flaming electrode puller (Model P-87, Sutter Instrument Co., Novato, CA, USA). Intracellular and extracellular recordings were obtained in stratum radiatum and pyramidale of CA1. Signals were recorded and amplified with an Axoprobe-1A (Axon Instruments, Foster City, CA, USA), fed into an A/D converter (Digidata 1200, Axon Instruments) digitized, stored and analysed off-line using pCLAMP 7 software from Axon Instruments in a Pentium PC.

Stimulation

In selected recordings (Figs. 6 and 8), electrical stimulation was applied to stratum radiatum Schaffer collateral-commissural fibres through a pair of insulated tungsten bipolar electrodes (stimulation range from 40 to 200 μA).

Figure 6. Pharmacological blockade of monosynaptic inhibitory responses by PTX and Bic.

Figure 6

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IPSPs and gGABAA underlying the stimulation-evoked monosynaptic inhibitory responses were evoked and analysed as described in Stelzer et al. (1994). Monosynaptic inhibition was isolated through a pharmacological blockade of excitation and GABAB-mediated inhibition (Davies et al. 1990). A, about 4 nS synaptic peak conductances were measured in the presence of CNQX (40 μm), d-AP5 (50 μm), saclofen (100 μm) and PTX (100 μm) (a). The addition of Bic (10 μm) reduced peak conductances to about 3 nS (b). Measurements were performed in pyramidal cell soma in stratum pyramidale of the CA1 minislice at Vrest. B, monosynaptic IPSPs in the presence of PTX or Bic at various concentrations: IPSPs were completely blocked by ≥ 5 μm Bic or ≥ 10 μm PTX. C, monosynaptic gGABAA during PTX and Bic application at various concentrations. D, comparison of the block of IPSPs and gGABAA by PTX at various concentrations.

Figure 8. Effects of interictal activity on the frequency of ictal events.

Figure 8

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A, ictal frequency in the presence of Bic (50-100 μm; depicted as average number of ictal events per 60 min: a, during Bic alone (Bic, n = 60), during Bic and weak stimulation evoking EPSPs (+ ortho EPSP, n = 19), with strong stimulation-evoking burst responses (+ ortho burst; n = 14) and in whole-slice recordings (Whole slice, n = 7). Stimulation was applied every 30 s in stratum radiatum and stimulation intensity was adjusted to generate EPSPs or burst responses, respectively. * Significant differences (reduction) in the ictal frequency compared with Bic alone (P < 0.05, ANOVA). Ab, representative burst responses in various experimental conditions. B, number of ictal events plotted as a function of the AHP efficacy (calculated as the number of interictal events ×∫AHP(dt) in 3 different protocols: Bic alone (n = 21, intracellular), whole-slice experiments (n = 4, intracellular) and during evoked burst responses (n = 5, intracellular). Ca, number of ictal events in the CA1 minislice: in the presence of DHPG alone (20-80 μm; DHPG, n = 21), during additional weak stimulation (+ ortho EPSP, n = 14), during additional strong stimulation (ortho burst, n = 12) and in the whole slice (n = 4). Cb, representative burst response during DHPG (40 μm). One of two concomitant intracellular recordings. D, prolonged discharges elicited by orthodromic stimulation (100 μs, 140 μA) in the presence of (R,S)-DHPG (20 μm). Arrows in inset mark the orthodromic EPSP that preceded the ictal-like event.

Drugs

Bicuculline-methiodide (Bic), picrotoxin (PTX) (from Sigma, St Louis, MO, USA), GABAZINE (GBZ), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), d-aminophosphonovalerate (d-AP5), (S)-4-carboxyphenylglycine (4CPG), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) and (RS)-3,5-dihydroxyphenyglycine ((RS)-3,5-DHPG) (from Tocris Cookson, Inc., Ballwin, MO, USA) were applied by bath perfusion.

RESULTS

Recordings in the CA1 minislice (Fig. 1B) showed spontaneous synchronized bursts (interictal-like events), but also sustained discharges similar to seizure-like (ictal) activity (Fig. 1A). Ictal events were seen in the presence of different antagonists of GABAA receptor function, the competitive antagonists Bic and GBZ, and the chloride channel blocker PTX (Fig. 1C).

Figure 1. Interictal and ictal activity in the CA1 minislice.

Figure 1

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A, concomitant intracellular recordings from pyramidal cells, somatic in stratum pyramidale (top trace) and dendritic in stratum radiatum (bottom trace), in the presence of 100 μm Bic. A partially synchronized interictal burst (left, marked by double-headed arrow) was followed by a long-lasting (about 7 s) ictal-like discharge (right). The interictal event is shown at a faster time scale at the bottom. All action potentials are truncated. B, schematic drawing of the CA1 minislice in which recordings were performed. The CA2/3 region and the subiculum-entorhinal cortex were removed by dissection as indicated by respective gaps. In some minislices (n = 7), the dentate gyrus was removed by a cut along the hippocampal fissure (dashed line). Ictal events were also generated in minislices in which about 20 % of CA1 was cut off, either adjacent to CA2 (n = 6) or adjacent to subiculum (n = 4). Extracellular recordings were carried out in stratum radiatum (2) and intracellular recordings (single or paired) in stratum pyramidale (1) and/or radiatum (2). C, duration of ictal activity in the presence of antagonists of GABAA receptor function: bicuculline-methiodide (Bic, n = 60 slices), picrotoxin (PTX, n = 22) and GABAZINE (GBZ, n = 7 slices). Duration of ictal activity (in s per 60 min) was calculated as the number of ictal events × duration of individual episodes.

Interictal and ictal states

Dependent on the concentration of antagonists of GABAA receptor function, two different states of epileptiform activity were observed. At lower concentrations (Bic, GBZ, 1-10 μm; PTX < 100 μm), interictal activity was prevalent (e.g. Fig. 2A at 5 μm Bic). Interictal-like activity (in some recordings with afterdischarges, e.g. Fig. 7Ab) (Traub et al. 1993) was observed in all 14 recordings carried out at these lower concentrations of GABAA antagonists. This type of spontaneous activity has been shown in many previous studies.

Figure 2. Interictal and ictal states.

Figure 2

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A, plot of the duration of spontaneous field events in individual recording. The duration measurements are illustrated by arrows in top recording traces. Bars mark the duration of Bic application. During controls (in the absence of Bic), no spontaneous field potentials were seen. Following Bic application (5 μm), interictal bursts (about 300-500 ms in duration) were seen (1). Following the application of 100 μm Bic, ictal events lasting about 4-6 s (2) were recorded. B, plot of a recording in which 100 μm Bic was applied 55 min after the start of the recording. After only two interictal events, ictal events appeared. Ictal activity persisted after Bic washout. Dashed lines in A and B mark the transition from interictal to ictal activity. C, plots of the averaged number of interictal and ictal events per 60 min, seen in the presence of low (1-10 μm, n = 14 slices) and high (50-100 μm, n = 60 slices) Bic concentrations.

Figure 7. Mixed interictal and ictal activity in CA1 recorded in the whole slice.

Figure 7

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A, simultaneous intracellular recordings both in stratum pyramidale, 200 μm apart, in the presence of 50 μm Bic (two consecutive traces are depicted). The first and last interictal events are depicted at slower speed in Ba and b, respectively. The two ictal events are marked by *.

However, at higher concentrations of GABAA antagonists (Bic, GBZ ≥ 50 μm; PTX ≥ 100 μm), epileptiform activity progressed to a state in which, almost exclusively, ictal-like events (4-17 s in duration) were observed (Figs 1A, 2A and 3Bc). Figure 2A illustrates the fact that interictal and ictal activity depended on the GABAA antagonist concentration. Interictal episodes appeared after the application of 5 μm Bic. Ictal-like events (between 4 and 6 s in duration) followed the increase of Bic to 100 μm. This recording exemplifies the typical observation that both forms of epileptiform activity were almost exclusively expressed at the respective lower or higher GABAA antagonist concentrations (see also Fig. 2C). When high concentrations of GABAA antagonists (e.g. 50-100 μm Bic) were applied from the beginning, ictal events were observed after only one or two interictal events and remained ictal-like for the remainder of the recording even after washout of Bic (Fig. 2b).

Figure 3. Transition to ictal activity.

Figure 3

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A, loss of AHP and emergence of ADP. Concomitant intracellular and field potential recordings after Bic (100 μm) application. The intracellular interictal event at enlarged voltage and slower time scale (a, bottom trace) shows a small remaining AHP during which a long-lasting ADP arises. b, the second spontaneous event in the same recording demonstrates (i) complete loss of AHP and (ii) growing ADP. B, consecutive episodes (a-c) of spontaneous field activity after PTX application (100 μm). The first episode (a) consisted of a short negative voltage deflection (‘interictal response’), the bottom trace (c) depicts a fully developed ictal event. The middle trace (b) depicts an intermediate stage characterized by the absence of the secondary burst (2) and fewer and less frequent oscillations during the tertiary burst (3).

Characteristics of ictal events

Ictal episodes consisted of three distinct components (‘bursts’; Fig. 3Bc; marked 1, 2 and 3) in agreement with the general model of in vitro ictal events proposed for the CA3 network (Traub et al. 1996). The following three criteria were applied to identify ictal events: (1) expression of all three bursts (which excluded, for example, the event shown in Fig. 3Ab in which the secondary burst was missing); (2) overall duration ≥ 4 s; (3) oscillatory activity during the teritary burst ≥ 3 Hz, at least in the initial stages. The duration criteria of > 4 s (ictal) and < 500 ms (interictal) allowed a clear separation of ictal and interictal activity. Spontaneous events which did not meet all the criteria of an ictal event (see above) and were longer than 500 ms are referred to as ‘intermediate events’ (e.g. events in Fig. 3Ab and also 3Bb). The trace in Fig. 3Bb was similar to ictal events in duration but had fewer afterdischarges (2-10) at lower frequency (0.5-2 Hz).

The primary burst was represented by the short (< 200 ms) high-frequency burst that is the interictal event in the absence of afterdischarges (compare panels Aa and Ab in Fig. 3). In field recordings, the primary burst consisted of a fast negative voltage deflection (time to peak < 50 ms; e.g. Fig. 3Ba). The intracellular correlate consisted of a sudden depolarization shift accompanied by high-frequency burst firing (50-100 Hz; Fig. 1A and Fig. 3Aa). Secondary bursts (labelled 2 in Fig. 3Bc), which were shown to represent a prolongation of the primary burst during sustained depolarization (Traub et al. 1996), lasted about 0.5 s, but due to overlap with tertiary bursts, their exact duration was difficult to determine.

The developing tertiary bursts which resembled the primary burst at a smaller scale (Fig. 3b) evolved through the loss of the burst AHP and, concomitantly, the development of a large afterdepolarization (ADP; Fig. 3A). The ADP envelope carried rhythmic short bursts in synchrony with the negative voltage deflections of the field (Fig. 3Ab) or in synchrony with those of simultaneously recorded cells (Fig. 1A). Discharges of the tertiary burst comprised about 80 % of ictal duration. The overall duration of fully developed ictal events in the disinhibited CA1 minislice ranged from 4 to 17 s (6.5 ± 1.1 s, mean ±s.e.m., n = 1213 episodes in 67 recordings).

Ictal events were followed by long-lasting after-hyperpolarizations (ictal AHP; not shown). The average duration of ictal AHPs was almost 90 times longer (22.5 ± 2.1 s; mean ±s.e.m., n = 362 in 27 slices versus 251 ± 72 ms, n = 318 events in 5 slices), but ictal AHP peak amplitudes (4.1 ± 0.3 mV) were only about half the size of interictal AHPs (8.7 ± 1.1 mV).

Synchronization

The synchronization mechanisms leading to the development of synchronized bursts (interictal activity) were similar to those described in the disinhibited CA3 region (Wong et al. 1986; Miles & Wong, 1987). This is illustrated in Fig. 4 during the washin of GABAA antagonists. Latent recurrent excitatory pathways became functional as revealed by the occurrence of unitary EPSPs in paired intracellular pyramidal cell recordings (Fig. 4A). The criteria of identifying unitary EPSPs were the latency between a presynaptic AP and the onset of an EPSP (between 0.3 and 1.4 ms) and the size of the postsynaptic depolarization (> 0.3 mV) within this latency range (Fig. 4A and B; Miles & Wong, 1986; Deuchars & Thomson, 1996). In the absence of GABAA antagonists, no detectable unitary EPSPs were seen in all 11 concomitant pyramidal-pyramidal cell recordings (e.g. Fig. 4Aa, Con). In contrast, unitary EPSPs were revealed in 3 of these 11 recordings shortly after the application of GABAA antagonists (Fig. 4Ab, Bic). The failure rate was 0.2 to 0.5. Typically, unitary EPSPs were followed by small IPSPs (Fig. 4A). Spontaneous synchronized synaptic potentials, typically large EPSPs followed by IPSPs, were observed soon after (Fig. 4Ca). Burst firing occurred first asynchronously (i.e. in individual cells without a corresponding event in another pyramidal cell, not shown), then in synchrony with population EPSPs (Fig. 4Cb) and then synchronously in many cells as indicated by (a) synchronous bursts in paired recordings (interictal event, Fig. 1A and Fig. 4Cc) or (b) field potentials (e.g. Fig. 3Bc).

Figure 4. Unitary EPSPs and synchronization leading to interictal activity.

Figure 4

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Concomitant recordings in two pyramidal cells. Ab (Bic), unitary EPSPs in cell 1 were seen following single action potentials in the presynaptic cell 10 min after Bic (10 μm) application. Bottom traces show presynaptic action potential and postsynaptic EPSP (marked by arrow) at faster time scale. During control recordings (Con, i.e. before Bic application; a), no EPSPs were detectable in response to action potentials in cell 2. Histograms in B show the number of EPSPs recorded in response to presynaptic action potential as a function of EPSP peak amplitudes (a) and as a function of the latency between the action potential peak (cell 2) and the onset of the EPSP in cell 1 (b). Ca, synchronized EPSP/IPSPs at 15 min of Bic washin; Cb, burst in cell 2 and giant EPSP/IPSP in cell 1 at 17 min of Bic washin. The EPSP in cell 1 preceded the burst; Cc, synchronized bursts (interictal event) in both cells 20 min after Bic application.

Disinhibition as the cause of ictal activity

Ictal activity was observed in 53 of 60 (88 %) recordings in the presence of Bic (50-100 μm), in 10 of 21 (48 %) recordings in the presence of PTX (100-200 μm) and in 4 of 7 (57 %) recordings in the presence of GBZ (50-100 μm). No additional experimental measures to increase excitability were applied: divalent cations ([Ca2+]o= 2 mm, [Mg2+]o= 1.7 mm), recording temperature (30-32 °C) and [K+]o (3 mm) were standard and GABAB-mediated inhibition was intact. The fact that all three GABAA antagonists were able to generate ictal activity provides strong evidence that the seizure-like activity was prompted solely by the blockade of GABAA receptor function in the small (ca 1-2 mm3) CA1 neuronal aggregate.

The clear separation of two states of epileptiform activity (interictal and ictal) as a function of GABAA antagonist concentrations (Fig. 2) indicates that synaptic inhibition was only partially blocked at the lower GABAA antagonist concentrations. This notion was supported by two additional lines of evidence: occurrence of fast IPSPs during the interictal state (Fig. 5) and dose-response curves of inhibition (Fig. 6). Figure 5Aa shows prominent IPSPs in recordings which exhibited synchronized bursts with afterdischarges (i.e. intermediate events). Both poly- and monosynaptic fast IPSPs were seen. Polysynaptic IPSPs (Fig. 4Ca and Fig. 5Ad; Miles & Wong, 1987) can probably be traced to the considerably lower firing threshold of CA1 interneurons (in comparison with their pyramidal cell counterparts) (Karnup & Stelzer, 1999). Monosynaptic IPSPs with faster kinetics (e.g. time to peak < 15 ms) were generated either asynchronously (i.e. in one of two simultaneous recordings, Fig. 5Ac and Bc) or synchronously (in two cells, Fig. 5Bb). Both types of IPSPs were mediated by GABAA receptors as they were blocked by higher concentrations of Bic (not shown). In summary, fast IPSPs were observed during the interictal and intermediate, but not the ictal state.

Figure 5. Fast IPSPs during the interictal state.

Figure 5

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Aa, traces of concomitant intracellular pyramidal cell recordings in the presence of 5 μm Bic. b, synchronized bursts with afterdischarges (box in a) are depicted at a faster time scale. c, single giant IPSP in cell 2; d, synchronized IPSPs following small synchronized EPSPs. B, simultaneous intracellular recordings in a different slice show fast IPSPs (times to peak < 25 ms; marked by boxes) in 5 μm Bic (a). Fast IPSPs were generated synchronously (b) or asynchronously in one cell (c).

Although GABAA-mediated IPSPs were preserved to some extent at the lower GABAA antagonist concentrations, the relation between the occurrence of IPSPs and keeping the network in the interictal state was not clear-cut: predominantly interictal activity was also seen in recordings in which fast IPSPs were abolished (e.g. at borderline concentrations in the presence of 10 μm Bic and in 11 of 21 recordings in the presence of 100-200 μm PTX).

In a series of experiments, postsynaptic GABAA receptor-mediated conductance changes (gGABAA) (Stelzer et al. 1994) were measured during the stimulation-evoked monosynaptic inhibitory response (Fig. 6). Inhibitory conductance changes (‘shunting’) are a separate measure of the GABAA response: in contrast to IPSP/IPSCs, they occur (a) in the immediate vicinity of the GABAA receptor, (b) at VCl (the chloride reversal potential) and (c) are faster than IPSP/IPSCs (Karnup & Stelzer, 1999). During the evoked GABAA response, both the monosynaptic IPSP (Davies et al. 1990) and gGABAA can be measured (see Fig. 6) (Stelzer et al. 1994). Pooled data in Fig. 6D and original recordings in Fig. 6A demonstrate that a remainder of gGABAA was effective in the absence of detectable IPSPs. For example, IPSPs were blocked by 5 μm Bic or 50 μm PTX (Fig. 6b), but the block of gGABAA was only about 75 % at these IPSP blocking concentrations (Fig. 6C). The monosynaptic inhibition protocol did not allow a direct comparison between states of excitability (interictal/ictal state) on the one hand and the status of fast synaptic inhibition on the other since synaptic excitation was blocked. However, by inference, the ictal activity occurred at GABAA antagonist concentrations that blocked gGABAA to > 80 %.

Burst AHPs

During the interictal state in the presence of low GABAA antagonist concentrations, interictal burst AHPs were intact (8.7 ± 1.1 mV peak amplitude; mean ±s.e.m., n = 318 events in 7 intracellular recordings; e.g. Fig. 4Cc; Table 1). During the ictal state (i.e. in the presence of high GABAA antagonist concentrations), burst AHPs of interictal events (i.e. in the one or two interictal events preceding ictal events in the minislice (Fig. 1A and Fig. 3b) and also in the few interictal events between ictal activity) were greatly reduced: AHP peak amplitudes were in all cases < 1.5 mV (0.4 ± 0.2 mV peak amplitude; mean ±s.e.m., n = 35 in 27 intracellular recordings; Fig. 1A and Fig. 3A; P < 0.001; ANOVA).

Table 1.

Status of AH

AHP (mV)
Interictal state
  Recurrent −8.7 ± 1.1 (n = 7)
  Afferent −9.2 ± 1.7 (n = 4)
Ictal state
  Recurrent −0.4 ± 0.2 * (n = 27)
  Afferent −8.1 ± 1.4 (n = 9)
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The status of burst AHP was measured in intracellular recordings as peak amplitude (means ±s.e.m.; n denotes the number of slices). Whole-slice and stimulation experiments were ‘lumped’ together in the respective pools of afferent interictal events.

Role of afferent interictal activity

In whole-slice recordings a mixture of interictal-ictal activity was recorded in CA1 cells during high GABAA antagonist concentrations (Fig. 7). The number of ictal events was reduced by almost 50 % compared with minislice recordings (6.7 ± 1.7 events per 60 min in the whole slice (mean, n = 7)versus 12.2 events in the minislice (n = 53); Fig. 8A). By another measure, the mean interval between ictal events was 5.6 min (n = 53) in the minislice but > 10 min in the whole slice.

It is assumed that the interictal events in whole-slice recordings had propagated from the CA2/3 region into CA1 (Schwartzkroin & Prince, 1978). Were these ‘afferent’ interictal events responsible for the reduced number of ictal events (Jensen & Yaari, 1988; Barbarosie & Avoli, 1997)? In a series of experiments, stimulation was applied during the ictal state to test this hypothesis. The histogram in Fig. 8A illustrates that the frequency of ictal activity was significantly reduced during stratum radiatum stimulation at high intensities to evoke burst responses (‘ortho burst’), but not at low stimulation intensities to generate orthodromic EPSPs (the number of ictal events during Bic alone, i.e. in the absence of stimulation, served as control). Intervals between ictal episodes (5.6 ± 0.8 min during Bic alone; mean ±s.e.m., n = 53) increased to 9.1 ± 0.9 min (n = 14) during evoked bursts (P < 0.05, ANOVA). Stimulation-evoked reduction of ictal activity was not observed after discontinuation of burst stimulation (not shown), which indicates that the effect was transient. The ictal-suppressing effect (in both recording protocols, whole-slice and interictal-like stimulation) was confined to a reduction of the frequency of events as all other characteristics of ictal activity (e.g. shape, depolarization envelope, duration of ictal events) were not affected.

Burst AHPs as ictal-preventing mechanism

Two lines of evidence suggest that the (preserved) burst AHP was a factor in the ictal-suppressing action of afferent input in the disinhibited slice. First, burst AHPs were largely preserved in the two protocols in which a reduction of the ictal frequency was seen: whole-slice recordings (Fig. 7) and during afferent stimulation. This is illustrated in Fig. 8Aa: on average, peak amplitudes were 7.4 ± 0.9 mV in whole-slice recordings (mean ±s.e.m., n = 233 events in 4 intracellular recordings) and 5.9 ± 0.6 mV during burst stimulation (n = 347 events in 5 intracellular recordings). The plot in Fig. 8B illustrates the ictal reduction as a function of the AHP taking into account both the frequency of interictal events and AHP size (through the variable ‘interictal number ×∫AHP(dt)’ as AHP factor).

Second, during the pharmacological stimulation of group I mGluR (by the agonist (RS)-3,5-dihydroxyphenyglycine ((RS)-3,5-DHPG or DHPG; 10-50 μm), afferent interictal events (evoked by burst stimulation or in whole-slice recordings) did not change the frequency of ictal events (Fig. 8Ca). During DHPG application, the burst AHP was eliminated and a burst afterdepolarization (ADP) was revealed instead (Fig. 8Cb) (Greene et al. 1994; Holmes et al. 1996). In another sign of contrasting efficacies of afferent input, orthodromic stimuli in the presence of DHPG, but not in the presence of GABAA antagonists, were able to trigger ictal events (Fig. 8D). Orthodromic stimulation in the presence of GABAA antagonists (at any concentration) could result in interictal-like events, but no stimulation-triggered ictal activity (at any stimulation intensity) was identified during > 40 h of recordings in the disinhibited slice.

Role of GluR

The pharmacological blockade of ionotropic GluR (AMPA or NMDA) prevented all synchronized activity. Any epileptiform activity in the disinhibited minislice, interictal events at lower and ictal events at higher levels of GABAA antagonists, were prevented or reversibly blocked when either CNQX (40 μm, n = 5) or d-AP5 (50 μm, n = 9) were added. This shows that, in agreement with many previous studies, the integrity of ionotropic glutamate receptors was essential for synchronization, but conclusions of a particular role beyond that (e.g. a possible role of NMDA receptors in the expression of afterdischarges or the transition to ictal activity) cannot be drawn from these results.

Group I mGluR was not a factor in the development of disinhibition-induced ictal activity

Several studies have shown a critical role of mGluR in the development of ictal activity (Holmes et al. 1996; Merlin & Wong, 1997; Camón et al. 1998; Wong et al. 1999). The question was asked whether the development of ictal activity during disinhibition depended on activation of group I mGluR. This was not the case. As shown in Fig. 9A, the induction of disinhibition-induced ictal activity was not prevented by the presence of specific group I mGluR antagonists. All parameters of disinhibition-induced ictal events (number per 60 min (Fig. 9Ba), duration of individual episodes (Fig. 9Bb) and the average frequency of oscillatory activity (Fig. 9Bc)) were statistically the same in the absence and presence of group I mGluR antagonists in 18 of 19 recordings (in the presence of 1 mm AIDA (Pellicciari et al. 1995) in 11 of 11; in the presence of 4CPG (0.3-1 mm) in 7 of 8). In addition, when AIDA or 4CPG were applied after the establishment of ictal events (in Bic, GBZ or PTX), no change in frequency of occurrence, duration or shape of ictal activity was observed (not shown).

Figure 9. Bic-induced ictal activity is not blocked by group I mGluR antagonism.

Figure 9

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A, in the presence of the group I mGluR antagonist AIDA (500 μm), the normal development and maintenance of ictal activity was observed. Recordings of the (only) interictal event (1) and of one representative ictal event (2) are depicted at the top. B, pooled data: a, plot of the number of ictal events per 60 min during Bic (50-100 μm) alone (n = 60) and Bic in combination with group I mGluR antagonists AIDA (0.5-1 mm; n = 11) and 4CPG (0.3-1 mm; n = 8). b, the average duration of individual ictal events; and c, the average oscillation frequency of ictal events.

Differential effects of (RS)-3,5-DHPG on ictal activity

Ictal promotion through group I mGluR stimulation

The pharmacological activation of group I mGluR (by DHPG, 10-50 μm) led initially to a mixture of interictal/ ictal events that was replaced by exclusive ictal-like events > 60 min after DHPG application (n = 21) (Fig. 10A). Similar observations have been made in whole-slice experiments (Merlin & Wong, 1997). The consecutive application of ictal-inducing concentrations of GABAA antagonists did not result in noticeable changes of the DHPG-induced activity: ictal events occurred at the same frequency and individual episodes were similar in shape and duration (seen in 5 of 6 recordings).

Figure 10. Differential effects of DHPG on ictal activity.

Figure 10

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A, group I mGluR stimulation: duration of epileptiform responses. (RS)-3,5-DHPG (50 μm) generated mixed interictal/ictal activity in the beginning which was then replaced by exclusive ictal activity. The addition of Bic (100 μm) did not affect the properties of DHPG-induced ictal activity. B, DHPG prevented the development of ictal activity in a group I mGluR-independent manner. Bicuculline (100 μm, Bic), applied after AIDA (1 mm) and (RS)-3,5-DHPG (300 μm) generated non-ictal events (< 1000 ms, 1). After washout of (RS)-3,5-DHPG, ictal events (> 5 s, 2) appeared in concert with interictal events. C, interictal event in the presence of AIDA, DHPG and Bic. D, concentration dependency of anticonvulsant DHPG action. Dose-response curve of ictal activity as a function of [DHPG] (in the presence of fixed amount of AIDA (or 4CPG, 0.3-1 mm) and Bic (50-100 μm).

Ictal prevention/reversal in a group I mGluR-independent fashion

The ictal-promoting action of DHPG (Fig. 10A) was probably due to the activation of group I mGluR (Merlin & Wong 1997; Wong et al. 1999). Data in Figs 10B-D and 11, however, demonstrate the opposite effect by DHPG, i.e. prevention and reversal of disinhibition-induced ictal activity through a pharmacologically different mechanism. Figure 10B shows that Bic - when added in the combined presence of (RS)-3,5-DHPG (300 μm) and the group I mGluR antagonist AIDA - did not result in the normally observed ictal activity: the CA1 network was kept in the ‘interictal state’ as long as DHPG was present. In the combined presence of DHPG (300 μm) and AIDA (1 mm), no spontaneous activity was seen for > 30 min indicating that the mGluR stimulatory action of DHPG was blocked. DHPG's ictal preventive action was a general observation: Bic (100 μm) resulted in interictal and intermediate events (all < 1 s in duration) in 11 of 13 minislice recordings. Ictal activity was observed after washout of DHPG (in 9 of these 11 recordings), regardless of the presence (Fig. 10b) or absence of group I mGluR antagonists at that point.

Figure 11. DHPG reversed ictal activity.

Figure 11

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A, ictal activity was generated by Bic (100 μm Bic). AIDA (1 mm) was present throughout. The addition of (RS)-3,5-DHPG (250 μm) (at t = 90 min) led to a gradual decline of ictal activity until exclusive interictal activity was generated (around t = 135 min). Ba, superimposed original traces of ictal event and interictal event after DHPG was added. b, subtraction of the traces in a illustrates that DHPG reversed the tertiary burst. C, original traces of ictal event before (a) and shortly after DHPG was added (b); c, interictal event > 40 min after DHPG was added.

Two pharmacological properties distinguished DHPG's ictal-preventing action from its stimulation of group I mGluR. First, ictal events were prevented in the presence of group I mGluR antagonists (Fig. 10b). Second, ictal prevention by DHPG required > 100 μm concentrations (Fig. 10D). In contrast, ictal promotion by DHPG was seen at concentrations between 10 and 50 μm (Fig. 10A).

Data in Fig. 11 and Fig. 12D and E illustrate that DHPG not only prevented the induction of Bic-induced ictal activity, but also reversed it. The reversal of ictal activity by DHPG was characterized by a gradual decline of the sustained depolarization during the tertiary burst and, concomitantly, the gradual shortening of the overall duration and the disappearance of afterdischarges (illustrated in Fig. 11Ca-c). In all recordings, the reversal of ictal activity was triggered after adding DHPG. Moreover, AIDA and/or 4CPG were present throughout. Taken together, these data confirm the notion that the block of the anticonvulsant activity was mediated by DHPG in a group I mGluR-independent fashion.

Figure 12. Summary of the ictal development in the disinhibited CA1 region.

Figure 12

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A, interictal event with AHP during the interictal state at low GABAA antagonist concentrations. B, interictal event preceding ictal activity (reduced AHP, emerging ADP). C, intermediate ictal event (blocked AHP, growing ADP). The emergence of the ADP (tertiary burst) was blocked by high DHPG plus mGluR antagonists (Fig. 11). D, full ictal event containing 3 bursts. E, reversal of tertiary block by DHPG (mGluR-independent mechanism).

The comparison of events before and after DHPG (by superimposition, Fig. 11Ba and by isolation of the component that was blocked by DHPG, Fig. 11Bb) indicates that DHPG had selectively blocked the tertiary burst of the ictal event. In 7 of 12 recordings, a near-complete block of the tertiary burst was implemented leaving an intermediate event consisting only of the primary and secondary burst of overall durations < 1 s (as in the recording in Fig. 11Cc). In 4 of 12 recordings, DHPG's block of the depolarization led to a shortening of the ictal event by somewhere between 1 and 4 s, and in only 1 of 12 recordings did DHPG reduce the overall duration to > 4 s (from an average 6.5 s to an average of 4.7 s after steady state). After washout of DHPG, the tertiary burst slowly re-emerged and ictal-like events re-appeared after > 90 min (n = 4, not shown). These data demonstrate that the block of the tertiary burst was reversible and contingent upon the presence of DHPG.

DISCUSSION

Disinhibition as the cause of ictal activity

A main question underlying this study is whether ictal events in the CA1 minislice were indeed caused by the removal of GABAA receptor-mediated inhibition. Numerous previous studies had not reported ictal activity in the slice upon using GABAA antagonists as the sole experimental variable. In addition, recent studies have shown effects of bicuculline-methiodide that were not related to its antagonism of GABAA receptor function, e.g. block of apamin-sensitive, Ca2+-activated K+ channels in several preparations including the hippocampal slice (Seutin & Johnson, 1999). To address these points, different GABAA antagonists, i.e. GBZ as an alternative competitive antagonist and the non-competitive antagonist PTX, were used. Based on the fact that each agent was alone capable of generating ictal activity (in the presence of respective suprathreshold concentrations), it can be concluded that the removal of GABAA-mediated inhibition was sufficient for the generation of ictal events in the CA1 minislice. Notably, the block of the interictal AHPs during the ictal state (Fig. 1A and Fig. 3A) was also observed during PTX- and GBZ-induced ictal activity and thus was related to the block of inhibition. However, it is conceivable that non-disinhibitory actions were responsible for the larger volume of ictal activity during Bic application (in comparison with GBZ and PTX, Fig. 1C).

Several lines of presented evidence, (a) GABAA antagonist concentration dependency of interictal/ictal activity (Fig. 2), (b) occurrence of IPSPs at the lower GABAA antagonist concentrations during the interictal state (Fig. 5), (c) gGABAA blockade at various GABAA antagonist concentrations (Fig. 6), indicate that the implementation of ictal activity required a more efficient level of GABAA antagonism. The dose-response data in Fig. 6C suggest that ictal activity emerged when the block of gGABAA was > 80 %. This could explain the absence of ictal activity in previous hippocampal slice studies in which either submaximal concentrations of competitive GABAA antagonists or non-competitive antagonists (e.g. PTX) were used. PTX was shown to implement only a partial (interictal/ictal borderline) blockade of gGABAA up to 1 mm concentrations, consistent with a non-competitive mechanism of action (Fig. 6).

Interictal activity

The functional significance of interictal activity is controversial: classifications range from ictal-preventing (Engel & Ackermann, 1980; Gotman, 1984; Swartzwelder et al. 1987; Jensen & Yaari, 1988; Barbarosie & Avoli, 1997), to physiologically occurring (Schneiderman, 1986; Schwartzkroin & Haglund, 1986) to ictal-preceding or ictal-promoting (Ralston, 1958; Alger, 1984).

Evidence is provided that afferent interictal events (in whole-slice recordings (Fig. 7) and also during evoked burst responses (Fig. 8A), reduced the frequency of ictal events confirming observations made in several previous studies in in vitro models of epilepsy (Swartzwelder et al. 1987; Jensen & Yaari, 1988) and also in the kindling model of epilepsy (Engel & Ackermann, 1980; Gotman, 1984). The preserved burst AHP (Fig. 8Ab) was probably a main factor since afferent interictal events devoid of the AHP (during the pharmacological activation of group I mGluR; Fig. 8C) lacked ictal suppressing efficacy. The underlying mechanism of the transiently increased ictal threshold can probably be found in AHP-induced ionic imbalances and pump restoration processes (in the duration range of tens of seconds), but the exact mechanism as to how AHP-mediated changes affected ictal generation remains to be established.

Although the interictal-induced reduction of ictal frequency was highly significant (50 % on average in whole-slice recordings), the interictal effects occurred after ictal activity was established and they did not change the overall excitability. Possible long-term interictal effects during the ictal development, i.e. before ictal expression, may be even more important. It was shown that a single burst in CA1 pyramidal cells could generate lasting changes of excitability during theta rhythm background (Huerta & Lisman, 1995). Did the one or two interictal events that preceded ictal activity (Figs 1A, 3Aa and B, 9A and 11A) act as a trigger of ictal activity? The question cannot be answered with certainty, but a strong argument could be found in the fact that in all 67 recordings in which ictal activity was observed, one or two interictal events had occurred just prior to the first ictus. The burst AHP in these ‘interictal’ or ‘pre-ictal’ events was greatly reduced (Figs 1A, 3A and 12b). In stark contrast, burst AHPs were intact when the network remained in the interictal state (Fig. 4Cc and 12A; Table 1). These data demonstrate that interictal activity per se and also strong GluR activation per se (giant EPSPs, frequent asynchronous and synchronous bursts; Fig. 4) were not sufficient to promote ictal development. The higher, ictal-promoting GABAA antagonist concentrations may have revealed additional mechanisms which led to the loss of the burst AHP (Fig. 3A and Fig. 12) (Spencer & Kandel, 1969; Alger, 1984; Schwartzkroin, 1986) and the growth of a sustained ADP (Fig. 3A; see below).

Cellular mechanisms

With regard to the lasting loss of the burst AHP, two main intracellular pathways were identified by previous studies: glutamate was shown to block the AHP via mGluR and possibly PKC (Malenka et al. 1986). Other agents implicated in the AHP loss (histamine, noradrenaline, dopamine and others) may have acted through cAMP accumulation and PKA activation (Haas & Konnerth, 1983).

Besides the AHP loss, the incremental growth of the prolonged ADP underlying the tertiary burst (Fig. 3A) was arguably the main mechanism in the transition to ictal activity. We showed that ictal activity developed in the presence of high concentrations of AIDA/4CPG (Fig. 9). Although AIDA and 4CPG are more specific mGlu1 antagonists, the 1 mm antagonist concentrations, notably 4CPG, also exert significant mGlu5 antagonism. This indicates that group I mGluR activation was not a factor in the development of ictal activity. But, in reverse, was the tertiary burst's block (by DHPG; Fig 10 and 11) mediated by group I mGluR activation? On a functional basis, activation of mGlu1 can be ruled out as it acts as a promoter of ictal activity (Wong et al. 1999). On the other hand, mGlu5 activation was shown to produce long-term depression in the juvenile hippocampus (Fitzjohn et al. 1999), a mechanism that could explain the loss of the tertiary component if it had occurred in the adult brain. This was not the case for two reasons: first, DHPG's anticonvulsant effect was not seen at the low (10-50 μm), mGluR-stimulating concentrations. Second, the DHPG-mediated block of the tertiary burst was implemented in the presence of group I mGluR antagonists (Fig. 9). Taken together, neither the development of the tertiary burst (Fig. 9A) nor its block by DHPG (Fig. 11) involved group I mGluR.

The pharmacological properties of DHPG's block of ictal activity point to a role of phospholipase D (PLD) (Pellegrini-Giampietro et al. 1996) as a signalling mechanism in ictal development. The main argument for this notion is the higher concentration dependency of DHPG's block of PLD: in biochemical experiments, DHPG blocked PLD with an EC50 of 70 μm (Pellegrini-Giampietro et al. 1996) whereas the EC50 to activate group I mGluR is < 10 μm. Our data show that > 100 μm DHPG was required to prevent and reverse ictal activity (Fig. 10D).

DHPG was shown to be a potent, but unspecific, antagonist of PLD activiation by not only blocking the mGluR-mediated PLD activation, but also that of several other potential physiological agonists (such as norepinephrine and cAMP (Albani-Torregrossa et al. 1999)). DHPG's lack of specificity, however, may have been advantageous in revealing its anticonvulsant action since the effects of glutamate alone (i.e. the activation of PLD-linked mGluR) may not have been sufficient in promoting ictal activity (see Fig. 4). Surely, a possible role of PLD activity in the maintenance of ictal activity remains to be established by future experiments. Such a hypothesis would, however, comfortably coexist with previous studies focusing on the essence of PKC activity in ictal generation since PLD-mediated long-term effects are contingent upon the presence of active PKC (Thompson et al. 1991; Pastorino et al. 2000).

Role of impaired GABAA receptor function in epilepsy

Ictal activity in this study was generated within ca 1-2 mm3 hippocampal slice (Fig. 1b) confirming similar observations of a small epileptic focus in other models of epilepsy both in vitro (Traynelis & Dingledine, 1988; Jensen & Yaari, 1988; Traub et al. 1996) and also in vivo (Bragin et al. 1997). The study adds further evidence in support of the notion that blocking fast synaptic inhibition is a sufficient condition for the generation of seizure-like activity. However, despite abundant evidence for disinhibition as an epileptogenic condition, it is unclear whether it is a factor in human temporal lobe epilepsy (Bernard et al. 1999). A main argument against disinhibition as the underlying cause of focal epilepsy is based on the findings that inhibitory parameters were potentiated in models of chronic epilepsy (Prince & Jacobs, 1998; Nusser et al. 1998). Two lines of data of the present study suggest that a measurement of ‘potentiated’ inhibitory responses does not necessarily rule out disinhibition as the causal mechanism of focal activity. First, recordings outside of the potentially miniscule focus will lead to inaccurate accounts of inhibitory efficacy since a potentiation of inhibition is likely to occur in the para-focal, non-epileptic tissue (through enhanced focal efferent activity regardless of the cause of focal activity, including disinhibition). Second, a potentiation of inhibition may occur within the focus through a strengthening of interneuron excitability: giant IPSPs (Fig. 5Ac) and synchronized IPSPs (Fig. 5Bb) represented potentiated inhibitory responses as either form of inhibition was rarely seen during control recordings. ‘Potentiated’ IPSPs during block of GABAA receptor function occur when (1) GABAA blockade is incomplete and (2) interneuron activity is intact or potentiated. These conditions lead to a shift of inhibitory efficacy from the postsynaptic to the presynaptic site through a combination of interneuron-enhancing mechanisms (caused by the block of GABAA receptor function): (a) intrinsic interneuron hyperexcitability, (b) synchronization of the inhibitory network (Schwartzkroin & Haglund, 1986) and (c) an enhanced excitatory drive from principal cells onto interneurons (Domann et al. 1991).

Acknowledgments

This study was supported by a grant from NINDS. We thank R. Bianchi and M. Stewart for critically reading the manuscript.

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