Cyclothiazide Induces Robust Epileptiform Activity in Rat Hippocampal Neurons Both in Vitro and in Vivo
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http://www.paper.edu.cn 中国科技论文在线 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 转载 中国科技论文在线 http://www.paper.edu.cn 606 J. Qi and others J Physiol 571.3 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 C 2006 The Authors. Journal compilation C 2006 The Physiological Society 中国科技论文在线 http://www.paper.edu.cn J Physiol 571.3 Cyclothiazide induces epileptiform activity 607 30 min recorded baseline of all responses, drugs or vehicles glutamatergic neurotransmission (Patneau et al.