Cyclothiazide induces robust epileptiform activity in rat hippocampal neurons both in vitro and in vivo
1 Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA
2 Lilly Research Centre, Eli Lilly & Co Ltd, Sunninghill Road, Windlesham, Surrey, GU20 6PH, UK
Abstract
Cyclothiazide (CTZ) is a potent blocker of AMPA receptor desensitization. We have recently demonstrated that CTZ also inhibits GABAA receptors. Here we report that CTZ induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo. We first found that chronic treatment of hippocampal cultures with CTZ (5 μM, 48 h) results in epileptiform activity in the majority of neurons (80%). The epileptiform activity lasts more than 48 h after washing off CTZ, suggesting a permanent change of the neural network properties after CTZ treatment. We then demonstrated in in vivo recordings that injection of CTZ (5 μmol in 5 μl) into the lateral ventricles of anaesthetized rats also induces spontaneous epileptiform activity in the hippocampal CA1 region. The epileptogenic effect of CTZ is probably due to its enhancing glutamatergic neurotransmission as shown by increasing the frequency and decay time of mEPSCs, and simultaneously inhibiting GABAergic neurotransmission by reducing the frequency of mIPSCs. Comparing to a well-known epileptogenic agent kainic acid (KA), CTZ affects neuronal activity mainly through modulating synaptic transmission without significant change of the intrinsic membrane excitability. Unlike KA, which induces significant cell death in hippocampal cultures, CTZ treatment does not result in any apparent neuronal death. Therefore, the CTZ-induced epilepsy model may provide a novel research tool to elucidate the molecular and cellular mechanisms of epileptogenesis without any complication from drug-induced cell death. The long-lasting epileptiform activity after CTZ washout may also make it a very useful model in screening antiepileptic drugs.
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Introduction
Cyclothiazide (CTZ) was originally known as a diuretic drug with antihypertensive effects (Julius et al. 1962; Schvartz et al. 1962). It was later found that CTZ is a potent blocker of AMPA receptor desensitization (Patneau et al. 1993; Trussell et al. 1993; Yamada & Tang, 1993; Zorumski et al. 1993; Barnes-Davies & Forsythe, 1995; Mennerick & Zorumski, 1995). CTZ also increases presynaptic glutamate release (Diamond & Jahr, 1995; Bellingham & Walmsley, 1999; Ishikawa & Takahashi, 2001). Our recent work has further demonstrated that CTZ can directly inhibit GABAA receptors as shown by both whole-cell and single-channel experiments (Deng & Chen, 2003). Thus, CTZ has a unique characteristic in acting simultaneously on two prominent synaptic transmission systems: it significantly enhances excitatory glutamatergic neurotransmission while suppressing inhibitory GABAergic neurotransmission. The net effect of CTZ on a neural network will be a significant tilt of the excitation–inhibition balance toward hyperexcitation. We therefore hypothesize that CTZ may work as an epileptogenic agent to induce epileptiform activity in central neurons.
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To test our hypothesis, we treated hippocampal CA1–CA3 cultures with CTZ either in short-term duration but high concentration (1–2 h, 20–50 μM), or chronically with low concentration (2–10 days, 5 μM). In both conditions, CTZ consistently induced robust epileptiform activity in cultured hippocampal neurons. More importantly, the epileptiform activity induced by chronic CTZ treatment lasts more than 48 h after washing off CTZ, suggesting a substantial change of neural network activities after CTZ treatment. To test whether the epileptogenic effect of CTZ is limited to in vitro cultures, we injected CTZ into the lateral ventricles of anaesthetized rats, and found that CTZ can also induce spontaneous epileptiform activity in the hippocampal CA1 region in vivo. The epileptogenic effect of CTZ is attributable to its modulation of glutamatergic and GABAergic neurotransmission without any significant change of the intrinsic membrane properties, because the spiking threshold and action potential firing rate did not change after CTZ treatment. In comparison to KA, which shows strong neurotoxicity, chronic CTZ treatment does not induce any significant cell death. Thus, a CTZ-induced epilepsy model may serve as a useful tool in epilepsy research with minimal side-effects on neuronal survival.
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Methods
Cell culture
Hippocampal cultures were prepared from new born Sprague-Dawley rats (P0–P1) as previously described (Deng & Chen, 2003; Chen et al. 2004). In brief, the hippocampal CA1–CA3 region was dissected out and incubated for 30 min in 0.05% trypsin–EDTA solution. After enzyme treatment, tissue blocks were triturated gently, and dissociated cells were plated onto a monolayer of astrocytes. The culture medium contained 500 ml MEM (Gibco), 5% fetal bovine serum (Hyclone), 10 ml B-27, 100 mg NaHCO3, 20 mMD-glucose, 0.5 mML-glutamine, and 25 u ml–1 penicillin/streptomycin. Cells were maintained in 5% CO2 incubator for 2–3 weeks. The adult rats were killed with CO2,and new-born pups were decapitated in accordance with animal protocols approved by the IACUC committee in Pennsylvania State University.
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Electrophysiology
Whole-cell recordings were performed in current- or voltage-clamp mode using a MultiClamp 700 A amplifier (Axon Instruments). Patch pipettes were pulled from borosilicate glass and fire polished (2–4 M). The recording chamber was continuously perfused with a bath solution consisting of (mM): 128 NaCl, 30 Glucose, 25 Hepes, 5 KCl, 2 CaCl2, 1 MgCl2, pH 7.3 adjusted with NaOH. The pipette solution for most of the experiments, such as recording action potentials, mEPSCs, and glutamate receptor responses, contained (mM): 125 K-gluconate, 10 KCl, 2 EGTA, 10 Hepes, 10 Tris-phosphocreatine, 4 MgATP, 0.5 Na2GTP, pH 7.3 adjusted with KOH. For mIPSCs and GABA-induced currents, pipettes were filled with (mM): 135 KCl, 10 Tris-phosphocreatine, 2 EGTA, 10 Hepes, 4 MgATP, 0.5 Na2GTP, pH 7.3 adjusted with KOH. The series resistance was typically 10–20 M and partially compensated by 30–50%. The membrane potential was held around –70 mV in both voltage-clamp and current-clamp recordings. Data were acquired using pClamp 9 software, sampled at 2–10 kHz, and filtered at 1 kHz. Off-line analysis was done with Clampfit 9 software (Axon Instruments). Miniature events were analysed using MiniAnalysis software (Synaptosoft). All data were expressed as mean ±S.E.M. and Student's t test was used for statistical analysis.
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Large depolarization shift resembling paroxysmal depolarization shift is defined here as 10 mV depolarization and 300 ms in duration. An epileptiform burst is defined by at least five consecutive action potentials overlaying on top of the large depolarization shift. When quantifying the percentage of neurons showing epileptiform activity, the criterion is at least four repeated epileptiform bursts occurring during 30 min of recording. Pyramidal shape neurons were selected for recordings. For each neuron recorded after kainic acid (KA) or CTZ treatment, epileptiform discharges were first verified under current-clamp conditions before recording miniature and whole-cell currents under voltage-clamp conditions.
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In vivo recordings
In vivo experiments were performed using adult male Sprague-Dawley rats (250–350 g body weight) and anaesthetized with urethane (1.2 g kg–1) under the Animals (Scientific Procedures) Act 1986 and approved by the local ethics committee. At the end of experiments, animals were killed with an overdose of urethane.
The femoral artery was cannulated to allow arterial blood pressure to be monitored. Animals were then mounted in a stereotaxic frame. A drill hole was made on the skull above the left side of the lateral ventricle (0.3 mm posterior to bregma, 1.3 mm lateral to the midline). A guide cannula was then placed 4 mm below the skull surface for drug delivery, and secured by the dental cement. For recording and stimulating, a large burr hole was made in the left side of the incised skull above the hippocampal area, and the dura was pierced and removed. For recording in the CA1 pyramidal cell layer, a tungsten electrode (0.5 M) was placed 3.5–4.2 mm posterior to bregma, 2.0–3.0 mm lateral to the midline. The depth of the recording electrode was approximately 2.0–2.5 mm below the brain surface as determined by the sudden change of electrical noise and the shape of the evoked field excitatory postsynaptic potential (fEPSPs) and population spike. A bipolar stimulating electrode was placed close to the CA3 region in order to stimulate the Shaffer collateral (3.8–4.5 mm posterior to bregma, 3.5–4.0 mm lateral to the midline, and 3.0–3.8 mm below the brain surface). Once both electrodes were in the right place, the fEPSPs and population spike (PS) were monitored for at least 30 min until a stable recording was achieved. The stimulation frequency was set at once per minute with single biphasic square-wave pulses of 0.2 ms duration and 700–900 μA (supramaximal, determined by input–output curve). In between stimulations, the baseline activity was recorded for evidence of spontaneous activity. Following a 30 min recorded baseline of all responses, drugs or vehicles were administered I.C.V. (intracerebral ventricle) at volume of 5 μl via the pre-implanted guide cannula into the lateral ventricle. Pharmacologically induced seizure-like activity was monitored for 3 h after CTZ injection, by observing the change of the evoked potential (as the single PS transforms into a multipeaked display) and the spontaneous seizure burst activity of CA1 pyramidal neurons (Wheal et al. 1998). The anaesthetic level was monitored and maintained throughout the course of the experiment, in particular after convulsant drug administration. On some occasions, the brain was taken for histological validation of the injection and recording/stimulating sites.
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Cell viability assay
A live/dead viability kit (Molecular Probes, Eugene, OR, USA) containing ethidium homodimer-1 and calcein-AM was used to examine cell viability. Ethidium homodimer-1 binds to cellular DNA in membrane-compromised cells and yields a strong red fluorescence in dead cells, but is unable to penetrate the intact plasma membrane of live cells. Calcein-AM is a membrane-permeable dye which can be cleaved by esterases in live cells and produces a uniform cytoplasmic green fluorescence. After drug treatment, 16–23 days in vitro (DIV) neurons were incubated in bath solution containing 1 μM calcein-AM and 4 μM ethidium homodimer-1 for 20 min at room temperature. Cell death rate was then measured by determining the percentage of ethidium homodimer-1-positive cells to total cell number (ethidium homodimer-1 and calcein-AM-positive cells). For each experimental treatment, at least seven fields of each coverslip were imaged, and cell death rate was averaged over a total of three different batches of experiments.
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Drugs
Cyclothiazide, kainic acid, bicuculline, and CNQX were purchased from Tocris. TTX was obtained from Sigma. All of the drugs were freshly diluted in bath solution to their final concentrations before experiments. Bath application of drugs was rapidly delivered through a micropipette (400–500 μm tip) positioned about 1000 μm away from recorded neurons at a 45 degree angle. The micropipette was connected to a Warner (Hamden, CT, USA) VC-6 drug delivery system.
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Results
Chronic treatment of hippocampal cultures with CTZ elicits robust epileptiform activity
We recently discovered that CTZ inhibits GABAergic neurotransmission in addition to its effect on glutamatergic neurotransmission (Patneau et al. 1993; Trussell et al. 1993; Yamada & Tang, 1993; Zorumski et al. 1993; Deng & Chen, 2003). The dual effects of CTZ led us to predict that CTZ will drive neural networks toward hyperexcitability. To test whether CTZ is capable of eliciting epileptiform activity in hippocampal neurons, we applied CTZ (5 μM) in the culture medium for 48 h. After CTZ treatment, neurons were transferred into a recording chamber and constantly perfused with normal bath solution without CTZ. For control, the same volume of vehicle (culture medium containing 0.01% DMSO) was added. We found that most of the control neurons showed only synaptic potentials and individual action potentials but no epileptiform bursts (Fig. 1A). In contrast, the majority of CTZ-treated neurons showed abnormally synchronized bursting activities, with high-frequency action potentials overlaying large depolarizing shifts (>10 mV) (Fig. 1B). These recurrent bursting activities are reminiscent of epileptiform activity in vivo (Prince & Connors, 1986).
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Aa and b, a representative current-clamp recording showing that chronic treatment with vehicle (0.01% DMSO) did not induce any epileptiform activity in cultured hippocampal neurons. Most of the action potentials are clearly separated, as shown in the expanded trace (Ab). Ba and b, a typical recording showing recurrent epileptiform bursts after chronic pretreatment with CTZ (5 μM, 48 h) in a pyramidal neuron. One of the epileptiform bursts in Ba was expanded in Bb, showing a train of action potentials overlaying a large depolarization shift. Ca and b, representative epileptiform burst activities induced by pretreatment (5 μM, 48 h) with kainic acid (KA), with an expanded trace shown in Cb. D, bar graphs showing the percentage of neurons manifesting epileptiform activity after pretreatment with CTZ (84.6%) and KA (60.9%). E, bar graphs showing the frequency of epileptiform bursts after pretreatment with CTZ (0.11 ± 0.01 Hz, n= 33) and KA (0.044 ± 0.012 Hz, n= 14; P < 0.001, Student's t test).
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We compared the ability of CTZ to induce epileptiform activity to previously established KA epilepsy model (Sarkisian, 2001; Omrani et al. 2003; Bausch & McNamara, 2004; Buckmaster, 2004). The same protocol (5 μM, 48 h) was used for chronic KA treatment. Similar to CTZ treatment, typical epileptiform bursts were also observed after KA treatment (Fig. 1C). Quantitative analysis showed a higher percentage of neurons with epileptiform activity (84.6%, n= 33 out of 39; Fig. 1D) and higher burst frequency (0.11 ± 0.01 Hz, n= 33; Fig. 1E) after CTZ treatment, compared to KA treatment (60.9%, n= 14 out of 23; burst frequency, 0.044 ± 0.012 Hz, n= 14, P < 0.001).
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To test how long the epileptiform activity may last after washout of CTZ, we transferred CTZ-pretreated coverslips into normal culture medium without CTZ for 24–48 h before performing patch-clamp recordings. Interestingly, the epileptiform activity persisted long after washout of CTZ. Figure 2 shows representative recordings with typical epileptiform activities after washout of CTZ for 24 h (Fig. 2A) or 48 h (Fig. 2B). Figure 2C shows the summarized data. Comparing with the control (14.7%, n= 34), the percentage of neurons showing epileptiform activity was 84.6% within 3 h after CTZ washout (n= 39), and remained 77.8% and 66.7% after washout of CTZ for 24 h or 48 h, respectively. These results suggest that CTZ-induced epileptiform activity is a long-term change of the neural network properties, which has become independent of CTZ itself.
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A, epileptiform activity recorded from a neuron pretreated with CTZ (5 μM, 48 h) and then transferred into CTZ-free culture medium for another 24 h. B, a representative recording showing persistent epileptiform activities even 48 h after transferring CTZ-pretreated coverslips into CTZ-free culture medium. C, summarized data showing the percentage of neurons with epileptiform activity after washout of CTZ. Compared with control (14.7%), the percentage of neurons showing epileptiform activity within 3 h, or after 24 h and 48 h washout of CTZ is 84.6%, 77.8% and 66.7%, respectively.
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Besides the chronic treatment with CTZ (5 μM, 48 h), we also tested whether short-term treatment with CTZ will induce epileptiform activity in hippocampal cultures. We found that a low concentration of CTZ (5 μM) was unable to induce epileptiform activity in most of the neurons within 2 h of treatment (Fig. 3). However, a high concentration of CTZ (20–50 μM) could effectively elicit epileptiform activities after a short-term treatment. Figure 3A shows typical epileptiform activities induced by 50 μM (1–2 h) and 20 μM (2 h) CTZ treatment. It is important to note that similar to chronic experiments, all recordings after short-term CTZ treatment were made in normal bath solution without CTZ. Bar graphs in Fig. 3B show the percentage of neurons with epileptiform activity after different CTZ-treatment conditions. Notice that 2 h treatment with 50 μM CTZ induced epileptiform activity in every neuron tested (100%; n= 13), indicating that CTZ is a powerful epileptogenic agent. In addition, we also observed epileptiform activities in hippocampal neurons after long-term (10 days) treatment with 5 μM CTZ (Fig. 3).
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A, typical epileptiform activities induced by short-term treatment with high concentration of CTZ (20–50 μM, 1–2 h), or by long-term treatment with low concentration of CTZ (5 μM, 10 days). Note that 2 h treatment with 5 μM CTZ (top trace) did not induce typical epileptiform activity in most neurons. B, bar graphs showing the percentage of neurons with epileptiform activity under different CTZ-treatment conditions. Note that 50 μM CTZ treatment for 2 h induced epileptiform activity in every neuron tested (n= 13).
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Together, these experiments demonstrate that CTZ-induced epileptiform activity, once elicited, will become the hallmark of neural network properties irrespective of the presence or absence of CTZ itself. Thus, the CTZ-induced in vitro epilepsy model will be useful in studying the long-term changes of neural networks after epileptogenesis.
CTZ induces spontaneous seizure activity in in vivo recordings
Can CTZ induce epileptiform activity in in vivo conditions We recorded neuronal activity in the hippocampal CA1 pyramidal layer from 13 urethane-anaesthetized rats. In all 13 rats studied, the evoked responses following stimulation of CA3 regions and/or Shaffer collaterals consisted of a large EPSP and a single PS during control recordings (Fig. 4Aa), and the baseline activity was virtually ‘silent’ (Fig. 4Ba). Following intracerebroventricular (I.C.V.) injection of CTZ (5 μmol in 5 μl), the single-peaked PS gradually transformed into a multiple-peaked event in all 10 rats tested. Figure 4Ab and Ac shows typical characteristic epileptiform activity in in vivo recordings (Wheal et al. 1998). The onset latency for the second peak appearance was 22 ± 3 min (ranging from 6 to 37 min). In nine out of these ten rats, more than two peaks occurred after stimulation. In eight out of the ten rats, spontaneous recurrent epileptiform bursts were observed after CTZ injection (Fig. 4Bb). The onset latency for spontaneous epileptiform bursts was 97 ± 17 min (ranging from 40 to 177 min). In two rats, epileptiform bursting activities did not occur. Over a 30 min analysis period, the mean epileptiform burst number was 7 ± 1 (ranging from 4 to 11). DMSO control experiments were performed in three rats. None of the control rats developed either multiple-peaked PS or spontaneous epileptiform bursting activities in the 3 h observation period after DMSO injection (5 μl, I.C.V.; data not shown).
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A, typical recordings showing that the evoked population spikes recorded from the CA1 pyramidal layer in urethane-anaesthetized rats transform from single peak at control (a) to multiple peaks (b and c, indicated by arrows) after CTZ injection (5 μmol, 5 μl, I.C.V.) ( indicates the stimulus artefact). B, a representative recording showing that spontaneous epileptiform bursts appeared 2 h after CTZ injection. In control conditions (a), there were only small baseline activities in CA1 pyramidal cells. Black dots indicate stimulus artefacts, the same as shown in Aa. After CTZ administration (b), large synchronized bursting activities appeared, with each large burst consisting of many smaller bursts of activities.
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Acute and chronic CTZ effects on the kinetics of mEPSCs and mIPSCs in hippocampal cultures
It is well-known that CTZ enhances glutamatergic neurotransmission by blocking the desensitization of AMPA receptors (Barnes-Davies & Forsythe, 1995; Diamond & Jahr, 1995; Mennerick & Zorumski, 1995; Bellingham & Walmsley, 1999; Ishikawa & Takahashi, 2001). It is unclear, however, whether the effects of CTZ on mEPSC kinetics are long-lasting and contribute to the prolonged excitability in the absence of CTZ. Moreover, the effects of acute and chronic CTZ on GABAergic neurotransmission and their contribution to CTZ-induced epileptiform activity are not well-understood. To investigate synaptic changes underlying CTZ induction of epileptiform activity, we compared acute and chronic CTZ effects on the kinetics of both mEPSCs and mIPSCs in cultured hippocampal neurons. The spontaneous mEPSCs were recorded in the presence of TTX (0.5 μM) and a specific GABAA receptor antagonist bicuculline (BIC, 20 μM). As illustrated in Fig. 5, the frequency of mEPSCs showed a significant increase after acute application (Fig. 5B) or chronic pretreatment (Fig. 5C) with CTZ (5 μM for both treatments). The decay time of averaged mEPSCs (bottom trace of Fig. 5A–C) also increased but only during acute application of CTZ, not after chronic pretreatment with CTZ. The quantified data indicated that the mEPSC frequency increased to 147 ± 11% during acute application of 5 μM CTZ (n= 20, P < 0.02) and to 193 ± 35% after chronic CTZ-treatment (n= 16, P < 0.05) (Fig. 5D). The mEPSC amplitude was not significantly changed after acute (107 ± 6%, n= 20; P > 0.3) or chronic (119 ± 12%, n= 16; P > 0.05) treatment with 5 μM CTZ (Fig. 5E). In addition, we analysed the kinetics of mEPSCs and found that the decay time constant was greatly increased during acute application of 5 μM CTZ (156 ± 12%, n= 10, P < 0.003), but there was no significant change after chronic pretreatment with CTZ (91 ± 10%, n= 14, P > 0.5) compared with control (Fig. 5F). Therefore, the CTZ effect on mEPSC kinetics is transient, only occurring during application of CTZ. However, the CTZ effect on presynaptic glutamate release as reflected by a significant increase of mEPSC frequency is a persistent change of neural network activity long after washout of CTZ.
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A–C, typical mEPSC traces recorded in the presence of TTX (0.5 μM) and BIC (20 μM) in control (A), acute application (B), and after chronic pretreatment (C) with CTZ (both 5 μM). Insets at the bottom of each panel are the averaged mEPSCs, showing a slowed decay phase after acute application of CTZ (B) but not chronic CTZ-pretreatment (C, after washout of CTZ). D, bar graphs showing that the normalized mEPSC frequency was significantly increased to 147 ± 11% during acute CTZ application (P < 0.02), and to 193 ± 35% after chronic CTZ pretreatment (P < 0.05). E, bar graphs showing that the normalized mEPSC amplitude was not significantly changed during acute application (107 ± 6%, P > 0.3) or after chronic pretreatment with CTZ (119 ± 12%, P > 0.05). F, kinetics analysis showing a significant increase in the normalized decay time constant during acute application of 5 μM CTZ (156 ± 12%, P < 0.003), but not after chronic pretreatment with CTZ (91 ± 10%, P > 0.5).
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We next examined the CTZ effect on mIPSCs. The spontaneous mIPSCs were recorded in the presence of TTX (0.5 μM) and the AMPA/kainate receptor antagonist CNQX (20 μM). As illustrated in Fig. 6, not only acute CTZ application (Fig. 6B) but also chronic CTZ (both 5 μM) treatment (Fig. 6C) significantly decreased the frequency of mIPSCs. Quantitative analysis showed that the mIPSC frequency decreased to 72 ± 5% (P < 0.0005, n= 15) during acute CTZ application, and to 50 ± 6% (P < 0.0002, n= 15) of the control after chronic CTZ treatment, respectively. However, the amplitude and the decay time constants (1 and 2) of mIPSCs did not change significantly during acute CTZ application and after chronic CTZ treatment at low concentration (5 μM) (Fig. 6E–G). At high concentration (50–100 μM), acute CTZ application decreased the amplitude of mIPSCs (data not shown, also see Deng & Chen, 2003).
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A–C, consecutive traces illustrating mIPSCs recorded in the presence of TTX (0.5 μM) and CNQX (20 μM) in control (A), during acute application (B), and after chronic pretreatment with CTZ (both 5 μM) (C). The mIPSC frequency showed an obvious decrease after both acute and chronic CTZ treatment. Insets at the bottom of each panel are the averaged mIPSCs, showing no apparent changes in the amplitude and kinetics after CTZ treatment. D, the normalized mIPSC frequency was significantly decreased to 72 ± 5% during acute application of CTZ (P < 0.0005) and to 50 ± 6% of the control after chronic CTZ pretreatment (P < 0.0002), respectively. E, there was no significant change in the normalized mIPSC amplitude after acute CTZ application (103 ± 4%, P > 0.5) or chronic CTZ pretreatment (92 ± 10%, P > 0.5). F and G, kinetics analysis showing no significant changes in the normalized decay time constants, 1 (F, P > 0.19 for acute and P > 0.1 for chronic CTZ treatment) and 2 (G, P > 0.86 for acute and P > 0.26 for chronic CTZ treatment), during acute application or after chronic CTZ-pretreatment.
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Taken together, acute CTZ application substantially enhances glutamatergic neurotransmission by increasing the frequency and decay time of mEPSCs, and meanwhile exerts a mild effect on GABAergic neurotransmission by decreasing the frequency of mIPSCs. After chronic CTZ pretreatment, the effects of CTZ on the frequency of mEPSCs and mIPSCs continued in the absence of CTZ, suggesting a long-lasting modulation of synaptic networks by chronic CTZ-treatment.
CTZ differs from KA in affecting neuronal excitability
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We next examined the effects of CTZ on neural network activity and intrinsic excitability and compared them with the effects of KA. We found that the frequency of spontaneous action potentials, a general index for neural network activity, was significantly increased in the presence of CTZ (Fig. 7A, n= 18). Bath application of 5 μM CTZ (upper trace) or 20 μM CTZ (lower trace) both dramatically increased the action potential firing. Note that the increase of action potentials by acute CTZ application was reversible, unlike the effect of long-term CTZ treatment (compare Fig. 7 with Figs 1–3). As expected, 20 μM CTZ has a stronger stimulatory effect than that induced by 5 μM CTZ, with the former showing higher firing frequency and longer persistency after washing off CTZ.
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A, bath application of 5 μM (upper trace) and 20 μM CTZ (lower trace) increased the firing frequency of spontaneous action potentials. As expected, 20 μM CTZ has a stronger stimulatory effect on neuronal firing. B, bath application of 5 μM KA (upper trace) also increased the frequency of action potentials, but was accompanied with an obvious membrane depolarization. After application of 20 μM KA (lower trace), a strong membrane depolarization resulted in inactivation of sodium channels and suppression of action potentials. A and B are from the same neuron. C, in the presence of TTX, spontaneous and evoked action potentials were totally blocked. CTZ (5 μM and 20 μM) application did not induce any membrane depolarization (upper traces), whereas KA (5 μM and 20 μM) application still induced significant membrane depolarization (lower traces). All recordings in C are from the same neuron.
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We compared the effects of CTZ versus KA on the spontaneous action potential firing at the same neuron (Fig. 7B). Bath application of 5 μM KA (upper trace, n= 8) significantly increased the frequency of action potentials which was also accompanied with an obvious membrane depolarization. During application of 20 μM KA in the same neuron, the membrane depolarization became so strong that it essentially inactivated sodium channels and suppressed action potentials (Fig. 7B, lower trace; n= 8). The difference between CTZ and KA on membrane depolarization can be best seen in the presence of TTX (0.5 μM) (Fig. 7C). After blocking action potentials, CTZ application (5–20 μM) did not induce any membrane potential changes (Fig. 7C, upper traces, n= 8), suggesting that CTZ itself does not activate membrane receptors or ionic channels. In contrast, KA application (5–20 μM) in the same neurons induced significant membrane depolarization (Fig. 7C; lower traces, n= 8), probably due to its direct activation of KA/AMPA receptors on the membrane. Thus, the cellular mechanism underlying the CTZ-induced epilepsy model is clearly different from that of the KA-induced epilepsy model.
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In order to clarify whether CTZ affects the neuronal intrinsic excitability, we used CdCl2 (300 μM) to eliminate network recurrent activities and examined the effect of CTZ on spiking threshold and neuronal intrinsic firing rate through injection of depolarizing currents. Figure 8A shows representative traces of current injection-induced action potentials in control (left), during acute CTZ application (middle), and after chronic CTZ pretreatment (right). The number of action potentials during 300 ms of current injection increased with the increase of depolarizing currents (Fig. 8A and C). The action potential threshold, shown in Fig. 8B, did not change significantly during acute application of CTZ (–37.4 ± 2.0 mV, n= 6, P > 0.17) and after chronic pretreatment with CTZ (–38.0 ± 3.8 mV, n= 6, P > 0.49), compared to the control (–35.1 ± 1.8 mV, n= 7). The membrane excitability plots in Fig. 8C show a linear correlation between the number of action potentials and the stimulus intensity (r > 0.9 in all groups). The slopes of each fitted line were very close to each other in control and CTZ-treatments (the slope is 0.036 in control; 0.034 during acute CTZ application; and 0.036 in chronic CTZ-treatment). These results indicate that CTZ does not affect neuronal intrinsic excitability.
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A, representative current-clamp recordings in control (left panel), during acute application of 50 μM CTZ (middle panel), and after pretreatment with 5 μM CTZ for 2 days (right panel). Each panel shows the number of action potentials during 300 ms of a series of current injections. The bath solution contained 300 μM CdCl2 to inhibit recurrent activity. Membrane potential was held at –70 mV, with 50 pA of stimulus increment during eight consecutive sweeps. B, bar graphs showing no significant change in the spiking threshold during acute application and after chronic pretreatment with CTZ. C, neuronal intrinsic excitability as determined from plots of the number of action potentials versus the stimulus intensity.
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CTZ-induced calcium influx is activity-dependent
The calcium signal has been implicated in epileptogenesis (Meyer, 1989; Sun et al. 2002). We next compared the effects of CTZ and KA on intracellular calcium changes. We employed Fura-2 ratio imaging to monitor the calcium transients after bath application of CTZ or KA (van den Pol et al. 1996). After loading with Fura-2, hippocampal neurons were challenged with CTZ (5–20 μM), KA (5–20 μM), and high concentration of potassium solution (Fig. 9). The 90 mM KCl stimulation was for positive control to make sure neurons were healthy and responsive. In control neurons, there were often some spontaneous Ca2+ spikes which are probably induced by spontaneous action potentials (Fig. 9A). After bath application of CTZ (upper traces) or KA (lower traces), the intracellular calcium concentration significantly increased, with higher concentration (20 μM) of CTZ and KA inducing stronger calcium influx (n= 97 in CTZ; n= 53 in KA). To suppress the complication from spontaneous action potential firing, we compared the effect of CTZ and KA in inducing calcium influx in the presence of TTX (Fig. 9B). Interestingly, no Ca2+ increase was induced by bath application of CTZ after blocking action potentials, suggesting that CTZ does not have any direct effect on calcium channel activation, and that the Ca2+ increase in the resting condition is dependent on neural network activities (Fig. 9B, upper trace; n= 47). In contrast, bath application of KA in the presence of TTX still effectively elicited calcium responses, especially at a concentration of 20 μM (Fig. 9B, lower trace; n= 82). These results suggest that CTZ-induced calcium influx is action potential dependent, while KA-induced calcium influx can be both action potential dependent and independent, with the latter resulting from direct activation of calcium channels by KA-induced membrane depolarization.
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A, bath application of CTZ (upper traces) and KA (lower traces) increased intracellular calcium concentration [Ca2+]i in normal bath solution; 90 mM KCl stimulation serves as positive control for neuronal calcium response. B, in the presence of TTX (0.5 μM), bath application of CTZ (upper traces) did not increase the [Ca2+]i, whereas KA still effectively induced calcium responses.
CTZ does not induce neurotoxicity in hippocampal cultures
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KA is known to be associated with serious neurotoxicity (Pollard et al. 1994; Sattler & Tymianski, 2001; Holopainen et al. 2004). In the KA-induced epilepsy model, it is difficult to determine whether the neuronal death is caused by a direct effect of KA or secondary to the KA-induced epileptiform activity. It is therefore important to examine whether chronic CTZ treatment will result in any neuronal death. We performed a series of cell viability assay using a cell death kit (LIVE/DEAD viability kit, Molecular Probes, Eugene, OR, USA). Live cells were stained with bath solution containing 1 μM calcein-AM (green) and 4 μM ethidium homodimer-1 (red) for 20 min at room temperature. Dead cells will allow ethidium homodimer-1 to permeate the plasma membranes and bind to the DNA to emit red fluorescence in the nucleus. For a clear view of live versus dead neurons, we overlaid phase images with ethidium homodimer-1-stained red fluorescent images under various conditions (Fig. 10). After chronic treatment with CTZ (5 or 20 μM, 48 h), most neurons looked healthy in morphology and were not stained by ethidium homodimer-1 (Fig. 10B–C). In contrast, after exposure to 5 or 20 μM KA for 24–48 h, neuronal death was apparent especially at higher concentration (20 μM) where most neurons were dead as stained by ethidium homodimer-1 (Fig. 10D and E). Quantitative data analysis showed that CTZ did not induce any significant increase of cell death compared to the control group (control, 3.8 ± 0.5%; 5 μM CTZ, 6.1 ± 1.3%, P > 0.07; 20 μM CTZ, 4.9 ± 1.3%, P > 0.3). However, chronic treatment with 5 μM KA (48 h) increased the neuronal death rate to 24.2 ± 4.9% (P < 0.002), and treatment with higher concentration of KA (20 μM, 24 h) resulted in an even more strikingly high death rate of 81.8 ± 6.6% (P < 0.0001) (Fig. 10F). These experiments indicate that unlike KA, which induces significant neuronal death, CTZ treatment is not associated with direct neurotoxicity. Thus, studying the cellular mechanisms of CTZ-induced epileptogenesis will not have any complications resulting from drug-induced cell death.
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A, phase images of cultured hippocampal neurons in control group. B and C, phase images of neurons treated with 5 μM CTZ for 48 h (B) or 20 μM CTZ for 24 h (C). There was no apparent cell death after CTZ treatment. D and E, overlaying images showing ethidium homodimer-1 staining of neurons treated by 5 μM KA for 48 h (D) or 20 μM KA for 24 h (E). Nuclei of dead cells exhibit red fluorescent signal. F, quantification of cell viability assay. CTZ has minimal effect on neuronal death, but both KA treatments (5 μM and 20 μM) significantly increased neuronal cell death rate (P < 0.001 for 5 μM and P < 0.0001 for 20 μM CTZ, respectively).
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Discussion
We have demonstrated here that CTZ is a novel epileptogenic agent which induces robust epileptiform activity in both hippocampal cultures and in vivo hippocampal recordings. The CTZ-induced epileptiform activity is a long-lasting phenomenon, permitting extensive studies of the molecular and cellular mechanisms of epileptogenesis. The capability of CTZ in inducing epileptiform activity relies upon its modulating synaptic neurotransmission without a direct effect on the intrinsic membrane excitability. Compared with KA, which directly activates KA and AMPA receptors and triggers neuronal cell death, CTZ acts as a neuromodulator of AMPA and GABAA receptors and is not associated with significant cell death. CTZ can induce epileptiform activity after both short-term and long-term treatment. Thus, the CTZ-induced epilepsy model is an important new addition to the current epilepsy models, offering great flexibility and low cytotoxicity for epilepsy research.
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CTZ has been known for more than a decade for its ability to block glutamate AMPA receptor desensitization and increase glutamate release (Partin et al. 1993; Trussell et al. 1993; Yamada & Tang, 1993; Zorumski et al. 1993; Diamond & Jahr, 1995; Bellingham & Walmsley, 1999; Ishikawa & Takahashi, 2001). Our recent work revealed that CTZ also acts as an inhibitor of GABAA receptors (Deng & Chen, 2003). The current in vitro as well as in vivo studies further indicate that CTZ can induce robust epileptiform activity in hippocampal neurons by shifting the excitation–inhibition balance toward hyperexcitation. Importantly, the epileptiform activity induced by CTZ is not a transient change of neural network activity, but rather it sustained for more than 48 h after washing off CTZ (Fig. 2), suggesting a permanent alteration of the functional output of neural networks after CTZ treatment. The continuous firing of epileptiform activity long after CTZ washout provides an ample time window for a thorough investigation of the network properties after epileptogenesis. It will also be a useful model to screen new antiepileptic drugs by testing which chemical compound(s) is capable of extinguishing the long-lasting epileptiform activity after CTZ-treatment.
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In addition to in vitro studies, we have also demonstrated that CTZ induces spontaneous epileptiform activity in in vivo recordings in the hippocampal CA1 region. We are currently engaged in experiments aiming to establish the in vivo CTZ epilepsy model. A recent work reported that microinjection of CTZ locally into the area tempestas in the anterior piriform cortex did not induce seizures (Fornai et al. 2005). This appeared to conflict with our in vivo studies. However, the injection site and the amount of CTZ used were drastically different between their studies and ours. Fornai et al. (2005) used 1.2 nmol in 200 nl CTZ for focal cortex injection, whereas we used 5 μmol in 5 μl CTZ for lateral ventricle injection. Thus, the CTZ concentration used by Fornai et al. (2005) may be lower than what we have used in our in vivo recordings. We will test whether the high-dose injection of CTZ into the lateral ventricles will induce behavioural seizures.
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The physiological relevance of the current in vitro CTZ epilepsy model is that a mild modulation of glutamatergic and GABAergic neurotransmission will lead to irreversible changes of the whole neural network. We have demonstrated that acute application of CTZ at a relatively low concentration (5 μM) increases presynaptic glutamate release but decreases presynaptic GABA release (Figs 5 and 6). Together with prolonging glutamate responses, acute application of CTZ results in a reversible increase of neuronal firing of action potentials (Fig. 7). However, after chronic CTZ treatment (5 μM, 48 h), neuronal activity will be transformed from single individual firing into hypersynchronized bursting activity. What underlies such activity transformation is clearly a physiological question and may shed new light on the mechanism of activity-dependent neural plasticity. Interestingly, raising the concentration of CTZ (20–50 μM) greatly shortens the time required for the onset of epileptiform activity (Fig. 3), suggesting that the degree of perturbation of glutamatergic and GABAergic neurotransmission is positively correlated with the speed of transforming individual firing into epileptiform bursting activity. The short latency of the onset of epileptiform activity after treatment with a high concentration of CTZ (20–50 μM) in cell cultures (1–2 h) is consistent with the onset latency for spontaneous epileptiform activity in in vivo recordings after I.C.V. injection of CTZ (40–177 min), suggesting a common mechanism and similar time course operating under both in vitro and in vivo conditions.
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Compared to the current, widely used, epilepsy models including KA, PTZ, pilocarpine, and kindling models, the mechanism of convulsant action of CTZ is clearly different. KA directly activates KA and AMPA receptors (Castillo et al. 1997; Wilding & Huettner, 1997). PTZ is an antagonist of GABAA receptors (Suzuki et al. 1999). Pilocarpine acts on cholinergic receptors (Honchar et al. 1983) and the kindling model relies upon high-frequency stimulation to directly excite neurons (Lothman & Williamson, 1993; Morimoto et al. 2004). CTZ acts as a neuromodulator which enhances glutamatergic but inhibits GABAergic neurotransmission (Trussell et al. 1993; Barnes-Davies & Forsythe, 1995; Diamond & Jahr, 1995; Mennerick & Zorumski, 1995; Deng & Chen, 2003; and present study). We have carefully compared the effect of CTZ with that of KA on membrane potential, calcium influx, and neuronal survival. We found that acute application of CTZ significantly increased spontaneous firing of action potentials, but did not induce membrane depolarization in the presence of TTX (Fig. 7). In contrast, KA depolarizes the membrane potential significantly in the presence of TTX, as expected. Likewise, CTZ does not induce calcium influx in the presence of TTX, but KA does induce significant calcium influx especially at a concentration of 20 μM (Fig. 10). The direct membrane depolarization and calcium influx induced by KA are probably related to the cell death after KA treatment (Fig. 10 of this study; Vornov et al. 1991; Holopainen et al. 2004). In contrast, our cell viability assay did not show any significant increase in the cell death rate after chronic CTZ treatment (Fig. 10). Thus, although both CTZ and KA induce epileptiform activity after chronic treatment, the ultimate outcome is quite different: KA treatment results in significant cell death, which may eventually lead to destruction of neural networks; in contrast, CTZ treatment only results in robust epileptiform activity without apparent cell death, which will allow neural networks to continuously function for a long time.
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2 Lilly Research Centre, Eli Lilly & Co Ltd, Sunninghill Road, Windlesham, Surrey, GU20 6PH, UK
Abstract
Cyclothiazide (CTZ) is a potent blocker of AMPA receptor desensitization. We have recently demonstrated that CTZ also inhibits GABAA receptors. Here we report that CTZ induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo. We first found that chronic treatment of hippocampal cultures with CTZ (5 μM, 48 h) results in epileptiform activity in the majority of neurons (80%). The epileptiform activity lasts more than 48 h after washing off CTZ, suggesting a permanent change of the neural network properties after CTZ treatment. We then demonstrated in in vivo recordings that injection of CTZ (5 μmol in 5 μl) into the lateral ventricles of anaesthetized rats also induces spontaneous epileptiform activity in the hippocampal CA1 region. The epileptogenic effect of CTZ is probably due to its enhancing glutamatergic neurotransmission as shown by increasing the frequency and decay time of mEPSCs, and simultaneously inhibiting GABAergic neurotransmission by reducing the frequency of mIPSCs. Comparing to a well-known epileptogenic agent kainic acid (KA), CTZ affects neuronal activity mainly through modulating synaptic transmission without significant change of the intrinsic membrane excitability. Unlike KA, which induces significant cell death in hippocampal cultures, CTZ treatment does not result in any apparent neuronal death. Therefore, the CTZ-induced epilepsy model may provide a novel research tool to elucidate the molecular and cellular mechanisms of epileptogenesis without any complication from drug-induced cell death. The long-lasting epileptiform activity after CTZ washout may also make it a very useful model in screening antiepileptic drugs.
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Introduction
Cyclothiazide (CTZ) was originally known as a diuretic drug with antihypertensive effects (Julius et al. 1962; Schvartz et al. 1962). It was later found that CTZ is a potent blocker of AMPA receptor desensitization (Patneau et al. 1993; Trussell et al. 1993; Yamada & Tang, 1993; Zorumski et al. 1993; Barnes-Davies & Forsythe, 1995; Mennerick & Zorumski, 1995). CTZ also increases presynaptic glutamate release (Diamond & Jahr, 1995; Bellingham & Walmsley, 1999; Ishikawa & Takahashi, 2001). Our recent work has further demonstrated that CTZ can directly inhibit GABAA receptors as shown by both whole-cell and single-channel experiments (Deng & Chen, 2003). Thus, CTZ has a unique characteristic in acting simultaneously on two prominent synaptic transmission systems: it significantly enhances excitatory glutamatergic neurotransmission while suppressing inhibitory GABAergic neurotransmission. The net effect of CTZ on a neural network will be a significant tilt of the excitation–inhibition balance toward hyperexcitation. We therefore hypothesize that CTZ may work as an epileptogenic agent to induce epileptiform activity in central neurons.
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To test our hypothesis, we treated hippocampal CA1–CA3 cultures with CTZ either in short-term duration but high concentration (1–2 h, 20–50 μM), or chronically with low concentration (2–10 days, 5 μM). In both conditions, CTZ consistently induced robust epileptiform activity in cultured hippocampal neurons. More importantly, the epileptiform activity induced by chronic CTZ treatment lasts more than 48 h after washing off CTZ, suggesting a substantial change of neural network activities after CTZ treatment. To test whether the epileptogenic effect of CTZ is limited to in vitro cultures, we injected CTZ into the lateral ventricles of anaesthetized rats, and found that CTZ can also induce spontaneous epileptiform activity in the hippocampal CA1 region in vivo. The epileptogenic effect of CTZ is attributable to its modulation of glutamatergic and GABAergic neurotransmission without any significant change of the intrinsic membrane properties, because the spiking threshold and action potential firing rate did not change after CTZ treatment. In comparison to KA, which shows strong neurotoxicity, chronic CTZ treatment does not induce any significant cell death. Thus, a CTZ-induced epilepsy model may serve as a useful tool in epilepsy research with minimal side-effects on neuronal survival.
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Methods
Cell culture
Hippocampal cultures were prepared from new born Sprague-Dawley rats (P0–P1) as previously described (Deng & Chen, 2003; Chen et al. 2004). In brief, the hippocampal CA1–CA3 region was dissected out and incubated for 30 min in 0.05% trypsin–EDTA solution. After enzyme treatment, tissue blocks were triturated gently, and dissociated cells were plated onto a monolayer of astrocytes. The culture medium contained 500 ml MEM (Gibco), 5% fetal bovine serum (Hyclone), 10 ml B-27, 100 mg NaHCO3, 20 mMD-glucose, 0.5 mML-glutamine, and 25 u ml–1 penicillin/streptomycin. Cells were maintained in 5% CO2 incubator for 2–3 weeks. The adult rats were killed with CO2,and new-born pups were decapitated in accordance with animal protocols approved by the IACUC committee in Pennsylvania State University.
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Electrophysiology
Whole-cell recordings were performed in current- or voltage-clamp mode using a MultiClamp 700 A amplifier (Axon Instruments). Patch pipettes were pulled from borosilicate glass and fire polished (2–4 M). The recording chamber was continuously perfused with a bath solution consisting of (mM): 128 NaCl, 30 Glucose, 25 Hepes, 5 KCl, 2 CaCl2, 1 MgCl2, pH 7.3 adjusted with NaOH. The pipette solution for most of the experiments, such as recording action potentials, mEPSCs, and glutamate receptor responses, contained (mM): 125 K-gluconate, 10 KCl, 2 EGTA, 10 Hepes, 10 Tris-phosphocreatine, 4 MgATP, 0.5 Na2GTP, pH 7.3 adjusted with KOH. For mIPSCs and GABA-induced currents, pipettes were filled with (mM): 135 KCl, 10 Tris-phosphocreatine, 2 EGTA, 10 Hepes, 4 MgATP, 0.5 Na2GTP, pH 7.3 adjusted with KOH. The series resistance was typically 10–20 M and partially compensated by 30–50%. The membrane potential was held around –70 mV in both voltage-clamp and current-clamp recordings. Data were acquired using pClamp 9 software, sampled at 2–10 kHz, and filtered at 1 kHz. Off-line analysis was done with Clampfit 9 software (Axon Instruments). Miniature events were analysed using MiniAnalysis software (Synaptosoft). All data were expressed as mean ±S.E.M. and Student's t test was used for statistical analysis.
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Large depolarization shift resembling paroxysmal depolarization shift is defined here as 10 mV depolarization and 300 ms in duration. An epileptiform burst is defined by at least five consecutive action potentials overlaying on top of the large depolarization shift. When quantifying the percentage of neurons showing epileptiform activity, the criterion is at least four repeated epileptiform bursts occurring during 30 min of recording. Pyramidal shape neurons were selected for recordings. For each neuron recorded after kainic acid (KA) or CTZ treatment, epileptiform discharges were first verified under current-clamp conditions before recording miniature and whole-cell currents under voltage-clamp conditions.
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In vivo recordings
In vivo experiments were performed using adult male Sprague-Dawley rats (250–350 g body weight) and anaesthetized with urethane (1.2 g kg–1) under the Animals (Scientific Procedures) Act 1986 and approved by the local ethics committee. At the end of experiments, animals were killed with an overdose of urethane.
The femoral artery was cannulated to allow arterial blood pressure to be monitored. Animals were then mounted in a stereotaxic frame. A drill hole was made on the skull above the left side of the lateral ventricle (0.3 mm posterior to bregma, 1.3 mm lateral to the midline). A guide cannula was then placed 4 mm below the skull surface for drug delivery, and secured by the dental cement. For recording and stimulating, a large burr hole was made in the left side of the incised skull above the hippocampal area, and the dura was pierced and removed. For recording in the CA1 pyramidal cell layer, a tungsten electrode (0.5 M) was placed 3.5–4.2 mm posterior to bregma, 2.0–3.0 mm lateral to the midline. The depth of the recording electrode was approximately 2.0–2.5 mm below the brain surface as determined by the sudden change of electrical noise and the shape of the evoked field excitatory postsynaptic potential (fEPSPs) and population spike. A bipolar stimulating electrode was placed close to the CA3 region in order to stimulate the Shaffer collateral (3.8–4.5 mm posterior to bregma, 3.5–4.0 mm lateral to the midline, and 3.0–3.8 mm below the brain surface). Once both electrodes were in the right place, the fEPSPs and population spike (PS) were monitored for at least 30 min until a stable recording was achieved. The stimulation frequency was set at once per minute with single biphasic square-wave pulses of 0.2 ms duration and 700–900 μA (supramaximal, determined by input–output curve). In between stimulations, the baseline activity was recorded for evidence of spontaneous activity. Following a 30 min recorded baseline of all responses, drugs or vehicles were administered I.C.V. (intracerebral ventricle) at volume of 5 μl via the pre-implanted guide cannula into the lateral ventricle. Pharmacologically induced seizure-like activity was monitored for 3 h after CTZ injection, by observing the change of the evoked potential (as the single PS transforms into a multipeaked display) and the spontaneous seizure burst activity of CA1 pyramidal neurons (Wheal et al. 1998). The anaesthetic level was monitored and maintained throughout the course of the experiment, in particular after convulsant drug administration. On some occasions, the brain was taken for histological validation of the injection and recording/stimulating sites.
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Cell viability assay
A live/dead viability kit (Molecular Probes, Eugene, OR, USA) containing ethidium homodimer-1 and calcein-AM was used to examine cell viability. Ethidium homodimer-1 binds to cellular DNA in membrane-compromised cells and yields a strong red fluorescence in dead cells, but is unable to penetrate the intact plasma membrane of live cells. Calcein-AM is a membrane-permeable dye which can be cleaved by esterases in live cells and produces a uniform cytoplasmic green fluorescence. After drug treatment, 16–23 days in vitro (DIV) neurons were incubated in bath solution containing 1 μM calcein-AM and 4 μM ethidium homodimer-1 for 20 min at room temperature. Cell death rate was then measured by determining the percentage of ethidium homodimer-1-positive cells to total cell number (ethidium homodimer-1 and calcein-AM-positive cells). For each experimental treatment, at least seven fields of each coverslip were imaged, and cell death rate was averaged over a total of three different batches of experiments.
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Drugs
Cyclothiazide, kainic acid, bicuculline, and CNQX were purchased from Tocris. TTX was obtained from Sigma. All of the drugs were freshly diluted in bath solution to their final concentrations before experiments. Bath application of drugs was rapidly delivered through a micropipette (400–500 μm tip) positioned about 1000 μm away from recorded neurons at a 45 degree angle. The micropipette was connected to a Warner (Hamden, CT, USA) VC-6 drug delivery system.
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Results
Chronic treatment of hippocampal cultures with CTZ elicits robust epileptiform activity
We recently discovered that CTZ inhibits GABAergic neurotransmission in addition to its effect on glutamatergic neurotransmission (Patneau et al. 1993; Trussell et al. 1993; Yamada & Tang, 1993; Zorumski et al. 1993; Deng & Chen, 2003). The dual effects of CTZ led us to predict that CTZ will drive neural networks toward hyperexcitability. To test whether CTZ is capable of eliciting epileptiform activity in hippocampal neurons, we applied CTZ (5 μM) in the culture medium for 48 h. After CTZ treatment, neurons were transferred into a recording chamber and constantly perfused with normal bath solution without CTZ. For control, the same volume of vehicle (culture medium containing 0.01% DMSO) was added. We found that most of the control neurons showed only synaptic potentials and individual action potentials but no epileptiform bursts (Fig. 1A). In contrast, the majority of CTZ-treated neurons showed abnormally synchronized bursting activities, with high-frequency action potentials overlaying large depolarizing shifts (>10 mV) (Fig. 1B). These recurrent bursting activities are reminiscent of epileptiform activity in vivo (Prince & Connors, 1986).
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Aa and b, a representative current-clamp recording showing that chronic treatment with vehicle (0.01% DMSO) did not induce any epileptiform activity in cultured hippocampal neurons. Most of the action potentials are clearly separated, as shown in the expanded trace (Ab). Ba and b, a typical recording showing recurrent epileptiform bursts after chronic pretreatment with CTZ (5 μM, 48 h) in a pyramidal neuron. One of the epileptiform bursts in Ba was expanded in Bb, showing a train of action potentials overlaying a large depolarization shift. Ca and b, representative epileptiform burst activities induced by pretreatment (5 μM, 48 h) with kainic acid (KA), with an expanded trace shown in Cb. D, bar graphs showing the percentage of neurons manifesting epileptiform activity after pretreatment with CTZ (84.6%) and KA (60.9%). E, bar graphs showing the frequency of epileptiform bursts after pretreatment with CTZ (0.11 ± 0.01 Hz, n= 33) and KA (0.044 ± 0.012 Hz, n= 14; P < 0.001, Student's t test).
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We compared the ability of CTZ to induce epileptiform activity to previously established KA epilepsy model (Sarkisian, 2001; Omrani et al. 2003; Bausch & McNamara, 2004; Buckmaster, 2004). The same protocol (5 μM, 48 h) was used for chronic KA treatment. Similar to CTZ treatment, typical epileptiform bursts were also observed after KA treatment (Fig. 1C). Quantitative analysis showed a higher percentage of neurons with epileptiform activity (84.6%, n= 33 out of 39; Fig. 1D) and higher burst frequency (0.11 ± 0.01 Hz, n= 33; Fig. 1E) after CTZ treatment, compared to KA treatment (60.9%, n= 14 out of 23; burst frequency, 0.044 ± 0.012 Hz, n= 14, P < 0.001).
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To test how long the epileptiform activity may last after washout of CTZ, we transferred CTZ-pretreated coverslips into normal culture medium without CTZ for 24–48 h before performing patch-clamp recordings. Interestingly, the epileptiform activity persisted long after washout of CTZ. Figure 2 shows representative recordings with typical epileptiform activities after washout of CTZ for 24 h (Fig. 2A) or 48 h (Fig. 2B). Figure 2C shows the summarized data. Comparing with the control (14.7%, n= 34), the percentage of neurons showing epileptiform activity was 84.6% within 3 h after CTZ washout (n= 39), and remained 77.8% and 66.7% after washout of CTZ for 24 h or 48 h, respectively. These results suggest that CTZ-induced epileptiform activity is a long-term change of the neural network properties, which has become independent of CTZ itself.
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A, epileptiform activity recorded from a neuron pretreated with CTZ (5 μM, 48 h) and then transferred into CTZ-free culture medium for another 24 h. B, a representative recording showing persistent epileptiform activities even 48 h after transferring CTZ-pretreated coverslips into CTZ-free culture medium. C, summarized data showing the percentage of neurons with epileptiform activity after washout of CTZ. Compared with control (14.7%), the percentage of neurons showing epileptiform activity within 3 h, or after 24 h and 48 h washout of CTZ is 84.6%, 77.8% and 66.7%, respectively.
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Besides the chronic treatment with CTZ (5 μM, 48 h), we also tested whether short-term treatment with CTZ will induce epileptiform activity in hippocampal cultures. We found that a low concentration of CTZ (5 μM) was unable to induce epileptiform activity in most of the neurons within 2 h of treatment (Fig. 3). However, a high concentration of CTZ (20–50 μM) could effectively elicit epileptiform activities after a short-term treatment. Figure 3A shows typical epileptiform activities induced by 50 μM (1–2 h) and 20 μM (2 h) CTZ treatment. It is important to note that similar to chronic experiments, all recordings after short-term CTZ treatment were made in normal bath solution without CTZ. Bar graphs in Fig. 3B show the percentage of neurons with epileptiform activity after different CTZ-treatment conditions. Notice that 2 h treatment with 50 μM CTZ induced epileptiform activity in every neuron tested (100%; n= 13), indicating that CTZ is a powerful epileptogenic agent. In addition, we also observed epileptiform activities in hippocampal neurons after long-term (10 days) treatment with 5 μM CTZ (Fig. 3).
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A, typical epileptiform activities induced by short-term treatment with high concentration of CTZ (20–50 μM, 1–2 h), or by long-term treatment with low concentration of CTZ (5 μM, 10 days). Note that 2 h treatment with 5 μM CTZ (top trace) did not induce typical epileptiform activity in most neurons. B, bar graphs showing the percentage of neurons with epileptiform activity under different CTZ-treatment conditions. Note that 50 μM CTZ treatment for 2 h induced epileptiform activity in every neuron tested (n= 13).
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Together, these experiments demonstrate that CTZ-induced epileptiform activity, once elicited, will become the hallmark of neural network properties irrespective of the presence or absence of CTZ itself. Thus, the CTZ-induced in vitro epilepsy model will be useful in studying the long-term changes of neural networks after epileptogenesis.
CTZ induces spontaneous seizure activity in in vivo recordings
Can CTZ induce epileptiform activity in in vivo conditions We recorded neuronal activity in the hippocampal CA1 pyramidal layer from 13 urethane-anaesthetized rats. In all 13 rats studied, the evoked responses following stimulation of CA3 regions and/or Shaffer collaterals consisted of a large EPSP and a single PS during control recordings (Fig. 4Aa), and the baseline activity was virtually ‘silent’ (Fig. 4Ba). Following intracerebroventricular (I.C.V.) injection of CTZ (5 μmol in 5 μl), the single-peaked PS gradually transformed into a multiple-peaked event in all 10 rats tested. Figure 4Ab and Ac shows typical characteristic epileptiform activity in in vivo recordings (Wheal et al. 1998). The onset latency for the second peak appearance was 22 ± 3 min (ranging from 6 to 37 min). In nine out of these ten rats, more than two peaks occurred after stimulation. In eight out of the ten rats, spontaneous recurrent epileptiform bursts were observed after CTZ injection (Fig. 4Bb). The onset latency for spontaneous epileptiform bursts was 97 ± 17 min (ranging from 40 to 177 min). In two rats, epileptiform bursting activities did not occur. Over a 30 min analysis period, the mean epileptiform burst number was 7 ± 1 (ranging from 4 to 11). DMSO control experiments were performed in three rats. None of the control rats developed either multiple-peaked PS or spontaneous epileptiform bursting activities in the 3 h observation period after DMSO injection (5 μl, I.C.V.; data not shown).
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A, typical recordings showing that the evoked population spikes recorded from the CA1 pyramidal layer in urethane-anaesthetized rats transform from single peak at control (a) to multiple peaks (b and c, indicated by arrows) after CTZ injection (5 μmol, 5 μl, I.C.V.) ( indicates the stimulus artefact). B, a representative recording showing that spontaneous epileptiform bursts appeared 2 h after CTZ injection. In control conditions (a), there were only small baseline activities in CA1 pyramidal cells. Black dots indicate stimulus artefacts, the same as shown in Aa. After CTZ administration (b), large synchronized bursting activities appeared, with each large burst consisting of many smaller bursts of activities.
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Acute and chronic CTZ effects on the kinetics of mEPSCs and mIPSCs in hippocampal cultures
It is well-known that CTZ enhances glutamatergic neurotransmission by blocking the desensitization of AMPA receptors (Barnes-Davies & Forsythe, 1995; Diamond & Jahr, 1995; Mennerick & Zorumski, 1995; Bellingham & Walmsley, 1999; Ishikawa & Takahashi, 2001). It is unclear, however, whether the effects of CTZ on mEPSC kinetics are long-lasting and contribute to the prolonged excitability in the absence of CTZ. Moreover, the effects of acute and chronic CTZ on GABAergic neurotransmission and their contribution to CTZ-induced epileptiform activity are not well-understood. To investigate synaptic changes underlying CTZ induction of epileptiform activity, we compared acute and chronic CTZ effects on the kinetics of both mEPSCs and mIPSCs in cultured hippocampal neurons. The spontaneous mEPSCs were recorded in the presence of TTX (0.5 μM) and a specific GABAA receptor antagonist bicuculline (BIC, 20 μM). As illustrated in Fig. 5, the frequency of mEPSCs showed a significant increase after acute application (Fig. 5B) or chronic pretreatment (Fig. 5C) with CTZ (5 μM for both treatments). The decay time of averaged mEPSCs (bottom trace of Fig. 5A–C) also increased but only during acute application of CTZ, not after chronic pretreatment with CTZ. The quantified data indicated that the mEPSC frequency increased to 147 ± 11% during acute application of 5 μM CTZ (n= 20, P < 0.02) and to 193 ± 35% after chronic CTZ-treatment (n= 16, P < 0.05) (Fig. 5D). The mEPSC amplitude was not significantly changed after acute (107 ± 6%, n= 20; P > 0.3) or chronic (119 ± 12%, n= 16; P > 0.05) treatment with 5 μM CTZ (Fig. 5E). In addition, we analysed the kinetics of mEPSCs and found that the decay time constant was greatly increased during acute application of 5 μM CTZ (156 ± 12%, n= 10, P < 0.003), but there was no significant change after chronic pretreatment with CTZ (91 ± 10%, n= 14, P > 0.5) compared with control (Fig. 5F). Therefore, the CTZ effect on mEPSC kinetics is transient, only occurring during application of CTZ. However, the CTZ effect on presynaptic glutamate release as reflected by a significant increase of mEPSC frequency is a persistent change of neural network activity long after washout of CTZ.
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A–C, typical mEPSC traces recorded in the presence of TTX (0.5 μM) and BIC (20 μM) in control (A), acute application (B), and after chronic pretreatment (C) with CTZ (both 5 μM). Insets at the bottom of each panel are the averaged mEPSCs, showing a slowed decay phase after acute application of CTZ (B) but not chronic CTZ-pretreatment (C, after washout of CTZ). D, bar graphs showing that the normalized mEPSC frequency was significantly increased to 147 ± 11% during acute CTZ application (P < 0.02), and to 193 ± 35% after chronic CTZ pretreatment (P < 0.05). E, bar graphs showing that the normalized mEPSC amplitude was not significantly changed during acute application (107 ± 6%, P > 0.3) or after chronic pretreatment with CTZ (119 ± 12%, P > 0.05). F, kinetics analysis showing a significant increase in the normalized decay time constant during acute application of 5 μM CTZ (156 ± 12%, P < 0.003), but not after chronic pretreatment with CTZ (91 ± 10%, P > 0.5).
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We next examined the CTZ effect on mIPSCs. The spontaneous mIPSCs were recorded in the presence of TTX (0.5 μM) and the AMPA/kainate receptor antagonist CNQX (20 μM). As illustrated in Fig. 6, not only acute CTZ application (Fig. 6B) but also chronic CTZ (both 5 μM) treatment (Fig. 6C) significantly decreased the frequency of mIPSCs. Quantitative analysis showed that the mIPSC frequency decreased to 72 ± 5% (P < 0.0005, n= 15) during acute CTZ application, and to 50 ± 6% (P < 0.0002, n= 15) of the control after chronic CTZ treatment, respectively. However, the amplitude and the decay time constants (1 and 2) of mIPSCs did not change significantly during acute CTZ application and after chronic CTZ treatment at low concentration (5 μM) (Fig. 6E–G). At high concentration (50–100 μM), acute CTZ application decreased the amplitude of mIPSCs (data not shown, also see Deng & Chen, 2003).
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A–C, consecutive traces illustrating mIPSCs recorded in the presence of TTX (0.5 μM) and CNQX (20 μM) in control (A), during acute application (B), and after chronic pretreatment with CTZ (both 5 μM) (C). The mIPSC frequency showed an obvious decrease after both acute and chronic CTZ treatment. Insets at the bottom of each panel are the averaged mIPSCs, showing no apparent changes in the amplitude and kinetics after CTZ treatment. D, the normalized mIPSC frequency was significantly decreased to 72 ± 5% during acute application of CTZ (P < 0.0005) and to 50 ± 6% of the control after chronic CTZ pretreatment (P < 0.0002), respectively. E, there was no significant change in the normalized mIPSC amplitude after acute CTZ application (103 ± 4%, P > 0.5) or chronic CTZ pretreatment (92 ± 10%, P > 0.5). F and G, kinetics analysis showing no significant changes in the normalized decay time constants, 1 (F, P > 0.19 for acute and P > 0.1 for chronic CTZ treatment) and 2 (G, P > 0.86 for acute and P > 0.26 for chronic CTZ treatment), during acute application or after chronic CTZ-pretreatment.
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Taken together, acute CTZ application substantially enhances glutamatergic neurotransmission by increasing the frequency and decay time of mEPSCs, and meanwhile exerts a mild effect on GABAergic neurotransmission by decreasing the frequency of mIPSCs. After chronic CTZ pretreatment, the effects of CTZ on the frequency of mEPSCs and mIPSCs continued in the absence of CTZ, suggesting a long-lasting modulation of synaptic networks by chronic CTZ-treatment.
CTZ differs from KA in affecting neuronal excitability
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We next examined the effects of CTZ on neural network activity and intrinsic excitability and compared them with the effects of KA. We found that the frequency of spontaneous action potentials, a general index for neural network activity, was significantly increased in the presence of CTZ (Fig. 7A, n= 18). Bath application of 5 μM CTZ (upper trace) or 20 μM CTZ (lower trace) both dramatically increased the action potential firing. Note that the increase of action potentials by acute CTZ application was reversible, unlike the effect of long-term CTZ treatment (compare Fig. 7 with Figs 1–3). As expected, 20 μM CTZ has a stronger stimulatory effect than that induced by 5 μM CTZ, with the former showing higher firing frequency and longer persistency after washing off CTZ.
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A, bath application of 5 μM (upper trace) and 20 μM CTZ (lower trace) increased the firing frequency of spontaneous action potentials. As expected, 20 μM CTZ has a stronger stimulatory effect on neuronal firing. B, bath application of 5 μM KA (upper trace) also increased the frequency of action potentials, but was accompanied with an obvious membrane depolarization. After application of 20 μM KA (lower trace), a strong membrane depolarization resulted in inactivation of sodium channels and suppression of action potentials. A and B are from the same neuron. C, in the presence of TTX, spontaneous and evoked action potentials were totally blocked. CTZ (5 μM and 20 μM) application did not induce any membrane depolarization (upper traces), whereas KA (5 μM and 20 μM) application still induced significant membrane depolarization (lower traces). All recordings in C are from the same neuron.
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We compared the effects of CTZ versus KA on the spontaneous action potential firing at the same neuron (Fig. 7B). Bath application of 5 μM KA (upper trace, n= 8) significantly increased the frequency of action potentials which was also accompanied with an obvious membrane depolarization. During application of 20 μM KA in the same neuron, the membrane depolarization became so strong that it essentially inactivated sodium channels and suppressed action potentials (Fig. 7B, lower trace; n= 8). The difference between CTZ and KA on membrane depolarization can be best seen in the presence of TTX (0.5 μM) (Fig. 7C). After blocking action potentials, CTZ application (5–20 μM) did not induce any membrane potential changes (Fig. 7C, upper traces, n= 8), suggesting that CTZ itself does not activate membrane receptors or ionic channels. In contrast, KA application (5–20 μM) in the same neurons induced significant membrane depolarization (Fig. 7C; lower traces, n= 8), probably due to its direct activation of KA/AMPA receptors on the membrane. Thus, the cellular mechanism underlying the CTZ-induced epilepsy model is clearly different from that of the KA-induced epilepsy model.
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In order to clarify whether CTZ affects the neuronal intrinsic excitability, we used CdCl2 (300 μM) to eliminate network recurrent activities and examined the effect of CTZ on spiking threshold and neuronal intrinsic firing rate through injection of depolarizing currents. Figure 8A shows representative traces of current injection-induced action potentials in control (left), during acute CTZ application (middle), and after chronic CTZ pretreatment (right). The number of action potentials during 300 ms of current injection increased with the increase of depolarizing currents (Fig. 8A and C). The action potential threshold, shown in Fig. 8B, did not change significantly during acute application of CTZ (–37.4 ± 2.0 mV, n= 6, P > 0.17) and after chronic pretreatment with CTZ (–38.0 ± 3.8 mV, n= 6, P > 0.49), compared to the control (–35.1 ± 1.8 mV, n= 7). The membrane excitability plots in Fig. 8C show a linear correlation between the number of action potentials and the stimulus intensity (r > 0.9 in all groups). The slopes of each fitted line were very close to each other in control and CTZ-treatments (the slope is 0.036 in control; 0.034 during acute CTZ application; and 0.036 in chronic CTZ-treatment). These results indicate that CTZ does not affect neuronal intrinsic excitability.
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A, representative current-clamp recordings in control (left panel), during acute application of 50 μM CTZ (middle panel), and after pretreatment with 5 μM CTZ for 2 days (right panel). Each panel shows the number of action potentials during 300 ms of a series of current injections. The bath solution contained 300 μM CdCl2 to inhibit recurrent activity. Membrane potential was held at –70 mV, with 50 pA of stimulus increment during eight consecutive sweeps. B, bar graphs showing no significant change in the spiking threshold during acute application and after chronic pretreatment with CTZ. C, neuronal intrinsic excitability as determined from plots of the number of action potentials versus the stimulus intensity.
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CTZ-induced calcium influx is activity-dependent
The calcium signal has been implicated in epileptogenesis (Meyer, 1989; Sun et al. 2002). We next compared the effects of CTZ and KA on intracellular calcium changes. We employed Fura-2 ratio imaging to monitor the calcium transients after bath application of CTZ or KA (van den Pol et al. 1996). After loading with Fura-2, hippocampal neurons were challenged with CTZ (5–20 μM), KA (5–20 μM), and high concentration of potassium solution (Fig. 9). The 90 mM KCl stimulation was for positive control to make sure neurons were healthy and responsive. In control neurons, there were often some spontaneous Ca2+ spikes which are probably induced by spontaneous action potentials (Fig. 9A). After bath application of CTZ (upper traces) or KA (lower traces), the intracellular calcium concentration significantly increased, with higher concentration (20 μM) of CTZ and KA inducing stronger calcium influx (n= 97 in CTZ; n= 53 in KA). To suppress the complication from spontaneous action potential firing, we compared the effect of CTZ and KA in inducing calcium influx in the presence of TTX (Fig. 9B). Interestingly, no Ca2+ increase was induced by bath application of CTZ after blocking action potentials, suggesting that CTZ does not have any direct effect on calcium channel activation, and that the Ca2+ increase in the resting condition is dependent on neural network activities (Fig. 9B, upper trace; n= 47). In contrast, bath application of KA in the presence of TTX still effectively elicited calcium responses, especially at a concentration of 20 μM (Fig. 9B, lower trace; n= 82). These results suggest that CTZ-induced calcium influx is action potential dependent, while KA-induced calcium influx can be both action potential dependent and independent, with the latter resulting from direct activation of calcium channels by KA-induced membrane depolarization.
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A, bath application of CTZ (upper traces) and KA (lower traces) increased intracellular calcium concentration [Ca2+]i in normal bath solution; 90 mM KCl stimulation serves as positive control for neuronal calcium response. B, in the presence of TTX (0.5 μM), bath application of CTZ (upper traces) did not increase the [Ca2+]i, whereas KA still effectively induced calcium responses.
CTZ does not induce neurotoxicity in hippocampal cultures
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KA is known to be associated with serious neurotoxicity (Pollard et al. 1994; Sattler & Tymianski, 2001; Holopainen et al. 2004). In the KA-induced epilepsy model, it is difficult to determine whether the neuronal death is caused by a direct effect of KA or secondary to the KA-induced epileptiform activity. It is therefore important to examine whether chronic CTZ treatment will result in any neuronal death. We performed a series of cell viability assay using a cell death kit (LIVE/DEAD viability kit, Molecular Probes, Eugene, OR, USA). Live cells were stained with bath solution containing 1 μM calcein-AM (green) and 4 μM ethidium homodimer-1 (red) for 20 min at room temperature. Dead cells will allow ethidium homodimer-1 to permeate the plasma membranes and bind to the DNA to emit red fluorescence in the nucleus. For a clear view of live versus dead neurons, we overlaid phase images with ethidium homodimer-1-stained red fluorescent images under various conditions (Fig. 10). After chronic treatment with CTZ (5 or 20 μM, 48 h), most neurons looked healthy in morphology and were not stained by ethidium homodimer-1 (Fig. 10B–C). In contrast, after exposure to 5 or 20 μM KA for 24–48 h, neuronal death was apparent especially at higher concentration (20 μM) where most neurons were dead as stained by ethidium homodimer-1 (Fig. 10D and E). Quantitative data analysis showed that CTZ did not induce any significant increase of cell death compared to the control group (control, 3.8 ± 0.5%; 5 μM CTZ, 6.1 ± 1.3%, P > 0.07; 20 μM CTZ, 4.9 ± 1.3%, P > 0.3). However, chronic treatment with 5 μM KA (48 h) increased the neuronal death rate to 24.2 ± 4.9% (P < 0.002), and treatment with higher concentration of KA (20 μM, 24 h) resulted in an even more strikingly high death rate of 81.8 ± 6.6% (P < 0.0001) (Fig. 10F). These experiments indicate that unlike KA, which induces significant neuronal death, CTZ treatment is not associated with direct neurotoxicity. Thus, studying the cellular mechanisms of CTZ-induced epileptogenesis will not have any complications resulting from drug-induced cell death.
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A, phase images of cultured hippocampal neurons in control group. B and C, phase images of neurons treated with 5 μM CTZ for 48 h (B) or 20 μM CTZ for 24 h (C). There was no apparent cell death after CTZ treatment. D and E, overlaying images showing ethidium homodimer-1 staining of neurons treated by 5 μM KA for 48 h (D) or 20 μM KA for 24 h (E). Nuclei of dead cells exhibit red fluorescent signal. F, quantification of cell viability assay. CTZ has minimal effect on neuronal death, but both KA treatments (5 μM and 20 μM) significantly increased neuronal cell death rate (P < 0.001 for 5 μM and P < 0.0001 for 20 μM CTZ, respectively).
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Discussion
We have demonstrated here that CTZ is a novel epileptogenic agent which induces robust epileptiform activity in both hippocampal cultures and in vivo hippocampal recordings. The CTZ-induced epileptiform activity is a long-lasting phenomenon, permitting extensive studies of the molecular and cellular mechanisms of epileptogenesis. The capability of CTZ in inducing epileptiform activity relies upon its modulating synaptic neurotransmission without a direct effect on the intrinsic membrane excitability. Compared with KA, which directly activates KA and AMPA receptors and triggers neuronal cell death, CTZ acts as a neuromodulator of AMPA and GABAA receptors and is not associated with significant cell death. CTZ can induce epileptiform activity after both short-term and long-term treatment. Thus, the CTZ-induced epilepsy model is an important new addition to the current epilepsy models, offering great flexibility and low cytotoxicity for epilepsy research.
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CTZ has been known for more than a decade for its ability to block glutamate AMPA receptor desensitization and increase glutamate release (Partin et al. 1993; Trussell et al. 1993; Yamada & Tang, 1993; Zorumski et al. 1993; Diamond & Jahr, 1995; Bellingham & Walmsley, 1999; Ishikawa & Takahashi, 2001). Our recent work revealed that CTZ also acts as an inhibitor of GABAA receptors (Deng & Chen, 2003). The current in vitro as well as in vivo studies further indicate that CTZ can induce robust epileptiform activity in hippocampal neurons by shifting the excitation–inhibition balance toward hyperexcitation. Importantly, the epileptiform activity induced by CTZ is not a transient change of neural network activity, but rather it sustained for more than 48 h after washing off CTZ (Fig. 2), suggesting a permanent alteration of the functional output of neural networks after CTZ treatment. The continuous firing of epileptiform activity long after CTZ washout provides an ample time window for a thorough investigation of the network properties after epileptogenesis. It will also be a useful model to screen new antiepileptic drugs by testing which chemical compound(s) is capable of extinguishing the long-lasting epileptiform activity after CTZ-treatment.
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In addition to in vitro studies, we have also demonstrated that CTZ induces spontaneous epileptiform activity in in vivo recordings in the hippocampal CA1 region. We are currently engaged in experiments aiming to establish the in vivo CTZ epilepsy model. A recent work reported that microinjection of CTZ locally into the area tempestas in the anterior piriform cortex did not induce seizures (Fornai et al. 2005). This appeared to conflict with our in vivo studies. However, the injection site and the amount of CTZ used were drastically different between their studies and ours. Fornai et al. (2005) used 1.2 nmol in 200 nl CTZ for focal cortex injection, whereas we used 5 μmol in 5 μl CTZ for lateral ventricle injection. Thus, the CTZ concentration used by Fornai et al. (2005) may be lower than what we have used in our in vivo recordings. We will test whether the high-dose injection of CTZ into the lateral ventricles will induce behavioural seizures.
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The physiological relevance of the current in vitro CTZ epilepsy model is that a mild modulation of glutamatergic and GABAergic neurotransmission will lead to irreversible changes of the whole neural network. We have demonstrated that acute application of CTZ at a relatively low concentration (5 μM) increases presynaptic glutamate release but decreases presynaptic GABA release (Figs 5 and 6). Together with prolonging glutamate responses, acute application of CTZ results in a reversible increase of neuronal firing of action potentials (Fig. 7). However, after chronic CTZ treatment (5 μM, 48 h), neuronal activity will be transformed from single individual firing into hypersynchronized bursting activity. What underlies such activity transformation is clearly a physiological question and may shed new light on the mechanism of activity-dependent neural plasticity. Interestingly, raising the concentration of CTZ (20–50 μM) greatly shortens the time required for the onset of epileptiform activity (Fig. 3), suggesting that the degree of perturbation of glutamatergic and GABAergic neurotransmission is positively correlated with the speed of transforming individual firing into epileptiform bursting activity. The short latency of the onset of epileptiform activity after treatment with a high concentration of CTZ (20–50 μM) in cell cultures (1–2 h) is consistent with the onset latency for spontaneous epileptiform activity in in vivo recordings after I.C.V. injection of CTZ (40–177 min), suggesting a common mechanism and similar time course operating under both in vitro and in vivo conditions.
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Compared to the current, widely used, epilepsy models including KA, PTZ, pilocarpine, and kindling models, the mechanism of convulsant action of CTZ is clearly different. KA directly activates KA and AMPA receptors (Castillo et al. 1997; Wilding & Huettner, 1997). PTZ is an antagonist of GABAA receptors (Suzuki et al. 1999). Pilocarpine acts on cholinergic receptors (Honchar et al. 1983) and the kindling model relies upon high-frequency stimulation to directly excite neurons (Lothman & Williamson, 1993; Morimoto et al. 2004). CTZ acts as a neuromodulator which enhances glutamatergic but inhibits GABAergic neurotransmission (Trussell et al. 1993; Barnes-Davies & Forsythe, 1995; Diamond & Jahr, 1995; Mennerick & Zorumski, 1995; Deng & Chen, 2003; and present study). We have carefully compared the effect of CTZ with that of KA on membrane potential, calcium influx, and neuronal survival. We found that acute application of CTZ significantly increased spontaneous firing of action potentials, but did not induce membrane depolarization in the presence of TTX (Fig. 7). In contrast, KA depolarizes the membrane potential significantly in the presence of TTX, as expected. Likewise, CTZ does not induce calcium influx in the presence of TTX, but KA does induce significant calcium influx especially at a concentration of 20 μM (Fig. 10). The direct membrane depolarization and calcium influx induced by KA are probably related to the cell death after KA treatment (Fig. 10 of this study; Vornov et al. 1991; Holopainen et al. 2004). In contrast, our cell viability assay did not show any significant increase in the cell death rate after chronic CTZ treatment (Fig. 10). Thus, although both CTZ and KA induce epileptiform activity after chronic treatment, the ultimate outcome is quite different: KA treatment results in significant cell death, which may eventually lead to destruction of neural networks; in contrast, CTZ treatment only results in robust epileptiform activity without apparent cell death, which will allow neural networks to continuously function for a long time.
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