Cell Calcium 57 (2015) 376–384

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Epileptic activity during early postnatal life in the AY-9944 model of atypical absence epilepsy Seungmoon Jung a , Yong Jeong a,∗∗ , Daejong Jeon a,b,∗ a

Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Department of Neurology, Comprehensive Epilepsy Center, Biomedical Research Institute, Seoul National University Hospital (SNUH), Seoul, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 7 November 2014 Received in revised form 31 March 2015 Accepted 1 April 2015 Available online 8 April 2015 Keywords: AY-9944 Paroxysmal depolarizing shift Atypical absence epilepsy Lennox–Gastaut syndrome

a b s t r a c t Atypical absence epilepsy (AAE) is an intractable disorder characterized by slow spike-and-wave discharges in electroencephalograms (EEGs) and accompanied by severe cognitive dysfunction and neurodevelopmental or neurological deficits in humans. Administration of the cholesterol biosynthesis inhibitor AY-9944 (AY) during the postnatal developmental period induces AAE in animals; however, the neural mechanism of seizure development remains largely unknown. In this study, we characterized the cellular manifestations of AY-induced AAE in the mouse. Treatment of brain slices with AY increased membrane excitability of hippocampal CA1 neurons. AY treatment also increased input resistance of CA1 neurons during early postnatal days (PND) 5–10. However, these effects were not observed during late PND (14–21) or in adulthood (7–10 weeks). Notably, AY treatment elicited paroxysmal depolarizing shift (PDS)-like epileptiform discharges during the early postnatal period, but not during late PND or in adults. The PDS-like events were not compromised by application of glutamate or GABA receptor antagonists. However, the PDS-like events were abolished by blockage of voltage-gated Na+ channels. Hippocampal neurons isolated from an in vivo AY model of AAE showed similar PDS-like epileptiform discharges. Further, AY-treated neurons from T-type Ca2+ channel ␣1G knockout (Cav 3.1−/− ) mice, which do not exhibit typical absence seizures, showed similar PDS-like epileptiform discharges. These results demonstrate that PDS-like epileptiform discharges during the early postnatal period are dependent upon Na+ channels and are involved in the generation of AY-induced AAE, which is distinct from typical absence epilepsy. Our findings may aid our understanding of the pathophysiological mechanisms of clinical AAE in individuals, such as those with Lennox–Gastaut syndrome. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Absence epilepsy is characterized by bilateral, synchronous spike-and-wave discharges (SWDs) in electroencephalogram (EEG) traces. Two types of absence epilepsy have been identified both clinically in children and experimentally in animals [1–4]. Typical absence epilepsy exhibits characteristic SWDs (clinically at 3 Hz and experimentally at 7–9 Hz), demonstrates paroxysmal loss of or decrease in consciousness with no voluntary movement during the ictus, and usually has a benign outcome after treatment with antiepileptic drugs [4–7]. Atypical absence epilepsy (AAE) is

∗ Corresponding author at: Department of Neurology, Comprehensive Epilepsy Center, Biomedical Research Institute, Seoul National University Hospital (SNUH), Seoul, Republic of Korea. Tel.: +82 220720121. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Jeong), [email protected] (D. Jeon). http://dx.doi.org/10.1016/j.ceca.2015.04.001 0143-4160/© 2015 Elsevier Ltd. All rights reserved.

clinically distinct from typical absence epilepsy [7–9] for the following reasons: (1) the SWD frequency is less than 3 Hz, (2) there is voluntary movement and partial consciousness during the ictus, (3) it is accompanied by severe cognitive and neurodevelopmental impairments, and (4) it is often intractable to antiepileptic drugs [1,2,7,10,11]. Typical absence epilepsy has been evaluated vigorously, especially using genetic models, for several decades and is now understood to be constrained within the thalamocortical circuitry and to involve Ca2+ currents through T-type Ca2+ channels or burst spikes mediated by these channels [12–18]. By contrast, epileptic activity in AAE involves limbic circuitry, including the hippocampus, as well as thalamocortical circuitry [1,19,20], and the neural mechanism underlying AAE is not fully understood despite its malignant outcome. AY-9944 (AY), a cholesterol biosynthesis inhibitor that inhibits the reduction of 7-dehydrocholesterol to cholesterol, has been used to produce an animal model of AAE [1,21–25]. Repeated administration of AY during postnatal development elicits recurrent

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atypical absence seizures in animals, and the AY model shows phenotypes that parallel clinical AAE in humans with Lennox–Gastaut syndrome (LGS) [26–28]: slow SDWs (5–6 Hz in rats and 4–5 Hz in mice) and similar ictal behaviors and pharmacological, cognitive, and developmental profiles [1,21,29–31]. Thus, the study of the AY model both in vivo and in vitro can provide valuable insights into the clinical conditions. However, even though the AY model is considered a valid model of AAE due to its clinical parallels, the neural mechanism underlying seizure development induced by AY administration remains largely unknown. One of the distinct features of AAE is the involvement of the hippocampus [1,20,29], and thus, it is plausible that administration of AY during the early postnatal period alters hippocampal neuronal activity. Therefore, to investigate the mechanism of seizure generation in the AY-induced AAE model, we performed electrophysiological and pharmacological experiments on mouse hippocampal slices at different developmental stages. To mimic the in vivo repeated administration of AY, we incubated hippocampal slices with AY for several hours and measured the electrophysiological properties of the AY-treated hippocampal CA1 neurons. We also performed prolonged recordings to capture any seizurelike events or epileptiform activity. In a second set of experiments, the same electrophysiological protocols were conducted on hippocampal CA1 neurons from animals administered AY repeatedly during the early postnatal period. In addition, the possible involvement of T-type Ca2+ channels was explored using Cav 3.1−/− mice, which are resistant to typical absence epilepsy [32]. This study provides characterization of epileptic activity during early postnatal development in the AY model and proposes a mechanism of epileptogenesis in AAE. 2. Materials and methods 2.1. Animals B6/129 F1 mice were obtained from breeding two inbred mice, C57BL/6J and 129S4/SvJae. For the experiments used with Cav 3.1−/− , Cav 3.1 heterozygous mice (Cav 3.1+/− ) were maintained in the two genetic backgrounds, and Cav 3.1−/− mice were generated by mating the Cav 3.1+/− mice. Mice at postnatal day (PND) 5–10 (early PND), PND 14–22 (late PND), and 7–10 weeks (adulthood) of age were used. Mice were housed with a 12/12-h light/dark cycle and ad libitum access to food and water. Animal care and experimental handlings were approved by animal ethics committee and were carried out according to the guidelines from the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology. 2.2. Brain slice preparation and AY treatment in vitro Hippocampal brain slices (310 ␮m-thick) were prepared as described in our previous report [33,34]. Hippocampal brain slices were horizontally prepared in cold artificial cerebrospinal fluid (ACSF) (in mM, 124 NaCl, 3.0 KCl, 1.23 NaH2 PO4 , 2.2 CaCl2 , 1.2 MgCl2 , 26 NaHCO3 , and 10 glucose, pH 7.4) with oxygenation (95% O2 , 5% CO2 ). After 1 h recovery in normal ACSF, slices were incubated in ACSF containing AY-9944 (AY, 5 ␮M, Tocris) for 2–4 h, and then patch-clamp recordings were performed. 2.3. Whole-cell patch-clamp recording Whole-cell patch-clamp recordings were performed as described in our previous report [33,34]. The recordings were measured from visually guided hippocampal CA1 neurons at 31 ◦ C using glass pipette electrodes (3–6 m). Pipette electrodes were filled with the following an internal solution (in mM, 135

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K-gluconate, 5 KCl, 2 MgCl2 , 5 EGTA, 10 HEPES, 0.5 CaCl2 , 5 Mg-ATP, and 0.3 Na-GTP) which was buffered to pH 7.4 with KOH. Action potentials (APs) were triggered by a step-current injection (30 pA steps) from −120 pA to +120 pA under current-clamp mode for 500 ms. Current-voltage relationships were obtained from values measured at 400 ms of hyperpolarizing pulses. Input resistance was determined from the slope of a linear fit of the relationship between the change in membrane potential (Vm ) and the magnitude of the injected current (−1 * Vm )/(injected current amplitude * 500 ms). Before measuring of prolonged membrane potential changes in cells, membrane potential of the cells was adjusted at −50 mV by positive current-injections (from 10 to 15 pA) under current-clamp recording, and neither further negative nor positive currents were applied to the cells during the recording. Drugs [50 ␮M AP5 (dl-2-amino-5-phosphonopentanoic acid, Tocris), NMDA receptor antagonist; 10 ␮M CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, Tocris), AMPA receptor antagonist; 1 ␮M TTX (Tetrodotoxin, Tocris), Na+ channel blocker; 20 ␮M bicuculline (Tocris), GABAA receptor antagonist; 10 ␮M CGP-55845 (Tocris), GABAB receptor antagonist; 20 ␮M nifedipine (Tocris), L-type Ca2+ channel blocker] were added to external bath ACSF solutions. In patch-clamp recordings, hippocampal CA1 pyramidal neurons were visually guided and selected on the basis of their intrinsic membrane properties including firing patterns as described previously (Supplementary Table and Fig. S1) [35–38]. Overall 17 cells were excluded from the experiments and data analysis (PND 5–6, n = 6; PND 7–8, n = 7, PND 9–10; n = 1, PND 14–22; n = 2, 7–10 weeks, n = 1). The cells did not maintain their resting potentials, could not generate APs (silent cells), or exhibited spontaneous spikes at resting membrane potentials. Cell viability with the treatment of drugs, such as TTX, was verified by step current injections in the end of each recording. Patch-clamp recordings were performed using a MultiClamp 700B amplifier and a Digidata1440 (Axon instruments, CA), and the data were analyzed using the pCLAMP version 10.2 (Axon Instruments). 2.4. AY treatment in vivo: AY model of AAE To make AY model of AAE, B6/129 F1 mice pups were treated with a subcutaneous dose of AY (7.5 mg/kg, Tocris) every day from PND 3–6, as described previously [21]. At PND 7 or 8 the mice were decapitated, and hippocampal slices were prepared in normal ACSF, and then patch-clamp recordings were performed. 2.5. Statistical analysis All data are shown as means ± standard error of the mean (SEM). Two-way ANOVA followed by post hoc comparisons was conducted using SPSS software (SPSS, Chicago, IL, USA). Student’s t-test was used to identify main effects. A p-value < 0.05 was considered to indicate statistical significance. 3. Results 3.1. Alterations in passive membrane properties by AY treatment during the early postnatal period Postnatal administration of AY induces AAE in animals [1,21–25]. To mimic the in vivo condition, acutely prepared brain slices from different postnatal ages were incubated in ACSF solution containing 5 ␮M AY for 2–4 h. First, we assessed neuronal excitability by measuring passive membrane properties such as input resistance and current-voltage relationships (Fig. 1A–I). The AY-treated neurons from PND 5–10 (PND 5–6, n = 11; PND 7–8, n = 16; PND 9–10, n = 8) displayed significantly greater changes in membrane potential with injected current compared with the

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Fig. 1. Effects of AY-treatment on the passive electrical properties of hippocampal CA1 neurons: PND 5–10. (A–C) At PND 5–6, (D–F) At PND 7–8, and (G–I) At PND 9–10. Representative traces (A, D, G) of changes in membrane potential with step-current injections from control (upper) and AY-treated (lower) hippocampal CA1 neurons. (B, E, H) Current–voltage relationship for neurons at each age. AY-treatment resulted in significantly greater changes in membrane potential. (C, F, I) Input resistance for neurons at each age. The input resistance was significantly increased by AY-treatment. *p < 0.05, Student’s t-test

AY non-treated CA1 neurons (control neurons; PND 5–6, n = 12; PND 7–8, n = 13; PND 9–10, n = 9; Fig. 1B, E and H). In addition, input resistance was increased in the AY-treated neurons from PND 5–10 (p < 0.05, Student’s t-test; Fig. 1C, F and I). Interestingly, AY-treated neurons exhibited abnormal firing patterns, including plateau potentials with depolarizing step-current injections (Fig. 1A, D, G, and Supplementary Fig. S1). However, AY did not affect current–voltage relationships or input resistance of CA1 neurons at PND 14–22 (n = 29; Fig. 2A–C) or at 7–10 weeks of age (adulthood, n = 8) compared with control neurons (PND 14–22, n = 24; adulthood, n = 9; Fig. 2D–F). Although reduced threshold of AP was observed in the AY-treated neurons from PND 5–6, AY did not affect other membrane properties such as resting membrane potential, membrane capacitance, and AP latency (Supplementary Table). Taken together, these results indicate that AY treatment increases the membrane excitability of hippocampal CA1 neurons, but only during the early developmental period.

3.2. AY-induced paroxysmal depolarizing shift (PDS)-like epileptiform discharges Subsequent to observing abnormal firing patterns, including plateau potentials upon depolarizing step-current injections (Fig. 1 and Supplementary Fig. S1), we measured prolonged spontaneous firings to determine whether epileptiform activity occurs in AY-treated CA1 neurons. Strikingly, PDS-like epileptiform discharges, which are abnormal, prolonged depolarizations with repetitive spikes (bursting), were observed in AY-treated CA1 neurons at PND 5–8 (67 of 106 neurons; Fig. 3A and B). A normalized histogram showing the distribution of changes in membrane potential demonstrates prolonged PDS-like events during the recordings (Fig. 3A and B, right). However, AYtreatment did not induce PDS-like epileptiform discharges in CA1 neurons at PND 14–22 or in adulthood (Supplementary Fig. S2).

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Fig. 2. Effects of AY treatment on the electrical properties of hippocampal CA1 neurons: PND 14–22 and adults. (A–C) At PND 14–22, (D–F) at adulthood (7–10 weeks). Representative traces (A and D) of changes in membrane potential with step-current injections from control (upper) and AY-treated (lower) hippocampal CA1 neurons. AY-treated CA1 neurons at PND 14–22 and adulthood showed current-voltage relationships (B and E) and input resistance (C and F) similar to control CA1 neurons.

3.3. AY-induced PDS-like epileptiform discharges are not due to abnormal synaptic activity We next characterized the PDS-like epileptiform discharges induced by AY treatment pharmacologically. First, we investigated whether the PDS-like events were affected by excitatory synaptic inputs. CNQX and AP-5 were used to block glutamatergic AMPA/kainate and NMDA receptors, respectively. Although the shape of individual PDS-like events were altered subtly by application of CNQX (n = 8) or CNQX plus AP-5 (n = 14), the PDS-like events were not abolished (Fig. 3C and D). This result indicates that glutamatergic transmission is not critical for the generation of AY-induced PDS-like epileptiform discharges. Because GABAmediated synaptic transmission is excitatory during the early developmental period [39], we examined PDS-like events after application of bicuculline (GABAA receptor antagonist, n = 6) and CGP-55845 (GABAB receptor antagonist, n = 6; Fig. 3E and F). As with the glutamate receptor antagonists, blockade of GABA-mediated synaptic transmission did not abolish the PDS-like events, indicating that GABAergic transmission is also not involved in the generation of AY-induced PDS-like epileptiform discharges. These results demonstrate that AY-induced PDS-like epileptiform discharges are not a result of abnormal synaptic inputs. 3.4. AY-induced PDS-like epileptiform discharges are dependent on Na+ channels Next, we investigated pharmacologically whether non-synaptic constituents or certain defects in individual cells were involved

in generation of the PDS-like epileptiform discharges. TTX was used to block voltage-gated Na+ channels. In the presence of CNQX and AP-5, the treatment of TTX completely abolished (n = 8/22; Fig. 4A and B) or hugely diminished (n = 14/22; Fig. 4C and D) the PDS-like epileptiform discharges in AYtreated neurons. This result indicates that AY-induced PDS-like epileptiform discharges are largely dependent upon Na+ channels.

3.5. PDS-like epileptiform discharges in hippocampal neurons from the AY model of AAE To examine whether the PDS-like events also occur in the in vivo AY model of AAE, we repeated the experiment using hippocampal neurons from AY model animals. PDS-like events were observed in 65.38% of neurons from AY-injected mice (n = 34/52; Fig. 5A). The same pharmacological experiments used with the in vitro slice model were performed. PDS-like events completely disappeared after the treatment of TTX in 46.15% of neurons (n = 6/13; Fig. 5B), and 53.84% of neurons (n = 7/13; Fig. 5C) showed largely diminished PDS-like events, consistent with the result of the in vitro slice model. In addition, input resistance was increased in neurons from the AY-treated mice compared to the control neurons (p < 0.05, Student’s t-test; Supplementary Fig. S3). This demonstrates that PDS-like epileptiform discharges also occur in CA1 neurons of the in vivo AY model of AAE and exhibit pharmacological property similar to those induced by in vitro AY-treatment.

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Fig. 3. Elicitation of PDS-like epileptiform discharges by AY treatment and the effects of excitatory and inhibitory synaptic inputs. (A and B) Representative traces of changes in membrane potential during prolonged recordings of control (A) and AY-treated (B) hippocampal CA1 neurons. A normalized histogram shows the distribution of changes in membrane potential. (C–F) Pharmacological inhibition of excitatory (C and D) and inhibitory (E and F) synaptic receptors. CNQX (C), AP5 (D), bicuculline (E), and CGP55845 (F) could not abolish the AY-induced PDS-like epileptiform discharges. Lines indicate each drug treatment.

3.6. The effects of AY on CA1 neurons of Cav 3.1−/− mice

4. Discussion

Lastly, we investigated whether ␣1G (Cav 3.1) T-type Ca2+ channels are involved in the production of AY-induced PDS-like epileptiform discharges. It has been reported that T-type Ca2+ channels are critical in typical absence epilepsy, and that Cav 3.1−/− mice are resistant to typical absence epilepsy [18,32]. Interestingly, PDS-like epileptiform discharges were also observed in AY-treated CA1 neurons of the Cav 3.1−/− mice (PND 5–8; 11 of 16 neurons; Fig. 5D). This suggests that ␣1G T-type Ca2+ channels are not necessary for the generation of AY-induced PDS-like epileptiform discharges.

In contrast to typical absence epilepsy, patients with AAE show severe cognitive and neurodevelopmental impairments and intractability to antiepileptic drugs [1,2,7,10,11]. Experimentally, an animal model of AAE can be developed using a single or repeated postnatal administration of AY, a cholesterol biosynthesis inhibitor that induces recurrent atypical absence seizures throughout the animal’s life [1,21–25]. However, the neural mechanism of seizure development as a result of AY administration during postnatal development is not well known. In this study, we found that AY-treatment increased membrane excitability, induced abnormal

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Fig. 4. AY-induced PDS-like epileptiform discharges were dependent on voltage-gated Na+ channels. (A–D) Sample traces of Na+ channel-dependent AY-induced PDS-like epileptiform discharges. (A and B) Treatment of TTX totally blocked PDS-like epileptiform discharges in AY-treated neurons. (C and D) Sample traces of reduced PDS-like epileptiform discharges after TTX treatment. Lines indicate each drug treatment.

firing patterns including plateau potentials upon depolarizing stepcurrent injections, and elicited PDS-like epileptiform discharges. In addition, all such effects were limited to the early postnatal period (PND 5–10). Further, we attempted to demonstrate the relationship between Cav 3.1 T-type Ca2+ channels and the AY-induced PDS-like epileptiform discharges of AAE. Paroxysmal activity is related to spontaneous seizures, and thus its study is essential to the field of epilepsy [40]. Excitation/inhibition (E/I) synaptic imbalance and changes in intrinsic cellular properties could generate paroxysmal activity [41–46]. PDS is a paroxysmal activity with stereotypical prolonged, high-amplitude depolarization with overriding action potentials [47–54]. Experiments performing both in vivo and in vitro intracellular recordings of electrophysiological activities such as slow oscillations and spike-wave discharges during a developing seizure revealed that the PDS is the intracellular correlate of the interictal and/or ictal spike of the electroencephalographic seizure [47–54]. Thus, it is considered to be a cellular manifestation of epilepsy. The generation of PDSs is likely driven by E/I synaptic imbalance, such as excessive synaptic excitation, and by undetermined alterations in

the intrinsic properties of the neuron, such as increased excitability due to intracellular changes in ionic concentrations [47–54]. Our pharmacological experiments revealed that AY-induced PDS-like epileptiform discharges are non-synaptic events largely dependent upon voltage-gated Na+ channels. Although not all neurons exhibited PDS-like epileptiform discharges following AY treatment, all neurons did show abnormal firing patterns, including plateau potentials upon depolarizing current injections. Thus, AY affects certain ion channels, such as voltage-gated Na+ channels in the neuronal membrane, and this renders individual cells endogenously more excitable, with the potential to produce PDS-like events. Brain slices must be treated with AY for longer than 2 h to induce such effects; when we applied AY to brain slices for only 30–60 min, we failed to observe either PDS-like events or changes in membrane excitability. Therefore, the cellular alterations induced by AY might not result from direct interaction between AY and ion channels, but AY may induce neurons to become readily excitable, which is enough to elicit pathological events such as PDS. In the present study, the mechanism(s) of AY-induced changes in electrophysiological properties of CA1 neurons was uncovered.

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Fig. 5. PDS-like epileptiform discharges in hippocampal CA1 neurons from an in vivo AY model of AAE and Cav 3.1−/− mice. (A) PDS-like epileptiform discharges similar to those of the in vitro AY model were observed in hippocampal CA1 neurons from an in vivo AY model of AAE. (B and C) As with the in vitro model, treatment with TTX abolished (B) or reduced PDS-like epileptiform discharges, indicating that PDS-like events are largely dependent upon Na+ channels. (D) Application of AY to CA1 neurons of Cav 3.1−/− mice also induced PDS-like epileptiform discharges.

AY inhibits the reduction of 7-dehydrocholesterol to cholesterol. Cholesterol plays a key role in regulating the properties of cell membranes, as it is involved in within-membrane organization, dynamics, membrane function, and sorting [61,62]. Cholesterol affects the formation of membrane microdomains, including lipid rafts, and modulates the normal functioning of membrane proteins such as enzymes, ion channels, and neurotransmitter receptors, which are important for neuronal excitability and synaptic transmission [47–53,63,64]. Thus it is possible that AY affected on the composition or expression level of membrane proteins including Na+ channels, which might lead to physiological abnormalities such as alteration in membrane excitability. Alternatively, inhibition of cholesterol biosynthesis impacts myelinogenesis of the central nervous system (CNS). Impaired cholesterol homeostasis can cause neuronal degeneration within the hippocampal formation and seizure activity [65,66], and it is also one of the leading causes of several neurodegenerative disorders [67–70]. During the early postnatal period, the CNS undergoes an enormous number of cellular processes, and a proper ratio of cholesterol to phospholipids is crucial for the physicochemical properties of mammalian CNS neurons [71,72]. Further studies to investigate how AY developmentally alter input resistance and neuronal firing and why the effects of AY are limited to early postnatal period are needed.

Many studies suggest that AAE is distinct from typical absence epilepsy, and that Cav 3.1 T-type Ca2+ channels contribute to SWDs in typical absence epilepsy [18,32]. However, there has been no study on the relationship between Cav 3.1 T-type Ca2+ channels and AAE. In this study, we found that AY-treated CA1 neurons of Cav 3.1−/− mice exhibited PDS-like epileptiform discharges similar to those of normal mice treated with AY. In addition, AY treatment in vivo resulted in the development of spontaneous, recurrent, and synchronous slow SWDs in Cav 3.1−/− mice (Supplementary Methods and Fig. S4). These results suggest that Cav 3.1 T-type Ca2+ channels are not required for the generation of AY-induced SWDs or AAE in animals. Although Cav 3.1 T-type Ca2+ channels are involved in the production of SWDs in thalamic neurons, we examined the effects of AY on hippocampal neurons, because the SWDs of AAE involve both thalamocortical and hippocampal circuitries, and because AAE is associated with severe impairment in learning and memory [1,20,29,73]. In vitro and in vivo studies on AY-induced SWDs in thalamic neurons of Cav 3.1−/− mice will further elucidate the relationship between Cav 3.1 T-type Ca2+ channels and AAE. Along with T-type Ca2+ channels, L-type Ca2+ channels are involved in SWDs and absence epilepsy. We also examined the involvement of L-type Ca2+ channels in AY-induced PDS-like epileptiform discharges with nifedipine (blocker of L-type Ca2+

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channel). The blockade of L-type Ca2+ channels did not abolish the PDS-like events (Supplementary Fig. S5), indicating that L-type Ca2+ channels are also not involved in the generation of AY-induced PDS-like epileptiform discharges. A limitation in our study is that TTX did not abolished the PDSlike epileptiform discharges in all of recorded neurons; the PDS-like events completely disappeared in around 40% of neurons after the treatment of TTX, but the remaining neurons showed reduced PDS-like events with the TTX treatment. It is possible that the concentration of TTX (i.e., 1 ␮M) used in our study was not enough to entirely block voltage-gated Na+ channels. Alternatively, other ion channels, such as voltage-gated Ca2+ channels, might be involved in the PDS-like epileptiform discharges. In fact, application of ACSF with a low Ca2+ concentration (0.2 mM) altered the AY-induced PDS-like events; the paroxysmal activity with stereotypical prolonged depolarization was decreased (Supplementary Fig. S6). A further study to investigate whether voltage-gated Ca2+ channels, except Cav 3.1 T-type and L-type, are involved in the generation of AY-induced PDS-like epileptiform discharges may be needed. There is also a limitation regarding to cell types in our study. Although we attempted to select cells showing intrinsic membrane properties corresponding to pyramidal neurons, we cannot rule out the possibility of intermingling with interneurons. In this study, we electrophysiologically and pharmacologically characterized the cellular effects of AY, an agent widely used for generating animal models of AAE. Our results suggest that AY elicits PDS-like epileptiform discharges during the early postnatal period. We also suggest that the generation of AY-induced PDSs is nonsynaptic and largely dependent on voltage-gated Na+ channels, but not on Cav 3.1 T-type Ca2+ channels. Our study is the first report of an in vitro model of AAE using AY. AAE is often a major component of LGS, an early-onset childhood malignant epileptic disorder. LGS is associated with mental retardation and severe behavioral problems and is largely refractory to antiepileptic medications [11,26–28,74–77]. Our results from an in vitro animal model study may aid our understanding of the pathogenesis of AAE in humans, and we believe that this in vitro model will assist in the discovery of antiepileptic drugs to control the seizures of LGS. Acknowledgements The authors thank to Minji Bang and Dr. Hee-Sup Shin for helping brain slice preparation and providing T-type Ca2+ channel ␣1G knockout (Cav 3.1−/− ) mice, respectively. This work was supported by the Korea Health 21 R&D grants (HI12C0035) funded by Ministry of Health and Welfare and by the Basic Science Research Program grant (NRF-2014R1A2A2A01002608) funded by Ministry of Science, ICT and Future Planning. S.J. was supported by NRF2013-Global Ph.D. Fellowship Program. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.04.001. References [1] M.A. Cortez, C. McKerlie, O.C. Snead 3rd, A model of atypical absence seizures: EEG, pharmacology, and developmental characterization, Neurology 56 (2001) 341–349. [2] J.R. Farwell, C.B. Dodrill, L.W. Batzel, Neuropsychological abilities of children with epilepsy, Epilepsia 26 (1985) 395–400. [3] D. Pinault, T.J. O’Brien, Cellular and network mechanisms of genetically determined absence seizures, Thalamus Relat. Syst. 3 (2005) 181–203. [4] O.C. Snead 3rd, Basic mechanisms of generalized absence seizures, Ann. Neurol. 37 (1995) 146–157.

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Epileptic activity during early postnatal life in the AY-9944 model of atypical absence epilepsy.

Atypical absence epilepsy (AAE) is an intractable disorder characterized by slow spike-and-wave discharges in electroencephalograms (EEGs) and accompa...
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