Cyclothiazide Induces Robust Epileptiform Activity in Rat Hippocampal Neurons Both in Vitro and in Vivo

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J Physiol 571.3 (2006) pp 605–618 605

Cyclothiazide induces robust epileptiform activity in rat hippocampal neurons both in vitro and in vivo

Jinshun Qi1, Yun Wang2, Min Jiang1, Philippa Warren2 and Gong Chen1

1Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA 2Lilly Research Centre, Eli Lilly & Co Ltd, Sunninghill Road, Windlesham, Surrey, GU20 6PH, UK

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.

(Resubmitted 17 December 2005; accepted after revision 12 January 2006; first published online 19 January 2006) Corresponding author G. Chen: Assistant Professor of Neurobiology, Department of Biology, 201 Life Sciences Building, Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA. Email: [email protected]

Cyclothiazide (CTZ) was originally known as a diuretic inhibition balance toward hyperexcitation. We therefore drug with antihypertensive effects (Julius et al. 1962; hypothesize that CTZ may work as an epileptogenic agent Schvartz et al. 1962). It was later found that CTZ is a potent to induce epileptiform activity in central neurons. blocker of AMPA receptor desensitization (Patneau et al. To test our hypothesis, we treated hippocampal 1993; Trussell et al. 1993; Yamada & Tang, 1993; Zorumski CA1–CA3 cultures with CTZ either in short-term et al. 1993; Barnes-Davies & Forsythe, 1995; Mennerick duration but high concentration (1–2 h, 20–50 μm), or & Zorumski, 1995). CTZ also increases presynaptic chronically with low concentration (2–10 days, 5 μm). glutamate release (Diamond & Jahr, 1995; Bellingham & In both conditions, CTZ consistently induced robust Walmsley, 1999; Ishikawa & Takahashi, 2001). Our recent epileptiform activity in cultured hippocampal neurons. work has further demonstrated that CTZ can directly More importantly, the epileptiform activity induced by inhibit GABAA receptors as shown by both whole-cell and chronic CTZ treatment lasts more than 48 h after washing single-channel experiments (Deng & Chen, 2003). Thus, off CTZ, suggesting a substantial change of neural CTZ has a unique characteristic in acting simultaneously network activities after CTZ treatment. To test whether on two prominent synaptic transmission systems: it the epileptogenic effect of CTZ is limited to in vitro significantly enhances excitatory glutamatergic neuro- cultures, we injected CTZ into the lateral ventricles of transmission while suppressing inhibitory GABAergic anaesthetized rats, and found that CTZ can also induce neurotransmission. The net effect of CTZ on a neural spontaneous epileptiform activity in the hippocampal network will be a significant tilt of the excitation– CA1 region in vivo. The epileptogenic effect of CTZ

C 2006 The Authors. Journal compilation C 2006 The Physiological Society DOI: 10.1113/jphysiol.2005.103812

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is attributable to its modulation of glutamatergic and MiniAnalysis software (Synaptosoft). All data were GABAergic neurotransmission without any significant expressed as mean ± s.e.m. and Student’s t test was used change of the intrinsic membrane properties, because the for statistical analysis. spiking threshold and action potential firing rate did not Large depolarization shift resembling paroxysmal change after CTZ treatment. In comparison to KA, which depolarization shift is defined here as ≥10 mV shows strong neurotoxicity, chronic CTZ treatment does depolarization and ≥300 ms in duration. An epileptiform not induce any significant cell death. Thus, a CTZ-induced burst is defined by at least five consecutive action epilepsy model may serve as a useful tool in epilepsy potentials overlaying on top of the large depolarization research with minimal side-effects on neuronal survival. shift. When quantifying the percentage of neurons showing epileptiform activity, the criterion is at least four repeated epileptiform bursts occurring during 30 min of Methods recording. Pyramidal shape neurons were selected for Cell culture recordings. For each neuron recorded after kainic acid (KA) or CTZ treatment, epileptiform discharges were first Hippocampal cultures were prepared from new born verified under current-clamp conditions before recording Sprague-Dawley rats (P0–P1) as previously described miniature and whole-cell currents under voltage-clamp (Deng & Chen, 2003; Chen et al. 2004). In brief, the conditions. 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 In vivo recordings gently, and dissociated cells were plated onto a mono- In vivo experiments were performed using adult male layer of astrocytes. The culture medium contained Sprague-Dawley rats (250–350 g body weight) and 500 ml MEM (Gibco), 5% fetal bovine serum (Hyclone), anaesthetized with urethane (1.2 g kg−1) under the md m 10 ml B-27, 100 mg NaHCO3,20m -glucose, 0.5 m Animals (Scientific Procedures) Act 1986 and approved l −1 -glutamine, and 25 u ml penicillin/streptomycin. Cells by the local ethics committee. At the end of experiments, were maintained in 5% CO2 incubator for 2–3 weeks. The animals were killed with an overdose of urethane. adult rats were killed with CO2,and new-born pups were The femoral artery was cannulated to allow arterial decapitated in accordance with animal protocols approved blood pressure to be monitored. Animals were then bytheIACUCcommitteeinPennsylvaniaStateUniversity. 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). Electrophysiology A guide cannula was then placed 4 mm below the skull Whole-cell recordings were performed in current- or surface for drug delivery, and secured by the dental voltage-clamp mode using a MultiClamp 700 A amplifier cement. For recording and stimulating, a large burr hole (Axon Instruments). Patch pipettes were pulled from was made in the left side of the incised skull above the borosilicate glass and fire polished (2–4 M). The hippocampal area, and the dura was pierced and removed. recording chamber was continuously perfused with For recording in the CA1 pyramidal cell layer, a tungsten a bath solution consisting of (mm): 128 NaCl, 30 electrode (0.5 M) was placed 3.5–4.2 mm posterior to Glucose, 25 Hepes, 5 KCl, 2 CaCl2, 1 MgCl2, pH 7.3 bregma, 2.0–3.0 mm lateral to the midline. The depth of adjusted with NaOH. The pipette solution for most of the recording electrode was approximately 2.0–2.5 mm the experiments, such as recording action potentials, below the brain surface as determined by the sudden mEPSCs, and glutamate receptor responses, contained change of electrical noise and the shape of the evoked field (mm): 125 K-gluconate, 10 KCl, 2 EGTA, 10 Hepes, excitatory postsynaptic potential (fEPSPs) and population 10 Tris-phosphocreatine, 4 MgATP, 0.5 Na2GTP, pH 7.3 spike. A bipolar stimulating electrode was placed close adjusted with KOH. For mIPSCs and GABA-induced to the CA3 region in order to stimulate the Shaffer currents, pipettes were filled with (mm): 135 KCl, 10 collateral (3.8–4.5 mm posterior to bregma, 3.5–4.0 mm Tris-phosphocreatine, 2 EGTA, 10 Hepes, 4 MgATP, 0.5 lateral to the midline, and 3.0–3.8 mm below the brain Na2GTP, pH 7.3 adjusted with KOH. The series resistance surface). Once both electrodes were in the right place, the was typically 10–20 M and partially compensated by fEPSPs and population spike (PS) were monitored for at 30–50%. The membrane potential was held around least 30 min until a stable recording was achieved. The −70 mV in both voltage-clamp and current-clamp stimulation frequency was set at once per minute with recordings. Data were acquired using pClamp 9 software, single biphasic square-wave pulses of 0.2 ms duration and sampled at 2–10 kHz, and filtered at 1 kHz. Off-line 700–900 μA (supramaximal, determined by input–output analysis was done with Clampfit 9 software (Axon curve). In between stimulations, the baseline activity was Instruments). Miniature events were analysed using recorded for evidence of spontaneous activity. Following a

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30 min recorded baseline of all responses, drugs or vehicles glutamatergic neurotransmission (Patneau et al. 1993; wereadministeredi.c.v.(intracerebralventricle)atvolume Trussell et al. 1993; Yamada & Tang, 1993; Zorumski of 5 μl via the pre-implanted guide cannula into the lateral et al. 1993; Deng & Chen, 2003). The dual effects of CTZ ventricle. Pharmacologically induced seizure-like activity led us to predict that CTZ will drive neural networks wasmonitoredfor3 hafterCTZinjection,byobservingthe toward hyperexcitability. To test whether CTZ is capable change of the evoked potential (as the single PS transforms of eliciting epileptiform activity in hippocampal neurons, into a multipeaked display) and the spontaneous we applied CTZ (5 μm) in the culture medium for 48 h. seizure burst activity of CA1 pyramidal neurons (Wheal After CTZ treatment, neurons were transferred into a et al. 1998). The anaesthetic level was monitored and recording chamber and constantly perfused with normal maintained throughout the course of the experiment, in bath solution without CTZ. For control, the same volume particular after convulsant drug administration. On some of vehicle (culture medium containing 0.01% DMSO) was occasions, the brain was taken for histological validation added. We found that most of the control neurons showed of the injection and recording/stimulating sites. only synaptic potentials and individual action potentials but no epileptiform bursts (Fig. 1A). In contrast, the Cell viability assay majority of CTZ-treated neurons showed abnormally synchronized bursting activities, with high-frequency A live/dead viability kit (Molecular Probes, Eugene, OR, action potentials overlaying large depolarizing shifts USA) containing ethidium homodimer-1 and calcein-AM (>10 mV) (Fig. 1B). These recurrent bursting activities was used to examine cell viability. Ethidium homodimer-1 are reminiscent of epileptiform activity in vivo (Prince & binds to cellular DNA in membrane-compromised cells Connors, 1986). and yields a strong red fluorescence in dead cells, but is We compared the ability of CTZ to induce epileptiform unable to penetrate the intact plasma membrane of live activity to previously established KA epilepsy model cells.Calcein-AMisamembrane-permeabledyewhichcan (Sarkisian, 2001; Omrani et al. 2003; Bausch & McNamara, be cleaved by esterases in live cells and produces a uniform 2004; Buckmaster, 2004). The same protocol (5 μm,48h) cytoplasmic green fluorescence. After drug treatment, was used for chronic KA treatment. Similar to CTZ 16–23 days in vitro (DIV) neurons were incubated in bath treatment, typical epileptiform bursts were also observed solution containing 1 μm calcein-AM and 4 μm ethidium after KA treatment (Fig. 1C). Quantitative analysis showed homodimer-1 for 20 min at room temperature. Cell death a higher percentage of neurons with epileptiform activity rate was then measured by determining the percentage of (84.6%, n = 33 out of 39; Fig. 1D) and higher burst ethidium homodimer-1-positive cells to total cell number frequency (0.11 ± 0.01 Hz, n = 33; Fig. 1E) after CTZ (ethidium homodimer-1 and calcein-AM-positive cells). treatment, compared to KA treatment (60.9%, n = 14 For each experimental treatment, at least seven fields out of 23; burst frequency, 0.044 ± 0.012 Hz, n = 14, of each coverslip were imaged, and cell death rate P < 0.001). was averaged over a total of three different batches of To test how long the epileptiform activity may last experiments. after washout of CTZ, we transferred CTZ-pretreated coverslips into normal culture medium without CTZ Drugs for 24–48 h before performing patch-clamp recordings. Interestingly, the epileptiform activity persisted long after Cyclothiazide, kainic acid, bicuculline, and CNQX were washout of CTZ. Figure 2 shows representative recordings purchased from Tocris. TTX was obtained from Sigma. All with typical epileptiform activities after washout of CTZ of the drugs were freshly diluted in bath solution to their for 24 h (Fig. 2A) or 48 h (Fig. 2B). Figure 2C shows the final concentrations before experiments. Bath application summarized data. Comparing with the control (14.7%, of drugs was rapidly delivered through a micropipette n = 34), the percentage of neurons showing epileptiform (400–500 μm tip) positioned about 1000 μm away from activity was 84.6% within 3 h after CTZ washout (n = 39), recorded neurons at a 45 degree angle. The micropipette and remained 77.8% and 66.7% after washout of CTZ was connected to a Warner (Hamden, CT,USA) VC-6 drug for 24 h or 48 h, respectively. These results suggest that delivery system. CTZ-induced epileptiform activity is a long-term change of the neural network properties, which has become independent of CTZ itself. Results Besides the chronic treatment with CTZ (5 μm, 48 h), Chronic treatment of hippocampal cultures with CTZ we also tested whether short-term treatment with CTZ will induce epileptiform activity in hippocampal cultures. elicits robust epileptiform activity We found that a low concentration of CTZ (5 μm) was We recently discovered that CTZ inhibits GABAergic unable to induce epileptiform activity in most of the neurotransmission in addition to its effect on neurons within 2 h of treatment (Fig. 3). However, a

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high concentration of CTZ (20–50 μm) could effectively studying the long-term changes of neural networks after elicit epileptiform activities after a short-term treatment. epileptogenesis. 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, CTZ induces spontaneous seizure activity all recordings after short-term CTZ treatment were made in in vivo recordings in normal bath solution without CTZ. Bar graphs in Fig. 3B show the percentage of neurons with epileptiform Can CTZ induce epileptiform activity in in vivo activity after different CTZ-treatment conditions. Notice conditions? We recorded neuronal activity in the that 2 h treatment with 50 μm CTZ induced epileptiform hippocampal CA1 pyramidal layer from 13 urethane- activity in every neuron tested (100%; n = 13), indicating anaesthetized rats. In all 13 rats studied, the evoked that CTZ is a powerful epileptogenic agent. In addition, responses following stimulation of CA3 regions and/or we also observed epileptiform activities in hippocampal Shaffer collaterals consisted of a large EPSP and a single neurons after long-term (10 days) treatment with 5 μm PS during control recordings (Fig. 4Aa), and the baseline CTZ (Fig. 3). activity was virtually ‘silent’ (Fig. 4Ba). Following intra- Together, these experiments demonstrate that cerebroventricular (i.c.v.) injection of CTZ (5 μmol in CTZ-induced epileptiform activity, once elicited, will 5 μl), the single-peaked PS gradually transformed into become the hallmark of neural network properties a multiple-peaked event in all 10 rats tested. Figure 4Ab irrespective of the presence or absence of CTZ itself. Thus, and Ac shows typical characteristic epileptiform activity the CTZ-induced in vitro epilepsy model will be useful in in in vivo recordings (Wheal et al. 1998). The onset latency

Figure 1. Chronic CTZ-treatment induces robust epileptiform activity in hippocampal cultures 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,48h)ina 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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for the second peak appearance was 22 ± 3 min (ranging Acute and chronic CTZ effects on the kinetics from 6 to 37 min). In nine out of these ten rats, more than of mEPSCs and mIPSCs in hippocampal cultures two peaks occurred after stimulation. In eight out of the ten rats, spontaneous recurrent epileptiform bursts were It is well-known that CTZ enhances glutamatergic neuro- observed after CTZ injection (Fig. 4Bb). The onset latency transmission by blocking the desensitization of AMPA for spontaneous epileptiform bursts was 97 ± 17 min receptors (Barnes-Davies & Forsythe, 1995; Diamond & (ranging from 40 to 177 min). In two rats, epileptiform Jahr, 1995; Mennerick & Zorumski, 1995; Bellingham & bursting activities did not occur. Over a 30 min analysis Walmsley, 1999; Ishikawa & Takahashi,2001). It is unclear, period, the mean epileptiform burst number was 7 ± 1 however, whether the effects of CTZ on mEPSC kinetics are (ranging from 4 to 11). DMSO control experiments were long-lasting and contribute to the prolonged excitability performed in three rats. None of the control rats developed in the absence of CTZ. Moreover, the effects of acute and either multiple-peaked PS or spontaneous epileptiform chronic CTZ on GABAergic neurotransmission and their bursting activities in the 3 h observation period after DMSO injection (5 μl, i.c.v.; data not shown).

Figure 2. CTZ-induced epileptiform activity persists long after washout of CTZ Figure 3. Both short- and long-term treatment with CTZ elicit A, epileptiform activity recorded from a neuron pretreated with CTZ epileptiform activities (5 μM, 48 h) and then transferred into CTZ-free culture medium for A, typical epileptiform activities induced by short-term treatment with another 24 h. B, a representative recording showing persistent high concentration of CTZ (20–50 μM, 1–2 h), or by long-term epileptiform activities even 48 h after transferring CTZ-pretreated treatment with low concentration of CTZ (5 μM, 10 days). Note that coverslips into CTZ-free culture medium. C, summarized data showing 2 h treatment with 5 μM CTZ (top trace) did not induce typical the percentage of neurons with epileptiform activity after washout of epileptiform activity in most neurons. B, bar graphs showing the CTZ. Compared with control (14.7%), the percentage of neurons percentage of neurons with epileptiform activity under different showing epileptiform activity within 3 h, or after 24 h and 48 h CTZ-treatment conditions. Note that 50 μM CTZ treatment for 2 h washout of CTZ is 84.6%, 77.8% and 66.7%, respectively. induced epileptiform activity in every neuron tested (n = 13).

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contribution to CTZ-induced epileptiform activity are not mEPSC amplitude was not significantly changed after well-understood. To investigate synaptic changes under- acute (107 ± 6%, n = 20; P > 0.3) or chronic (119 ± 12%, lying CTZ induction of epileptiform activity, we compared n = 16; P > 0.05) treatment with 5 μm CTZ (Fig. 5E). acute and chronic CTZ effects on the kinetics of both In addition, we analysed the kinetics of mEPSCs and mEPSCs and mIPSCs in cultured hippocampal neurons. found that the decay time constant was greatly increased The spontaneous mEPSCs were recorded in the presence during acute application of 5 μm CTZ (156 ± 12%, of TTX (0.5 μm) and a specific GABAA receptor antagonist n = 10, P < 0.003), but there was no significant change bicuculline (BIC, 20 μm). As illustrated in Fig. 5, the after chronic pretreatment with CTZ (91 ± 10%, n = 14, frequency of mEPSCs showed a significant increase P > 0.5) compared with control (Fig. 5F). Therefore, the after acute application (Fig. 5B) or chronic pretreatment CTZ effect on mEPSC kinetics is transient, only occurring (Fig. 5C) with CTZ (5 μm for both treatments). The decay during application of CTZ. However, the CTZ effect on time of averaged mEPSCs (bottom trace of Fig. 5A–C) presynaptic glutamate release as reflected by a significant also increased but only during acute application of increase of mEPSC frequency is a persistent change of CTZ, not after chronic pretreatment with CTZ. The neural network activity long after washout of CTZ. quantified data indicated that the mEPSC frequency We next examined the CTZ effect on mIPSCs. The increased to 147 ± 11% during acute application of spontaneous mIPSCs were recorded in the presence of 5 μm CTZ (n = 20, P < 0.02) and to 193 ± 35% after TTX (0.5 μm) and the AMPA/kainate receptor antagonist chronic CTZ-treatment (n = 16, P < 0.05) (Fig. 5D). The CNQX (20 μm). As illustrated in Fig. 6, not only acute

Figure 4. CTZ induces spontaneous epileptiform activity in hippocampal CA1 region in in vivo recordings 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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CTZ application (Fig. 6B) but also chronic CTZ (both mEPSCs and mIPSCs continued in the absence of CTZ, 5 μm) treatment (Fig. 6C) significantly decreased the suggesting a long-lasting modulation of synaptic networks frequency of mIPSCs. Quantitative analysis showed that by chronic CTZ-treatment. 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 CTZ differs from KA in affecting neuronal excitability treatment, respectively. However, the amplitude and the decay time constants (τ 1 and τ 2) of mIPSCs did We next examined the effects of CTZ on neural network not change significantly during acute CTZ application activity and intrinsic excitability and compared them and after chronic CTZ treatment at low concentration with the effects of KA. We found that the frequency (5 μm) (Fig. 6E–G). At high concentration (50–100 μm), of spontaneous action potentials, a general index for acute CTZ application decreased the amplitude of neural network activity, was significantly increased in the mIPSCs (data not shown, also see Deng & Chen, presence of CTZ (Fig. 7A, n = 18). Bath application of 2003). 5 μm CTZ (upper trace) or 20 μm CTZ (lower trace) Taken together, acute CTZ application substantially both dramatically increased the action potential firing. enhances glutamatergic neurotransmission by increasing Note that the increase of action potentials by acute the frequency and decay time of mEPSCs, and meanwhile CTZ application was reversible, unlike the effect of exerts a mild effect on GABAergic neurotransmission by long-term CTZ treatment (compare Fig. 7 with Figs 1–3). decreasing the frequency of mIPSCs. After chronic CTZ As expected, 20 μm CTZ has a stronger stimulatory pretreatment, the effects of CTZ on the frequency of effect than that induced by 5 μm CTZ, with the former

Figure 5. Acute and chronic CTZ modulation of mEPSCs in hippocampal cultures 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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showing higher firing frequency and longer persistency receptors on the membrane. Thus, the cellular mechanism after washing off CTZ. underlying the CTZ-induced epilepsy model is clearly We compared the effects of CTZ versus KA on the different from that of the KA-induced epilepsy model. spontaneous action potential firing at the same neuron In order to clarify whether CTZ affects the neuronal (Fig. 7B). Bath application of 5 μm KA (upper trace, intrinsic excitability, we used CdCl2 (300 μm) to eliminate n = 8) significantly increased the frequency of action network recurrent activities and examined the effect of potentials which was also accompanied with an obvious CTZ on spiking threshold and neuronal intrinsic firing membrane depolarization. During application of 20 μm rate through injection of depolarizing currents. Figure 8A KA in the same neuron, the membrane depolarization shows representative traces of current injection-induced became so strong that it essentially inactivated sodium action potentials in control (left), during acute CTZ channels and suppressed action potentials (Fig. 7B,lower application (middle), and after chronic CTZ pretreatment trace; n = 8). The difference between CTZ and KA (right). The number of action potentials during 300 ms on membrane depolarization can be best seen in the of current injection increased with the increase of presence of TTX (0.5 μm) (Fig. 7C). After blocking action depolarizing currents (Fig. 8A andC).The actionpotential potentials, CTZ application (5–20 μm) did not induce any threshold, shown in Fig. 8B, did not change significantly membrane potential changes (Fig. 7C, upper traces, n = during acute application of CTZ (−37.4 ± 2.0 mV, 8), suggesting that CTZ itself does not activate membrane n = 6, P > 0.17) and after chronic pretreatment with receptors or ionic channels. In contrast, KA application CTZ (−38.0 ± 3.8 mV, n = 6, P > 0.49), compared to (5–20 μm) in the same neurons induced significant the control (−35.1 ± 1.8 mV, n = 7). The membrane membrane depolarization (Fig. 7C; lower traces, n = excitability plots in Fig. 8C show a linear correlation 8), probably due to its direct activation of KA/AMPA between the number of action potentials and the stimulus

Figure 6. Acute and chronic CTZ modulation of mIPSCs in hippocampal cultures 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, τ 1 (F, P > 0.19 for acute and P > 0.1 for chronic CTZ treatment) and τ 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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intensity (r > 0.9 in all groups). The slopes of each neuronal death. Weperformed a series of cell viability assay fitted line were very close to each other in control and using a cell death kit (LIVE/DEAD viability kit, Molecular CTZ-treatments (the slope is 0.036 in control; 0.034 Probes, Eugene, OR, USA). Live cells were stained with during acute CTZ application; and 0.036 in chronic bath solution containing 1 μm calcein-AM (green) and CTZ-treatment). These results indicate that CTZ does not 4 μm ethidium homodimer-1 (red) for 20 min at room affect neuronal intrinsic excitability. 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 CTZ-induced calcium influx is activity-dependent of live versus dead neurons, we overlaid phase images with ethidium homodimer-1-stained red fluorescent The calcium signal has been implicated in epileptogenesis images under various conditions (Fig. 10). After chronic (Meyer, 1989; Sun et al. 2002). We next compared the effects of CTZ and KA on intracellular calcium changes. Weemployed 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 thecomplicationfromspontaneousactionpotentialfiring, 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,lowertrace; 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 Figure 7. CTZ increases action potential firing but does not depolarization. directly induce membrane depolarization 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 CTZ does not induce neurotoxicity firing. B, bath application of 5 μM KA (upper trace) also increased the in hippocampal cultures frequency of action potentials, but was accompanied with an obvious membrane depolarization. After application of 20 μM KA (lower KA is known to be associated with serious neurotoxicity trace), a strong membrane depolarization resulted in inactivation of (Pollard et al. 1994; Sattler & Tymianski, 2001; Holopainen sodium channels and suppression of action potentials. A and B are et al. 2004). In the KA-induced epilepsy model, it is difficult from the same neuron. C, in the presence of TTX, spontaneous and μ μ to determine whether the neuronal death is caused by evoked action potentials were totally blocked. CTZ (5 M and 20 M) application did not induce any membrane depolarization (upper a direct effect of KA or secondary to the KA-induced traces), whereas KA (5 μM and 20 μM) application still induced epileptiform activity. It is therefore important to examine significant membrane depolarization (lower traces). All recordings in C whether chronic CTZ treatment will result in any are from the same neuron.

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treatment with CTZ (5 or 20 μm, 48 h), most neurons Discussion looked healthy in morphology and were not stained by ethidium homodimer-1 (Fig. 10B–C). In contrast, after We have demonstrated here that CTZ is a novel exposure to 5 or 20 μm KA for 24–48 h, neuronal death epileptogenic agent which induces robust epileptiform was apparent especially at higher concentration (20 μm) activity in both hippocampal cultures and in vivo where most neurons were dead as stained by ethidium hippocampal recordings. The CTZ-induced epileptiform homodimer-1 (Fig. 10D and E). Quantitative data analysis activity is a long-lasting phenomenon, permitting showed that CTZ did not induce any significant increase extensive studies of the molecular and cellular mechanisms of cell death compared to the control group (control, of epileptogenesis. The capability of CTZ in inducing 3.8 ± 0.5%; 5 μm CTZ, 6.1 ± 1.3%, P > 0.07; 20 μm epileptiform activity relies upon its modulating synaptic CTZ, 4.9 ± 1.3%, P > 0.3). However, chronic treatment neurotransmission without a direct effect on the with 5 μm KA (48 h) increased the neuronal death intrinsic membrane excitability. Compared with KA, rate to 24.2 ± 4.9% (P < 0.002), and treatment with which directly activates KA and AMPA receptors and higher concentration of KA (20 μm, 24 h) resulted in triggers neuronal cell death, CTZ acts as a neuro- an even more strikingly high death rate of 81.8 ± 6.6% modulator of AMPA and GABAA receptors and is not (P < 0.0001) (Fig. 10F). These experiments indicate that associated with significant cell death. CTZ can induce unlike KA, which induces significant neuronal death, epileptiform activity after both short-term and long-term CTZ treatment is not associated with direct neurotoxicity. treatment. Thus, the CTZ-induced epilepsy model is an Thus, studying the cellular mechanisms of CTZ-induced important new addition to the current epilepsy models, epileptogenesis will not have any complications resulting offering great flexibility and low cytotoxicity for epilepsy from drug-induced cell death. research.

Figure 8. Both acute application and chronic pretreatment with CTZ did not affect the intrinsic excitability of neurons 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 has been known for more than a decade for its the functional output of neural networks after CTZ ability to block glutamate AMPA receptor desensitization treatment. The continuous firing of epileptiform activity and increase glutamate release (Partin et al. 1993; Trussell long after CTZ washout provides an ample time window et al. 1993; Yamada & Tang, 1993; Zorumski et al. 1993; for a thorough investigation of the network properties Diamond & Jahr, 1995; Bellingham & Walmsley, 1999; after epileptogenesis. It will also be a useful model to Ishikawa & Takahashi, 2001). Our recent work revealed screen new antiepileptic drugs by testing which chemical that CTZ also acts as an inhibitor of GABAA receptors compound(s) is capable of extinguishing the long-lasting (Deng & Chen, 2003). The current in vitro as well as epileptiform activity after CTZ-treatment. in vivo studies further indicate that CTZ can induce Inadditiontoinvitrostudies,wehavealsodemonstrated robust epileptiform activity in hippocampal neurons by that CTZ induces spontaneous epileptiform activity in shifting the excitation–inhibition balance toward hyper- in vivo recordings in the hippocampal CA1 region. We excitation. Importantly, the epileptiform activity induced are currently engaged in experiments aiming to establish by CTZ is not a transient change of neural network activity, the in vivo CTZ epilepsy model. A recent work reported but rather it sustained for more than 48 h after washing that microinjection of CTZ locally into the area tempestas off CTZ (Fig. 2), suggesting a permanent alteration of in the anterior piriform cortex did not induce seizures

Figure 9. CTZ-induced intracellular calcium increase is activity dependent A, bath application of CTZ (upper traces) and KA (lower traces) increased intracellular calcium 2+ concentration [Ca ]i in normal bath solution; 90 mM KCl stimulation serves as positive control for neuronal calcium response. B,inthe presence of TTX (0.5 μM), bath application of 2+ CTZ (upper traces) did not increase the [Ca ]i, whereas KA still effectively induced calcium responses.

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(Fornai et al. 2005). This appeared to conflict with our in of neuronal firing of action potentials (Fig. 7). However, vivo studies. However, the injection site and the amount after chronic CTZ treatment (5 μm, 48 h), neuronal of CTZ used were drastically different between their activity will be transformed from single individual firing studies and ours. Fornai et al. (2005) used 1.2 nmol in into hypersynchronized bursting activity. What underlies 200 nl CTZ for focal cortex injection, whereas we used such activity transformation is clearly a physiological 5 μmol in 5 μl CTZ for lateral ventricle injection. Thus, question and may shed new light on the mechanism the CTZ concentration used by Fornai et al. (2005) of activity-dependent neural plasticity. Interestingly, may be lower than what we have used in our in vivo raising the concentration of CTZ (20–50 μm) greatly recordings. We will test whether the high-dose injection shortens the time required for the onset of epileptiform of CTZ into the lateral ventricles will induce behavioural activity (Fig. 3), suggesting that the degree of perturbation seizures. of glutamatergic and GABAergic neurotransmission is The physiological relevance of the current in vitro CTZ positively correlated with the speed of transforming epilepsy model is that a mild modulation of glutamatergic individual firing into epileptiform bursting activity. The and GABAergic neurotransmission will lead to irreversible short latency of the onset of epileptiform activity after changes of the whole neural network. We have treatment with a high concentration of CTZ (20–50 μm) demonstrated that acute application of CTZ at a relatively in cell cultures (1–2 h) is consistent with the onset latency low concentration (5 μm) increases presynaptic glutamate for spontaneous epileptiform activity in in vivo recordings release but decreases presynaptic GABA release (Figs 5 after i.c.v. injection of CTZ (40–177 min), suggesting a and 6). Together with prolonging glutamate responses, common mechanism and similar time course operating acute application of CTZ results in a reversible increase under both in vitro and in vivo conditions.

Figure 10. CTZ has less neurotoxicity than KA 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)or20μM 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)or20μM 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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Compared to the current, widely used, epilepsy models Chen G, Harata N & Tsien RW (2004). Paired-pulse depression including KA, PTZ, pilocarpine, and kindling models, the of unitary quantal amplitude at single hippocampal mechanism of convulsant action of CTZ is clearly different. synapses. PNAS 101, 1063–1068. KA directly activates KA and AMPA receptors (Castillo Deng LB & Chen G (2003). Cyclothiazide potently inhibits etal.1997;Wilding&Huettner,1997).PTZisanantagonist gamma-aminobutyric acid type A receptors in addition to enhancing glutamate responses. ProcNatlAcadSciUSA of GABA receptors (Suzuki et al. 1999). Pilocarpine acts A 100, 13025–13029. on cholinergic receptors (Honchar et al. 1983) and the Diamond JS & Jahr CE (1995). Asynchronous release of kindling model relies upon high-frequency stimulation synaptic vesicles determines the time course of the AMPA to directly excite neurons (Lothman & Williamson, 1993; receptor-mediated EPSC. Neuron 15, 1097–1107. Morimoto et al. 2004). CTZ acts as a neuromodulator Fornai F, Busceti CL, Kondratyev A & Gale K (2005). AMPA which enhances glutamatergic but inhibits GABAergic receptor desensitization as a determinant of vulnerability to neurotransmission (Trussell et al. 1993; Barnes-Davies focally evoked status epilepticus. EurJNeurosci21, & Forsythe, 1995; Diamond & Jahr, 1995; Mennerick 455–463. & Zorumski, 1995; Deng & Chen, 2003; and present Holopainen IE, Jarvela J, Lopez-Picon FR, Pelliniemi LJ & study). We have carefully compared the effect of CTZ Kukko-Lukjanov TK (2004). Mechanisms of kainate-induced with that of KA on membrane potential, calcium influx, region-specific neuronal death in immature organotypic hippocampal slice cultures. Neurochem Int 45, 1–10. and neuronal survival. We found that acute application of Honchar MP, Olney JW & Sherman WR (1983). Systemic CTZ significantly increased spontaneous firing of action cholinergic agents induce seizures and brain damage in potentials, but did not induce membrane depolarization in lithium-treated rats. Science 220, 323–325. the presence of TTX (Fig. 7). In contrast, KA depolarizes IshikawaT&Takahashi T (2001). Mechanisms underlying the membrane potential significantly in the presence of presynaptic facilitatory effect of cyclothiazide at the calyx of TTX, as expected. Likewise, CTZ does not induce calcium Held of juvenile rats. J Physiol 533, 423–431. influx in the presence of TTX, but KA does induce Julius S, Weller JM & Hoobler SW (1962). A comparative study significant calcium influx especially at a concentration of of several thiazides with special reference to the diuretic and 20 μm (Fig. 10). The direct membrane depolarization and anti-hypertensive effects of cyclothiazide. Curr Ther Res Clin calcium influx induced by KA are probably related to the Exp 4, 57–63. cell death after KA treatment (Fig. 10 of this study; Vornov Lothman EW & Williamson JM (1993). Rapid kindling with recurrent hippocampal seizures. Epilepsy Res 14, 209–220. et al. 1991; Holopainen et al. 2004). In contrast, our cell MennerickS&Zorumski CF (1995). Presynaptic influence on viability assay did not show any significant increase in the time course of fast excitatory synaptic currents in the cell death rate after chronic CTZ treatment (Fig. 10). cultured hippocampal cells. JNeurosci15, 3178–3192. Thus, although both CTZ and KA induce epileptiform Meyer FB (1989). Calcium, neuronal hyperexcitability and activity after chronic treatment, the ultimate outcome is ischemic injury. Brain Res Brain Res Rev 14, 227–243. quite different: KA treatment results in significant cell Morimoto K, Fahnestock M & Racine RJ (2004). Kindling and death, which may eventually lead to destruction of neural status epilepticus models of epilepsy: rewiring the brain. Prog networks; in contrast, CTZ treatment only results in robust Neurobiol 73, 1–60. epileptiform activity without apparent cell death, which Omrani A, Fathollahi Y, Almasi M, Semnanian S, Mohammad will allow neural networks to continuously function for a S & Firoozabadi P (2003). Contribution of ionotropic long time. glutamate receptors and voltage-dependent calcium channels to the potentiation phenomenon induced by transient pentylenetetrazol in the CA1 region of rat References hippocampal slices. Brain Res 959, 173–181. Partin KM, Patneau DK, Winters CA, Mayer ML & Buonanno Barnes-Davies M & Forsythe ID (1995). Pre- and postsynaptic A (1993). Selective modulation of desensitization at AMPA glutamate receptors at a giant excitatory synapse in rat versus kainate receptors by cyclothiazide and concanavalin auditory brainstem slices. J Physiol 488, 387–406. A. Neuron 11, 1069–1082. Bausch SB & McNamara JO (2004). Contributions of mossy Patneau DK, Vyklicky L Jr & Mayer ML (1993). Hippocampal fiber and CA1 pyramidal cell sprouting to dentate granule neurons exhibit cyclothiazide-sensitive rapidly desensitizing cell hyperexcitability in kainic acid-treated hippocampal slice responses to kainate. JNeurosci13, 3496–3509. cultures. J Neurophysiol 92, 3582–3595. Pollard H, Charriaut-Marlangue C, Cantagrel S, Represa A, Bellingham MC & Walmsley B (1999). A novel presynaptic Robain O, MoreauJ&Ben-AriY(1994). Kainate-induced inhibitory mechanism underlies paired pulse depression at a apoptotic cell death in hippocampal neurons. Neuroscience fast central synapse. Neuron 23, 159–170. 63, 7–18. Buckmaster PS (2004). Laboratory animal models of temporal Prince DA & Connors BW (1986). Mechanisms of interictal lobe epilepsy. Comp Med 54, 473–485. epileptogenesis. Adv Neurol 44, 275–299. Castillo PE, Malenka RC & Nicoll RA (1997). Kainate receptors Sarkisian MR (2001). Overview of the current animal models mediate a slow postsynaptic current in hippocampal CA3 for human seizure and epileptic disorders. Epilepsy Behav 2, neurons. Nature 388, 182–186. 201–216.

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Sattler R & Tymianski M (2001). Molecular mechanisms of Wheal HV, Bernard C, Chad JE & Cannon RC (1998). Pro- glutamate receptor-mediated excitotoxic neuronal cell death. epileptic changes in synaptic function can be accompanied Mol Neurobiol 24, 107–129. by pro-epileptic changes in neuronal excitability. Trends Schvartz N, Torosdag S, Fertig H, Fletcher L Jr, Quan RB, Neurosci 21, 167–174. Schwartz MS & Bryant JM (1962). Cyclothiazide, a new Wilding TJ & Huettner JE (1997). Activation and diuretic-antihypertensive drug. Curr Ther Res Clin Exp 4, desensitization of hippocampal kainate receptors. JNeurosci 437–440. 17, 2713–2721. Sun DA, Sombati S, Blair RE & DeLorenzo RJ (2002). Yamada KA & Tang CM (1993). Benzothiadiazides inhibit rapid Calcium-dependent epileptogenesis in an in vitro model of glutamate receptor desensitization and enhance stroke-induced ‘epilepsy’. Epilepsia 43, 1296–1305. glutamatergic synaptic currents. JNeurosci13, 3904–3915. Suzuki T, Shimizu N, Tsuda M, Soma M & Misawa M (1999). Zorumski CF, Yamada KA, Price MT & Olney JW (1993). A Role of metabotropic glutamate receptors in the benzodiazepine recognition site associated with the hypersusceptibility to pentylenetetrazole-induced seizure non-NMDA glutamate receptor. Neuron 10, 61–67. during diazepam withdrawal. Eur J Pharmacol 369, 163–168. Trussell LO, ZhangS&Raman IM (1993). Desensitization of AMPA receptors upon multiquantal neurotransmitter release. Neuron 10, 1185–1196. Acknowledgements van den Pol AN, Obrietan K & Chen G (1996). Excitatory actions of GABA after neuronal trauma. JNeurosci16, We are grateful to Dr David Lodge for critical reading of the 4283–4292. earlier version of the manuscript and invaluable comments. We Vornov JJ, Tasker RC & Coyle JT (1991). Direct observation of also thank Chen Laboratory members for helpful discussion. the agonist-specific regional vulnerability to glutamate, This work is supported by the National Science Foundation NMDA, and kainate neurotoxicity in organotypic (0236429) and a start-up fund provided by the Pennsylvania hippocampal cultures. Exp Neurol 114, 11–22. State University to G.C.

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