brain research 1622 (2015) 204–216
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Research Report
Cav3.1 T-type calcium channel modulates the epileptogenicity of hippocampal seizures in the kainic acid-induced temporal lobe epilepsy model Chong-Hyun Kima,b,n a
Center for Neuroscience, Brain Science Institute, Korea Institute of Science & Technology, Seoul 136-791, Republic of Korea Department of Neuroscience, Korea University of Science & Technology, Daejeon 305-333, Republic of Korea
b
ar t ic l e in f o
abs tra ct
Article history:
The molecular mechanism of temporal lobe epilepsy has not been clearly identified. T-type
Accepted 12 June 2015
calcium channels play a role in burst firing in neurons and have been implicated in several
Available online 23 June 2015
seizure models. In this study, the role of Cav3.1 T-type (α1G) calcium channel has been
Keywords:
investigated in the kainic acid (KA)-induced temporal lobe epilepsy model (TLE) by using
Seizure
conventional α1G knock-out (ko) mice. After intraperitoneal (i.p.) administration or
Kainic acid
intrahippocampal injection of KA, depth hippocampal and cortical electroencephalogram
Cav3.1 α1G T-type calcium channel
(EEG) and behavioral monitoring were recorded, and timm and Nissl staining of brain
Status epilepticus
sections were made later. Seizure was mainly identified by EEG signals, rather than
Temporal lobe epilepsy.
behaviorally, with analytic criteria. During the acute status epilepticus (SE) period, both the duration and the frequency of hippocampal seizures were significantly reduced and increased, respectively, in αlG ko mice compared to those of wild type mice. Epileptogenicity, the total period of seizures (hr 1), was also significantly reduced in α1G ko mice. However, the latency of seizure occurrence was not significantly different between wild type and ko mice. These differential effects were not observed in cortical seizures. Furthermore, the injection of KA caused a strong increase in δ rhythm power spectrum density (PSD) of EEG in αlG ko mice compared to that in wild type mice. The results with conventional ko mice indicate that α1G T-type calcium channel plays a modulatory role in the duration and frequency of hippocampal seizures as well as the epileptogenicity of KAinduced TLE in mice, mostly during acute periods. & 2015 Elsevier B.V. All rights reserved.
n Corresponding author at: Center for Neuroscience, Brain Science Institute, Korea Institute of Science & Technology, Hwarang-Ro 14 Gil 5, Seongbuk-Gu, Seoul, 136-791, Korea. Tel.: þ82 2 958 6953; fax: þ82 2 958 6937. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.brainres.2015.06.015 0006-8993/& 2015 Elsevier B.V. All rights reserved.
brain research 1622 (2015) 204–216
1.
Introduction
TLE originates mainly from hippocampus and is one of the most prevalent types of epileptic seizures in human (Sloviter, 1994; Wieser, 2004). The molecular mechanism of TLE is not clearly known and animal models have been developed to elucidate the underlying mechanism of epileptogenicity of TLE. Rodent TLE models are induced by kindling or administration of KA or pilocarpine (PC) (Buckmaster and Dudek, 1997; Bouilleret et al., 1999; Leite et al., 2002; Loscher, 2002; Morimoto et al., 2004). In adult mice, KA is applied i.p. or directly into dorsal hippocampus to induce acute SE and then spontaneous chronic recurrent seizures later (Ben-Ari and Cossart, 2000; Riban et al., 2002). SE causes newly bursting neurons to be formed and many of regular firing neurons to be converted to burst firing neurons (Sanabria et al., 2001). Most hippocampal pyramidal neurons fire action potentials in a regular mode under normal conditions (Sanabria et al., 2001; Jensen et al., 1994). However, SE causes newly bursting neurons to be formed and many of regular firing neurons to be converted to burst firing neurons (Sanabria et al., 2001). It further shows that increase of intrinsic neuronal bursting is correlated with the appearance of TLE. To identify the molecular basis of the change in the neuronal firing property in seizure models, functions of many types of ion channels have been studied (Scheffer and Berkovic, 2003; Nelson et al., 2006). T-type calcium channels are low-voltage activated calcium channels and have three isoforms, α1G, H and I (Huguenard, 1996; Perez-Reyes, 2003). Their activation causes most of low-threshold calcium spikes that can trigger Naþdependent action potential firing as well as other low-voltage activated calcium-dependent processes (Yunker and McEnery, 2003; Huc et al., 2009). Because of the intrinsic contribution to neuronal firing, their role in the transition of firing modes has been studied in neurons of many brain regions such as hippocampus (Magee and Carruth, 1999), cerebellum (Molineux et al., 2006), thalamus and cortex (Perez-Reyes, 2003; Steriade, 2001; Crunelli et al., 2006). Another role of T-type calcium channels has also been suggested in epileptic seizure models. T- and P/Q-type calcium channels appear to contribute to seizure genesis and susceptibility in idiopathic generalized epilepsies (Khosravani and Zamponi, 2006). Absence epilepsy models mutated in high-voltage activated calcium channels have revealed upregulation of T-type calcium current (Zhang et al., 2002a; Song et al., 2004). In the genetic model of absence epilepsy rats from Strasbourg (GAERS), levels of α1G and α1H mRNA were also up-regulated in thalamic neurons (Tsakiridou et al., 1995; Talley et al., 1999). The epileptic activity itself can increase T-type calcium currents of dentate granule cells in human patient and KA or PC-induced epileptic rats (Beck et al., 1998) and of CA1 neurons in kindling study (Faas et al., 1996). Even single episode of SE can increase T-type calcium current of CA1 pyramidal neurons (Su et al., 2002; Yaari et al., 2007; Becker et al., 2008) or alter thalamic α1H channel density in the rodent PC-induced TLE model (Graef et al.,
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2009). These results suggest correlative links between seizure models and T-type channel function. The specific function of each isoform of T-type calcium channel has been revealed in some seizure models by using ko or transgenic mouse due to the lack of isoform-specific inhibitors of T-type calcium channels. Deletion of α1G calcium channel caused resistance to a GABAB-receptor agonistinduced absence seizure generation (Song et al., 2004; Kim et al., 2001). Moreover overexpression of α1G calcium channel in BAC transgenic mouse induced pure absence epilepsy (Emst et al., 2009). Functional polymorphism of α1G and α1H gene was shown in a juvenile absence syndrome (Singh et al., 2007) and in childhood absence epilepsy (Chen et al., 2003; Vitko et al., 2005; Heron et al., 2007; Powell et al., 2009), respectively. Different from absence epilepsy, there have been few attempts on the role of specific T-type calcium channel in TLE models. Recently, Becker et al. (2008) showed that removal of α1H calcium channel reduced the bursting of CA1 neurons and chronic spontaneous seizure frequency in PC-induced model. In this study, I have examined a role of α1G calcium channel in KA-induced TLE model using α1G ko mice for both acute and chronic periods up to 8 months. The results suggest that α1G T-type calcium channel plays a modulatory role in the epileptogenicity of hippocampal seizures.
2.
Results
2.1.
KA induces SE in α1G ko mice
KA has been used to induce TLE models of rodents. However, the developmental time course of TLE induced by KA has not been thoroughly observed from the initial appearance of SE to the chronic periods. In this study, in order to observe both acute and chronic effects of KA induced seizures in TLE mouse models, two ways of KA administration were used. One is the local intrahippocampal injection of KA, revealing direct effects of KA on seizures from the affected area of hippocampus. The intrahippocampal injection does, however, require the recovery period for EEG recordings from the surgery, preventing the stable measurement of acute effects on EEG. Therefore, to record the acute effects of KA, single i.p. injection has been used, which also allows the chronic recordings. KA i.p. injection caused SE in both wild type and αlG ko mice. Seizure can be identified behaviorally or by EEG signal patterns. Although mouse behavior was monitored simultaneously during EEG recording, it was difficult to observe consistent correlation between EEG pattern and mouse behavior, probably due to the difference in the resolution of analyzing signals from these two types of data. Thereby EEG signal was chosen as the main parameter for the data analysis and the description of the results was based on EEG analysis otherwise mentioned in this study. To quantify and compare the power of the EEG signal of specific rhythmic band, PSD analysis was used. First of all, to show the EEG raw data from one mouse after KA treatment for the whole recording period, representative recordings covering both acute and chronic periods had been selected from two types,
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Fig. 1 – Single KA i.p. injection induces acute epileptic seizures and shifts the PSD of depth hippocampal EEG from both male A) wild type and B) αlG ko mice. (a)–(h) and (i)–(q) are each from independent EEG recordings, scale bars: 5 min (except 10 min for g, h, p, and q) and 1 mV. (a) and (j) 30 min Control EEG trace. The peak frequency of PSD is 0.6 Hz. Black lines in (i) and (r). (b) and (k) EEG recordings starting at 5 min after KA injection. (c) and (l) 39 min after KA injection. The animals are inactive during most of the recording period. (d) and (m) 72 min after KA injection. Green lines in (i) and (r). The animals still rarely move. (e) and (n) 113 min after KA injection. (f) and (o) 144 min after KA injection. The peak of PSD is at 1–3 Hz range. (g) and (p) 40 days after KA injection. Brown lines in (i) and (r). (h) and (q) 178 days after KA injection. (i) and (r) PSD plots from each EEG recording shown in A and B. Numeric labeling indicates the region of trace that si expanded in Fig. 2.
respectively. Fig. 1 shows example hippocampal depth EEG recording traces from one wild type (A) and an αlG ko mouse (B), measured at the same time. The control baseline EEG recording was collected before i.p. KA injection (a and j). The EEG seizure spikes appeared shortly (b and k) after KA administration. PSD started to increase (c and l), shown as red line (i and r). The peak of enhanced PSDs was found mostly in δ rhythm range and then seizure subsides slowly (see also Fig. 7A and B). During chronic periods, PSD decreased and returned close to that of control (h, q). To show raw EEG traces much visually identifiable manner, Fig. 2 shows expanded views of the selected regions of Fig. 1 EEG traces. During the control recording period, mice move around and explore the environment, generating θ rhythm as a dominant EEG rhythm (A1, B1). After i.p. KA injection, mice start to move less along with the appearance of seizure spikes (A2, B2), and then epileptic discharges occur frequently (A3and A4, B3 and B4). Ictal spike waves at 1–2 Hz range and interictal spike waves at 30–40 Hz occur (A3–7, B3–8). EEG traces on 40 days (A9 and A10, B11) and 178 days (A11–12, B12) after KA injection show much reduced epileptic activity.
The cortical EEG was recorded simultaneously as a kind of internal control since TLE is known to be originated mainly from hippocampus. Example cortical EEG recordings are shown in Fig. 3. In these mice, the time course of cortical EEG seizure occurrence and its development after KA injection is similar to that of hippocampal EEG. The cortical EEG seizure spikes appeared similarly after KA administration compared to hippocampal EEG seizure spikes in wild type and ko mice, respectively, though the cortical EEG from the wild type looks a bit late in developing seizure spikes and smaller in the magnitude of seizures. PSD was enhanced in most rhythm ranges and the peak PSD was found in δ rhythm range in general. Acute seizures subside slowly like hippocampal seizures. During chronic periods, PSD decreased and returned close to that of control like as hippocampal seizure PSDs. The analysis of seizure properties in detail below, however, shows the difference between hippocampal EEG seizures and cortical EEG ones (Fig. 5). Expanded views of the selected regions of Fig. 3 EEG traces are shown (Fig. 4). Epileptic discharges, ictal- and interictal seizures also appear acutely after KA injection.
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Fig. 2 – Expanded view of EEG traces shown in Fig. 1A and B. The trace number indicates the region of the raw trace shown in Fig. 1. 30–40 Hz rhythms appear in traces #3, 4, 5, 7 of A and #3, 5, 6, 7, 8 of B. Scale bars: 3 s (except 1.5 s in trace #1), 1 mV.
2.2. The duration, frequency and epileptogeneis of KAinduced hippocamapal seizures during SE period are affected in α1G ko mice To examine the effect of KA on the seizure generation, the onset latency of the seizure was measured up to 6 h after KA administration (see Experimental Procedure). Plots of latency and duration of hippocampal and cortical seizures generated after KA injection (t¼0) are shown in Fig. 5 A and E, respectively. Red circle indicates the first seizure of each recording. The latency and duration of the first seizures from α1G ko mice were not significantly different from those of wild type (B and F). When all seizures were considered, the average duration of hippocampal EEG seizures was significantly reduced in α1G ko mice by 58% during SE compared to that of wild type mice, without a significant change in the latency of seizures (C and D). The hippocampal seizure frequency per hour of recording (hr 1) was significantly increased by 64% in α1G ko mice compared to that of wild type mice. The changes in the duration of seizures and the seizure frequency (hr 1) gives a theoretical reduction of epileptogenicity, the total seizure duration per hour (hr 1), by 31% (0.42n1.64¼ 0.69), which is close to the observed reduction (28%, see Fig. 5C legend) of the total hippocampal seizure duration (hr 1) in α1G ko mice (C). Epileptogenicity in α1G ko mice decreases though the seizure frequency increases. In case of cortical seizures, the duration,
frequency, latency of seizures and total seizure duration were not changed in α1G ko mice (G and H). To see the relationship between latency and duration of seizures, pearson correlation coefficient values were calculated. The values of all coefficients (r) were smaller than 70.1, indicating non-significant relationships between latency and duration of seizures (þ/þ, hippocampus, r¼ 0.0165, p¼ 0.84; /, hippocampus, r¼ 0.0612, p¼ 0.38; þ/þ, cortex, r¼0.0564, p¼ 0.51; / , cortex, r¼ 0.00372, p¼0.96). To see the effect of KA on the chronic seizures, overnight EEG recordings on the same mouse were repeated at 1–4 week intervals up to 8 months. The duration of EEG seizure was still significantly smaller in α1G ko mice compared to that of wild type mice (Fig. 6A and B). The frequency of seizure occurrence was maintained a bit higher in α1G ko mice, revealed by K-S test (Fig. 6C and D). These results suggest that the effect of the absence of α1G calcium channel on seizure duration has been kept for the chronic period.
2.3. KA increases EEG rhythmic band powers, especially δ, more in α1G ko mice during SE EEG rhythms are used to define the status of certain brain functions and animal behaviors (Engel et al., 2001; Buzsaki et al., 2003). Though the appearance and pattern of seizure generation were quite variable from mouse to mouse as
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Fig. 3 – Single KA i.p. injection induces acute epileptic seizures and shifts the PSD of depth cortical EEG from both male A) wild type and B) αlG ko mice. Scale bars: 5 min (except 10 min in g, h, p, and q) and 1 mV. (a)–(r) Experimental conditions are the same as in Fig. 1. Numeric labeling indicates the region of trace that is expanded in Fig. 4.
described previously (Williams et al., 2009), KA injection seems to cause an acute increase in the PSD of most frequency ranges examined (Supplementary Fig. 2). In order to see how EEG rhythms develop and progress, the frequency of the peak PSD of each recording was plotted during either acute or chronic periods, respectively (Fig. 7). All PSD peaks were detected at frequencies lower than 10 Hz. The acute effects of KA on hippocampal EEG are shown in Figure 7A and B. The frequencies of peak PSDs from control EEG recordings marked at t¼ 0 are mainly in δ rhythm range in both types of mice (þ/þ, 74%; / , 76%) and the remains are mostly in θ rhythm range, which is described as a mixed pattern (δþθ). However, after KA injection, the frequencies of peak PSDs appear only in δ rhythm range in α1G ko mice, starting less than 10 min ( / , 100%), while the mixed pattern is still observed in wild-type mice (þ/þ, 71% in δ). When K-S test on the distribution of peak PSD frequency was further applied before and after 20 min period between wild type and ko, respectively, significant difference was shown during the late phase (see Figure legends 7A and B). In cortical EEGs, the percentage of recordings having PSD peak frequency in δ rhythm range within 10 min after KA injection is 85% and then stabilizes about 81% up to 160 min in wild-type mice (Fig. 7E). In α1G ko mice, it is 81% at 10 min period and then reduces a bit to 79% (Fig. 7F). The result indicates that the shift of the peak frequency of PSD to δ rhythm band occurs
clearly in hippocampal EEG but does little in cortical EEG PSD in α1G ko mice. K-S test on the peak PSD frequency distribution between wild type and ko indicated significant difference during the early phase of acute period (see legends of Fig. 7E and F). The chronic effects up to 8 months are also shown in Fig. 7C, D, G and H. In wild-type mice PSD peak frequencies of both hippocampal and cortical EEG data are mostly in δ rhythm range at 25 days after KA injection and then the mixed pattern of the peak frequency distribution appears through unknown mechanisms (Fig. 7C). K-S test on the peak PSD frequency distribution between wild type and ko showed marginal significant difference only during the early phase (o25 days) of chronic period of cortical EEG, suggesting that peak PSD frequencies seemed to occur quicker in the cortical area of α1G ko mice (Fig. 7D).
2.4. Cellular and structural pathology within hippocampus To check the phenotypes on hippocampal structural organization and mossy fiber sprouting (Nadler et al., 1980; Sutula et al., 1988; Buckmaster and Dudek, 1997; Bouilleret et al., 1999; Riban et al., 2002), timm and nissl staining were done on mouse brains after EEG recordings (see Supplementary Materials and Methods). The histological phenotypes were observed in the small fraction (o20%, WT, 1/6 mice; KO, 2/11
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Fig. 4 – Expanded view of EEG traces shown in Fig. 3A and B. The trace number indicates the region of the raw trace shown in Fig. 3. 30–40 Hz rhythms appear in traces #3, 4, 6, 7 in A and #3, 4, 5, 6, 7, 9 in B. Scale bar: 3 s (except 1.5 s in trace #1), 1 mV.
mice) of both wild type and ko mice, respectively. Consistent with previous studies, degeneration of CA1 neurons and dispersion of dentate gyrus granule cell layers with severe reorganization of hippocampal structures were observed (Supplementary Fig. 4). The staining results show little correlation with EEG patterns as previously suggested (Supplementary Fig. 5).
3.
Discussion
3.1. T-type calcium channels contribute to some types of epileptic seizure models This study finds a specific role of α1G T-type calcium channel on hippocampal seizures generated in rodent KA-induced TLE model, mainly via analysis of conventional ko mice. Three types of T-type calcium channels exist in the hippocampal and cortical neurons (Talley et al., 1999; McKay et al., 2006) and their involvement in specific types of seizure models have been shown, such as α1G channel in a GABABreceptor antagonist-induced absence seizure model (Kim et al., 2001) and a juvenile absence syndrome (Singh et al., 2007) and α1H channel in childhood absence epilepsy (Chen et al., 2003; Vitko et al., 2005; Heron et al., 2007; Powell et al., 2009). When all T-type currents are completely blocked with
specific pan-T-type calcium channel blockers in an absence epilepsy model using GAERS strain, seizure still occurs but with a reduction in seizure duration (TP Snutch, unpublished observations). Those results suggest that EEG seizure can be generated even without T-type calcium channels on its GAERS seizure model. Current study shows that α1G calcium channel contribute partially to epileptic seizure duration in KA-induced TLE model. Therefore, all of these studies together indicate the complexity of the underlying mechanisms of even a single seizure model.
3.2. Cav3.1 channel might be negatively related with the δ rhythm generation When KA is administered, first, δ rhythm PSD increases immediately and it returns faster in α1G ko mice than does in wild-type, while other rhythms are being kept enhanced in general (Supplementary Fig. 3). Secondly, mouse slows down and keeps the movement minimal including during severe SE EEG seizures except with convulsive behaviors. Since drowsiness or slow-wave stage in non-REM sleep is strongly correlated with δ rhythm (Crunelli et al., 2006; Zhu et al., 2006; Destexhe et al., 2007), it is plausible that α1G ko mice might get into these stages faster after KA injection compared to wild type mice.
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Fig. 5 – Latency, duration and frequency of the KA-induced acute hippocampal (A–D) and cortical (E–H) EEG seizures in wild type and αlG ko mice. þ/þ, Open circles, n ¼7 animals; / , filled circles, n ¼ 7 animals. p values at two-tailed t-test. (A, E) Plots of duration of each seizure along with its latency. (B, F) Plots of latency and duration of the first EEG seizures. In hippocampal EEGs, latency (þ/þ, 38.5710.8 min, n¼ 7 seizures; / , 35.7714.1 min, n ¼ 7 seizures, p¼ 0.87); duration (þ/þ, 2.4871.40 min, n¼ 7; / , 0.8570.33 min, p¼ 0.28). In cortical EEGs, latency (þ/þ, 40.0711.5 min, n ¼ 7 seizures; / , 34.6713.6 min, n ¼7 seizures, p¼ 0.77); duration (þ/þ, 0.9270.43 min, n ¼7; / , 0.5270.14 min, p ¼0.40). (C, G) Plots of latency, duration and frequency of seizures during SE. In hippocampal EEGs, latency (þ/þ, 114.275.4 min, total 146 seizures; / , 116.075.5 min, total 213 seizures, p¼ 0.82); duration (þ/þ, 4.7870.88 min; / , 2.0270.49 min, 15 recordings, p¼ 0.007); frequency (þ/þ, 11.071.9/hr; / , 18.172.5/hr, 19 recordings, p¼ 0.038); total seizure duration (þ/þ, 36.0573.07 min/hr recording; / , 25.8073.29 min/hr recording, p¼ 0.033). Total seizure number/total recording time (þ/þ, 167/15.48hr, / , 213/15.64 hr). In cortical EEG, latency (þ/þ, 102.576.1 min, total 144 seizures; /, 113.175.9 min, total 200 seizures, p¼ 0.220); duration (þ/þ, 3.7470.69 min; / , 3.0470.70 min, 15 recordings, p¼ 0.49); frequency (þ/þ, 11.071.2/hr; /, 16.372.4/hr, 19 recordings, p¼ 0.081); total seizure duration (þ/þ, 33.1073.67 min/hr recording; /, 31.4873.63 min/hr recording, p¼ 2.037). Total seizure number/total recording time (þ/þ, 144/15.48 hr, /, 200/15.64 hr). (D, H) Cumulative probability plots of seizure latency, duration and frequency. # indicates a significant difference of Kolmogorov-Smirnov test in the distribution of duration of hippocampal EEG (K-S, two-tailed, D ¼ 0.012).
3.3. Relationship between EEG discharge of TLE models and mossy fiber sprouting Hippocampal structural reorganization was observed in small fraction of mice tested (o20% in either type) with timm and nissl staining in this study. One reason might be too short a latency period to observe the histological phenotypes. A second possibility is that this study shows a typical pathological feature of TLE suggested previously, such that the high variability in the structural organization either even within one structure or across the limbic system (Bertram, 2003), or
little correlation between mossy fiber sprouting and EEG discharge (Bragin et al., 1999; Zhang et al., 2002b; Hunt et al., 2013). A third possibility is that the mouse strain, C57BL/6J, one of parental strain of our null mouse, has been shown to be resistant to cell death and synaptic reorganization despite seizure activity after KA injection (McKhann et al., 2003). Therefore, it remains to be seen how α1G T-type channel is exactly involved in the cellular hippocampal organization and mossy fiber growth. Since this study focuses mainly on the EEG phenotype analysis from the conventional ko mice, the conclusions of
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Fig. 6 – Duration and frequency of the KA-induced chronic hippocampal (A, B) and cortical (C, D) EEG seizures in wild type and αlG ko mice. EEGs fromZ 4 days after the KA-injection were analyzed. Both ## and # indicates where K-S test results on the cumulative distribution shape were shown significantly different, Do0.01 and Do0.05, respectively. A) Left, Bar graph of duration of hippocampal seizures per recording (þ/þ, 0.6470.14 min, n ¼76 recordings from 17 animals, total 7630 seizures; /, 0.3870.09 min, n ¼94 recordings from 17 animals, total 10555 seizures, two-tailed t-test, p¼ 0.114). Right, Cumulative probability plot of hippocampal seizure duration. K-S test shows significant difference (Do0.05). B) Left, Bar graph of duration of cortical seizures per recording (þ/þ, 1.2270.25 min, n¼ 78 recordings from 17 animals, total 9181 seizures; / , 0.6370.05 min, n ¼96 recordings from 17 animals, total 13268 seizures, two-tailed t-test, p¼ 0.0117). * indicates a significant ttest (po0.5). Right, Cumulative probability plot of cortical seizure duration. K-S test shows significant difference (Do0.05). C) Left, Bar graph of frequency of hippocampal seizures per recording (þ/þ, 14.671.8/hr; / , 15.671.9/hr, two-tailed t-test, p¼ 0.648). Right, Cumulative probability plot of hippocampal seizure frequency. K-S test shows significant difference (Do0.01). D) Left, Bar graph of frequency of cortical seizures per recording (þ/þ, 25.972.2/hr; / , 29.572.0/hr, two-tailed t-test, p¼ 0.231). Right, Cumulative probability plot of cortical seizure frequency. K-S test shows significant difference (Do0.05).
this study have to be considered more carefully. First, because EEG recording electrode does get signals from multiple regions of brain, it cannot exclude the possibility that the differential hippocampal EEG effects observed might contain signals generated from other areas such as thalamus (Anderson et al., 2005; Crunelli et al., 2006). Moreover, since conventional α1G calcium channel ko mice might establish compensatory phenotypes during development, more study is needed to make a direct causal relationship between α1G calcium channel and the phenotypes observed in this study, i.e. on the possible compensatory role of Cav3.2 expression, which has many diverse functions in addition to seizure (Chemin et al., 2002; Becker et al., 2008; Gangarossa et al., 2014). More refined methods for hippocampal-specific and timely-deletion or expression of α1G calcium channel should be adopted for directly identifying the role of α1G calcium channel in rodent TLE models.
3.4.
Conclusions
It is shown that α1G is mainly expressed in hippocampal interneurons (Vinet and Sik, 2006) and calretinin-containing hippocampal interneurons are involved in TLE by
synchronizing dendritic inhibitory interneurons (Toth and Magloczky, 2014). Therefore, it will be interesting whether α1G are expressed in calretining-containing hippocampal interneurons and what changes are made at both cellular physiology and neural circuit activity level after α1G is deleted in specific, i.e. calretinin-expressing, interneurons. Furthermore, it is also interesting to compare role of α1G with α1H (Cav3.2), which is known to be involved in pilocarpineinduced TLE. Since the increase of intrinsic neuronal bursting is correlated with the appearance of TLE (Sanabria et al., 2001), possible functions of diverse ion channels in TLE models have been pursued (Sheffer and Berkovic, 2003; Nelson et al., 2006). This study asks whether α1G T-type calcium channel plays a critical role in KA-induced TLE model using α1G ko mice since Cav3.1 calcium channel is known to have a critical role in seizure generation in a GABAB-receptor agonist-induced absence seizure model (Song et al., 2004; Kim et al., 2001) and in BAC transgenic mouse induced pure absence epilepsy (Ernst et al., 2009). The result suggests, first, that α1G calcium channel contributes for the maintenance of individual hippocampal seizures as well as for the epileptogenicity during KAinduced SE without affecting the latency of seizures. Secondly,
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it shows that deletion of α1G calcium channel causes an increase of the seizure frequency via an unknown mechanism. Shorter but more frequent hippocampal seizures are generated in α1G ko mice though 10epileptogenicity is reduced. Thirdly, these effects of α1G calcium channel on KA-induced seizures during SE are specific for hippocampal seizures with little effects on cortical seizure properties. In summary this study suggests that α1G calcium channel is involved in the regulation of individual hippocampal seizure duration, the seizure frequency and PSD of EEG rhythms, especially δ rhythm. The result supports the idea that α1G T-type calcium channel play a role in the modulation of epileptogenicity and seizure properties in KA-induced mouse TLE model and will contribute to the efforts of finding the appropriate clinical targets for the treatment and prevention of TLE seizure in the future, though it needs to show direct causal links between α1G and TLE models with more refined methods such as cell type-specific siRNA or conditional knock-down of α1G.
4.
Experimental procedure
4.1.
Animals and drug administration
The generation of α1G T-type calcium channel ko mice was previously described (Kim et al., 2001). α1G null mutation was maintained in two genetic backgrounds, either 129S4/svJae or C57BL/6J. Heterozygote mutants obtained from the chimera were backcrossed into each genetic background more than 10 times ( Z N10). The hybrid F1 α1G/ and wild-type littermate control mice were generated by mating heterozygotes from each of the two genetic backgrounds. These hybrid F1 mice were used for the experiment and were maintained with free access to food and water under a 12 h light/dark cycle, with the light cycle beginning at 8:00 AM. Experiments were done on 3–4 month old adult mice. All animal procedures were in accordance with the regulation of the institutional guideline (KIST, Seoul, Korea). Single i.p. injection of KA (10 mg/kg bw, Tocris, in 0.9% NaCl) has been used for acute and chronic recordings, but this dose sometimes caused fatality. For chronic observation and to prolong the SE while minimizing mortality, lower dose of KA (7 mg/kg bw) injection was made three times (1 /day), to both genotypes (n ¼7) (Hellier et al., 1998). For intrahippocampal injection for chronic recording only, mice were stereotaxically
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positioned and KA (50 mg/kg, 140 Nl) was applied into the right CA1 area of the dorsal hippocampus using a micropump (AP¼ 1.8 mm, ML¼ 1.6 mm, DV¼ 1.9 mm) under general anesthesia (0.2% avertin, 20 ml/kg i.p., Sigma, USA) over 10 min period or the same amount of saline as control. All of the acute recording data were from i.p. injected mice since the intrahippocampal injection after surgery does not allow the immediate acute recording. In case of the chronic recording data, most data were from i.p. injected mice (in WT, 10/10 (100%) from i.p.; in KO, 6/8 from i.p.) and from male mice (in WT, 8/10 from male; in KO, 7/8 from male). Wild type control mice without any treatment were also recorded (n¼ 2, one male and one female mouse).
4.2.
Electrode implantation
For EEG recordings, total three electrodes were implanted on to the mouse brain. One monopolar tungsten teflon-coated electrode (150 μm; WPI, USA) was implanted at the injected ipsilateral hippocampus and the screw electrode (Φ ¼ 1 mm) into the contralateral side of cortical area. A reference screw electrode was also inserted over the cerebellum. All electrodes were fixed to the skull with cyanoacrylate and dental acrylic cement. The mice were returned to the cage after recovery from the anesthesia. The position of the tungsten electrode was verified with Nissl or Timm staining of the brain sections (See Supplementary methods).
4.3.
EEG acquisition and analysis
After recovery from the surgery, EEG was recorded from the awakening mouse in a cage or 500 ml beaker, where thin wires were connected from the electrodes of mouse head to the amplifier. Grass quad AC amplifier (model QP511; Grass technologies, West Warwick, RI, USA), Digidata 1322 Digitizer and pClamp 9.2 software (Molecular Devices, Sunnyvale, CA, USA) were used for EEG acquisition. Raw EEG data was amplified by 5000 and, to remove drift signal and low frequency noise, band-pass filtered at 0.3–100 Hz, digitized at 200 Hz sampling rate, and stored on PC for off-line analysis. Mouse behaviors during acute EEG experiments were digitally recorded with a webcam (Microsoft LifeCam VX-3000). All data were analyzed single-blind test. PSD was calculated by using Fast Fourier Transformation and pwelch method (hanning window, window length: 512,
Fig. 7 – Plots showing the acute (A, B, E, and F) and chronic (C, D, G, and H) effects of KA i.p. injection on the peak frequency of PSD of each EEG recording. t¼ 0, the time or day of KA injection. (A, B) Peak frequency of PSD from each acute hippocampal EEG recording done before and after KA injection (þ/þ, n¼ 56 recordings from 11 animals; / , n ¼45 from 8 animals). K-S test on the cumulative distribution of peak PSD frequency for the early phase (o20 min) between þ/þ and / , D¼ 0.5809; for the late phase ( Z20 min), D ¼0.0066, suggesting significant difference in420 min period (C, D) Peak frequency of PSD from each chronic hippocampal recording (þ/þ, n¼ 95 from 17 animals; / , n ¼ 106 from 17 animals). K-S test between þ/þ and / for the early phase (o25days), D ¼0.1136; for the late phase ( Z25days), D ¼0.6045, suggesting non-significant difference for all chronic periods (E, F) Peak frequency of PSD from each acute cortical EEG recording measured before and after KA injection (þ/þ, n ¼ 58 from 11 animals; / , n ¼ 45 from 8 animals). K-S test between þ/þ and / for the early phase (o20 min), D ¼0.0082; for the late phase (Z 20 min), D ¼0.03386, suggesting clear significant difference in o20 min period, (G, H) Peak frequency of PSD of each chronic cortical EEG recording (þ/þ, n¼ 93 from 17 animals; / , n ¼ 108 from 17 animals). K-S test between þ/þ and / for the early phase (o25days), D¼ 0.0108; for the late phase ( Z25days), D ¼0.1037, suggesting marginal significant difference for o25days period.
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overlap: 256, nfft: 1024) in Clampfit 9.2. PSD of raw EEG data was initially normalized by subtracting its PSD at 50 Hz. The frequency bands were categorized by five rhythmic band ranges: 0.5–4 Hz(δ), 4–8 Hz(θ), 8–13 Hz(α), 13 30 Hz(β) and430 Hz(γ). The time–frequency analysis of EEG data from i.p. injection experiments was made to observe any change of the PSD amplitude at each specified band with short-time Fourier transform (window: hanning, window length: 200, overlap: 50, nfft: 1024) (Supplementary Fig. 1). To see the effect of KA, the PSD of total or each frequency band EEG data was secondly normalized to that of the control by subtracting the average PSD value of total or each frequency band of control EEG, respectively. Therefore, zero PSD indicates no change of PSD after KA injection compared to that of control EEG. Seizure was defined when two criteria were met: 1) the average of the total PSD is larger than that of control EEG, i.e. 40 value after the second PSD normalization step, and 2) the period fulfilling the first condition is 410 s. For chronic EEG data from intrahippocampal injection experiments, the analysis criteria are the same as ones for i.p. EEG data above, except adopting the average of all i.p. control data as the control. The chronic period was counted as the days after KA administration (day¼ 0).
4.4.
Statistical analysis
Group comparisons of latency, frequency and duration of seizure, the change of PSD peak for acute and chronic recording data were performed by two-tailed Student's t-test at α¼ 0.05 for independent samples and by the KolmogorovSmirnov test.
Competing interests The author declares no competing financial interests.
Authors' contributions CHK designed the experiments and acquired, analyzed, interpreted the data. CHK wrote the manuscript and has been accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Acknowledgments This work was supported by the grant from the KIST Institutional Program (Project no. 2E25210) to CHK. The author thanks Jahyun Kim, Dr. Eunjin Hwang and Dr. Jihyun Choi for the help on the programming for data analysis, and Soyeon Um for the help on the staining experiment, and Dr. Hee-Sup Shin for the helpful discussion.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.06.015.
r e f e r e n c e s
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Research Report
Cav3.1 T-type calcium channel modulates the epileptogenicity of hippocampal seizures in the kainic acid-induced temporal lobe epilepsy model Chong-Hyun Kima,b,n a
Center for Neuroscience, Brain Science Institute, Korea Institute of Science & Technology, Seoul 136-791, Republic of Korea Department of Neuroscience, Korea University of Science & Technology, Daejeon 305-333, Republic of Korea
b
ar t ic l e in f o
abs tra ct
Article history:
The molecular mechanism of temporal lobe epilepsy has not been clearly identified. T-type
Accepted 12 June 2015
calcium channels play a role in burst firing in neurons and have been implicated in several
Available online 23 June 2015
seizure models. In this study, the role of Cav3.1 T-type (α1G) calcium channel has been
Keywords:
investigated in the kainic acid (KA)-induced temporal lobe epilepsy model (TLE) by using
Seizure
conventional α1G knock-out (ko) mice. After intraperitoneal (i.p.) administration or
Kainic acid
intrahippocampal injection of KA, depth hippocampal and cortical electroencephalogram
Cav3.1 α1G T-type calcium channel
(EEG) and behavioral monitoring were recorded, and timm and Nissl staining of brain
Status epilepticus
sections were made later. Seizure was mainly identified by EEG signals, rather than
Temporal lobe epilepsy.
behaviorally, with analytic criteria. During the acute status epilepticus (SE) period, both the duration and the frequency of hippocampal seizures were significantly reduced and increased, respectively, in αlG ko mice compared to those of wild type mice. Epileptogenicity, the total period of seizures (hr 1), was also significantly reduced in α1G ko mice. However, the latency of seizure occurrence was not significantly different between wild type and ko mice. These differential effects were not observed in cortical seizures. Furthermore, the injection of KA caused a strong increase in δ rhythm power spectrum density (PSD) of EEG in αlG ko mice compared to that in wild type mice. The results with conventional ko mice indicate that α1G T-type calcium channel plays a modulatory role in the duration and frequency of hippocampal seizures as well as the epileptogenicity of KAinduced TLE in mice, mostly during acute periods. & 2015 Elsevier B.V. All rights reserved.
n Corresponding author at: Center for Neuroscience, Brain Science Institute, Korea Institute of Science & Technology, Hwarang-Ro 14 Gil 5, Seongbuk-Gu, Seoul, 136-791, Korea. Tel.: þ82 2 958 6953; fax: þ82 2 958 6937. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.brainres.2015.06.015 0006-8993/& 2015 Elsevier B.V. All rights reserved.
brain research 1622 (2015) 204–216
1.
Introduction
TLE originates mainly from hippocampus and is one of the most prevalent types of epileptic seizures in human (Sloviter, 1994; Wieser, 2004). The molecular mechanism of TLE is not clearly known and animal models have been developed to elucidate the underlying mechanism of epileptogenicity of TLE. Rodent TLE models are induced by kindling or administration of KA or pilocarpine (PC) (Buckmaster and Dudek, 1997; Bouilleret et al., 1999; Leite et al., 2002; Loscher, 2002; Morimoto et al., 2004). In adult mice, KA is applied i.p. or directly into dorsal hippocampus to induce acute SE and then spontaneous chronic recurrent seizures later (Ben-Ari and Cossart, 2000; Riban et al., 2002). SE causes newly bursting neurons to be formed and many of regular firing neurons to be converted to burst firing neurons (Sanabria et al., 2001). Most hippocampal pyramidal neurons fire action potentials in a regular mode under normal conditions (Sanabria et al., 2001; Jensen et al., 1994). However, SE causes newly bursting neurons to be formed and many of regular firing neurons to be converted to burst firing neurons (Sanabria et al., 2001). It further shows that increase of intrinsic neuronal bursting is correlated with the appearance of TLE. To identify the molecular basis of the change in the neuronal firing property in seizure models, functions of many types of ion channels have been studied (Scheffer and Berkovic, 2003; Nelson et al., 2006). T-type calcium channels are low-voltage activated calcium channels and have three isoforms, α1G, H and I (Huguenard, 1996; Perez-Reyes, 2003). Their activation causes most of low-threshold calcium spikes that can trigger Naþdependent action potential firing as well as other low-voltage activated calcium-dependent processes (Yunker and McEnery, 2003; Huc et al., 2009). Because of the intrinsic contribution to neuronal firing, their role in the transition of firing modes has been studied in neurons of many brain regions such as hippocampus (Magee and Carruth, 1999), cerebellum (Molineux et al., 2006), thalamus and cortex (Perez-Reyes, 2003; Steriade, 2001; Crunelli et al., 2006). Another role of T-type calcium channels has also been suggested in epileptic seizure models. T- and P/Q-type calcium channels appear to contribute to seizure genesis and susceptibility in idiopathic generalized epilepsies (Khosravani and Zamponi, 2006). Absence epilepsy models mutated in high-voltage activated calcium channels have revealed upregulation of T-type calcium current (Zhang et al., 2002a; Song et al., 2004). In the genetic model of absence epilepsy rats from Strasbourg (GAERS), levels of α1G and α1H mRNA were also up-regulated in thalamic neurons (Tsakiridou et al., 1995; Talley et al., 1999). The epileptic activity itself can increase T-type calcium currents of dentate granule cells in human patient and KA or PC-induced epileptic rats (Beck et al., 1998) and of CA1 neurons in kindling study (Faas et al., 1996). Even single episode of SE can increase T-type calcium current of CA1 pyramidal neurons (Su et al., 2002; Yaari et al., 2007; Becker et al., 2008) or alter thalamic α1H channel density in the rodent PC-induced TLE model (Graef et al.,
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2009). These results suggest correlative links between seizure models and T-type channel function. The specific function of each isoform of T-type calcium channel has been revealed in some seizure models by using ko or transgenic mouse due to the lack of isoform-specific inhibitors of T-type calcium channels. Deletion of α1G calcium channel caused resistance to a GABAB-receptor agonistinduced absence seizure generation (Song et al., 2004; Kim et al., 2001). Moreover overexpression of α1G calcium channel in BAC transgenic mouse induced pure absence epilepsy (Emst et al., 2009). Functional polymorphism of α1G and α1H gene was shown in a juvenile absence syndrome (Singh et al., 2007) and in childhood absence epilepsy (Chen et al., 2003; Vitko et al., 2005; Heron et al., 2007; Powell et al., 2009), respectively. Different from absence epilepsy, there have been few attempts on the role of specific T-type calcium channel in TLE models. Recently, Becker et al. (2008) showed that removal of α1H calcium channel reduced the bursting of CA1 neurons and chronic spontaneous seizure frequency in PC-induced model. In this study, I have examined a role of α1G calcium channel in KA-induced TLE model using α1G ko mice for both acute and chronic periods up to 8 months. The results suggest that α1G T-type calcium channel plays a modulatory role in the epileptogenicity of hippocampal seizures.
2.
Results
2.1.
KA induces SE in α1G ko mice
KA has been used to induce TLE models of rodents. However, the developmental time course of TLE induced by KA has not been thoroughly observed from the initial appearance of SE to the chronic periods. In this study, in order to observe both acute and chronic effects of KA induced seizures in TLE mouse models, two ways of KA administration were used. One is the local intrahippocampal injection of KA, revealing direct effects of KA on seizures from the affected area of hippocampus. The intrahippocampal injection does, however, require the recovery period for EEG recordings from the surgery, preventing the stable measurement of acute effects on EEG. Therefore, to record the acute effects of KA, single i.p. injection has been used, which also allows the chronic recordings. KA i.p. injection caused SE in both wild type and αlG ko mice. Seizure can be identified behaviorally or by EEG signal patterns. Although mouse behavior was monitored simultaneously during EEG recording, it was difficult to observe consistent correlation between EEG pattern and mouse behavior, probably due to the difference in the resolution of analyzing signals from these two types of data. Thereby EEG signal was chosen as the main parameter for the data analysis and the description of the results was based on EEG analysis otherwise mentioned in this study. To quantify and compare the power of the EEG signal of specific rhythmic band, PSD analysis was used. First of all, to show the EEG raw data from one mouse after KA treatment for the whole recording period, representative recordings covering both acute and chronic periods had been selected from two types,
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Fig. 1 – Single KA i.p. injection induces acute epileptic seizures and shifts the PSD of depth hippocampal EEG from both male A) wild type and B) αlG ko mice. (a)–(h) and (i)–(q) are each from independent EEG recordings, scale bars: 5 min (except 10 min for g, h, p, and q) and 1 mV. (a) and (j) 30 min Control EEG trace. The peak frequency of PSD is 0.6 Hz. Black lines in (i) and (r). (b) and (k) EEG recordings starting at 5 min after KA injection. (c) and (l) 39 min after KA injection. The animals are inactive during most of the recording period. (d) and (m) 72 min after KA injection. Green lines in (i) and (r). The animals still rarely move. (e) and (n) 113 min after KA injection. (f) and (o) 144 min after KA injection. The peak of PSD is at 1–3 Hz range. (g) and (p) 40 days after KA injection. Brown lines in (i) and (r). (h) and (q) 178 days after KA injection. (i) and (r) PSD plots from each EEG recording shown in A and B. Numeric labeling indicates the region of trace that si expanded in Fig. 2.
respectively. Fig. 1 shows example hippocampal depth EEG recording traces from one wild type (A) and an αlG ko mouse (B), measured at the same time. The control baseline EEG recording was collected before i.p. KA injection (a and j). The EEG seizure spikes appeared shortly (b and k) after KA administration. PSD started to increase (c and l), shown as red line (i and r). The peak of enhanced PSDs was found mostly in δ rhythm range and then seizure subsides slowly (see also Fig. 7A and B). During chronic periods, PSD decreased and returned close to that of control (h, q). To show raw EEG traces much visually identifiable manner, Fig. 2 shows expanded views of the selected regions of Fig. 1 EEG traces. During the control recording period, mice move around and explore the environment, generating θ rhythm as a dominant EEG rhythm (A1, B1). After i.p. KA injection, mice start to move less along with the appearance of seizure spikes (A2, B2), and then epileptic discharges occur frequently (A3and A4, B3 and B4). Ictal spike waves at 1–2 Hz range and interictal spike waves at 30–40 Hz occur (A3–7, B3–8). EEG traces on 40 days (A9 and A10, B11) and 178 days (A11–12, B12) after KA injection show much reduced epileptic activity.
The cortical EEG was recorded simultaneously as a kind of internal control since TLE is known to be originated mainly from hippocampus. Example cortical EEG recordings are shown in Fig. 3. In these mice, the time course of cortical EEG seizure occurrence and its development after KA injection is similar to that of hippocampal EEG. The cortical EEG seizure spikes appeared similarly after KA administration compared to hippocampal EEG seizure spikes in wild type and ko mice, respectively, though the cortical EEG from the wild type looks a bit late in developing seizure spikes and smaller in the magnitude of seizures. PSD was enhanced in most rhythm ranges and the peak PSD was found in δ rhythm range in general. Acute seizures subside slowly like hippocampal seizures. During chronic periods, PSD decreased and returned close to that of control like as hippocampal seizure PSDs. The analysis of seizure properties in detail below, however, shows the difference between hippocampal EEG seizures and cortical EEG ones (Fig. 5). Expanded views of the selected regions of Fig. 3 EEG traces are shown (Fig. 4). Epileptic discharges, ictal- and interictal seizures also appear acutely after KA injection.
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Fig. 2 – Expanded view of EEG traces shown in Fig. 1A and B. The trace number indicates the region of the raw trace shown in Fig. 1. 30–40 Hz rhythms appear in traces #3, 4, 5, 7 of A and #3, 5, 6, 7, 8 of B. Scale bars: 3 s (except 1.5 s in trace #1), 1 mV.
2.2. The duration, frequency and epileptogeneis of KAinduced hippocamapal seizures during SE period are affected in α1G ko mice To examine the effect of KA on the seizure generation, the onset latency of the seizure was measured up to 6 h after KA administration (see Experimental Procedure). Plots of latency and duration of hippocampal and cortical seizures generated after KA injection (t¼0) are shown in Fig. 5 A and E, respectively. Red circle indicates the first seizure of each recording. The latency and duration of the first seizures from α1G ko mice were not significantly different from those of wild type (B and F). When all seizures were considered, the average duration of hippocampal EEG seizures was significantly reduced in α1G ko mice by 58% during SE compared to that of wild type mice, without a significant change in the latency of seizures (C and D). The hippocampal seizure frequency per hour of recording (hr 1) was significantly increased by 64% in α1G ko mice compared to that of wild type mice. The changes in the duration of seizures and the seizure frequency (hr 1) gives a theoretical reduction of epileptogenicity, the total seizure duration per hour (hr 1), by 31% (0.42n1.64¼ 0.69), which is close to the observed reduction (28%, see Fig. 5C legend) of the total hippocampal seizure duration (hr 1) in α1G ko mice (C). Epileptogenicity in α1G ko mice decreases though the seizure frequency increases. In case of cortical seizures, the duration,
frequency, latency of seizures and total seizure duration were not changed in α1G ko mice (G and H). To see the relationship between latency and duration of seizures, pearson correlation coefficient values were calculated. The values of all coefficients (r) were smaller than 70.1, indicating non-significant relationships between latency and duration of seizures (þ/þ, hippocampus, r¼ 0.0165, p¼ 0.84; /, hippocampus, r¼ 0.0612, p¼ 0.38; þ/þ, cortex, r¼0.0564, p¼ 0.51; / , cortex, r¼ 0.00372, p¼0.96). To see the effect of KA on the chronic seizures, overnight EEG recordings on the same mouse were repeated at 1–4 week intervals up to 8 months. The duration of EEG seizure was still significantly smaller in α1G ko mice compared to that of wild type mice (Fig. 6A and B). The frequency of seizure occurrence was maintained a bit higher in α1G ko mice, revealed by K-S test (Fig. 6C and D). These results suggest that the effect of the absence of α1G calcium channel on seizure duration has been kept for the chronic period.
2.3. KA increases EEG rhythmic band powers, especially δ, more in α1G ko mice during SE EEG rhythms are used to define the status of certain brain functions and animal behaviors (Engel et al., 2001; Buzsaki et al., 2003). Though the appearance and pattern of seizure generation were quite variable from mouse to mouse as
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Fig. 3 – Single KA i.p. injection induces acute epileptic seizures and shifts the PSD of depth cortical EEG from both male A) wild type and B) αlG ko mice. Scale bars: 5 min (except 10 min in g, h, p, and q) and 1 mV. (a)–(r) Experimental conditions are the same as in Fig. 1. Numeric labeling indicates the region of trace that is expanded in Fig. 4.
described previously (Williams et al., 2009), KA injection seems to cause an acute increase in the PSD of most frequency ranges examined (Supplementary Fig. 2). In order to see how EEG rhythms develop and progress, the frequency of the peak PSD of each recording was plotted during either acute or chronic periods, respectively (Fig. 7). All PSD peaks were detected at frequencies lower than 10 Hz. The acute effects of KA on hippocampal EEG are shown in Figure 7A and B. The frequencies of peak PSDs from control EEG recordings marked at t¼ 0 are mainly in δ rhythm range in both types of mice (þ/þ, 74%; / , 76%) and the remains are mostly in θ rhythm range, which is described as a mixed pattern (δþθ). However, after KA injection, the frequencies of peak PSDs appear only in δ rhythm range in α1G ko mice, starting less than 10 min ( / , 100%), while the mixed pattern is still observed in wild-type mice (þ/þ, 71% in δ). When K-S test on the distribution of peak PSD frequency was further applied before and after 20 min period between wild type and ko, respectively, significant difference was shown during the late phase (see Figure legends 7A and B). In cortical EEGs, the percentage of recordings having PSD peak frequency in δ rhythm range within 10 min after KA injection is 85% and then stabilizes about 81% up to 160 min in wild-type mice (Fig. 7E). In α1G ko mice, it is 81% at 10 min period and then reduces a bit to 79% (Fig. 7F). The result indicates that the shift of the peak frequency of PSD to δ rhythm band occurs
clearly in hippocampal EEG but does little in cortical EEG PSD in α1G ko mice. K-S test on the peak PSD frequency distribution between wild type and ko indicated significant difference during the early phase of acute period (see legends of Fig. 7E and F). The chronic effects up to 8 months are also shown in Fig. 7C, D, G and H. In wild-type mice PSD peak frequencies of both hippocampal and cortical EEG data are mostly in δ rhythm range at 25 days after KA injection and then the mixed pattern of the peak frequency distribution appears through unknown mechanisms (Fig. 7C). K-S test on the peak PSD frequency distribution between wild type and ko showed marginal significant difference only during the early phase (o25 days) of chronic period of cortical EEG, suggesting that peak PSD frequencies seemed to occur quicker in the cortical area of α1G ko mice (Fig. 7D).
2.4. Cellular and structural pathology within hippocampus To check the phenotypes on hippocampal structural organization and mossy fiber sprouting (Nadler et al., 1980; Sutula et al., 1988; Buckmaster and Dudek, 1997; Bouilleret et al., 1999; Riban et al., 2002), timm and nissl staining were done on mouse brains after EEG recordings (see Supplementary Materials and Methods). The histological phenotypes were observed in the small fraction (o20%, WT, 1/6 mice; KO, 2/11
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Fig. 4 – Expanded view of EEG traces shown in Fig. 3A and B. The trace number indicates the region of the raw trace shown in Fig. 3. 30–40 Hz rhythms appear in traces #3, 4, 6, 7 in A and #3, 4, 5, 6, 7, 9 in B. Scale bar: 3 s (except 1.5 s in trace #1), 1 mV.
mice) of both wild type and ko mice, respectively. Consistent with previous studies, degeneration of CA1 neurons and dispersion of dentate gyrus granule cell layers with severe reorganization of hippocampal structures were observed (Supplementary Fig. 4). The staining results show little correlation with EEG patterns as previously suggested (Supplementary Fig. 5).
3.
Discussion
3.1. T-type calcium channels contribute to some types of epileptic seizure models This study finds a specific role of α1G T-type calcium channel on hippocampal seizures generated in rodent KA-induced TLE model, mainly via analysis of conventional ko mice. Three types of T-type calcium channels exist in the hippocampal and cortical neurons (Talley et al., 1999; McKay et al., 2006) and their involvement in specific types of seizure models have been shown, such as α1G channel in a GABABreceptor antagonist-induced absence seizure model (Kim et al., 2001) and a juvenile absence syndrome (Singh et al., 2007) and α1H channel in childhood absence epilepsy (Chen et al., 2003; Vitko et al., 2005; Heron et al., 2007; Powell et al., 2009). When all T-type currents are completely blocked with
specific pan-T-type calcium channel blockers in an absence epilepsy model using GAERS strain, seizure still occurs but with a reduction in seizure duration (TP Snutch, unpublished observations). Those results suggest that EEG seizure can be generated even without T-type calcium channels on its GAERS seizure model. Current study shows that α1G calcium channel contribute partially to epileptic seizure duration in KA-induced TLE model. Therefore, all of these studies together indicate the complexity of the underlying mechanisms of even a single seizure model.
3.2. Cav3.1 channel might be negatively related with the δ rhythm generation When KA is administered, first, δ rhythm PSD increases immediately and it returns faster in α1G ko mice than does in wild-type, while other rhythms are being kept enhanced in general (Supplementary Fig. 3). Secondly, mouse slows down and keeps the movement minimal including during severe SE EEG seizures except with convulsive behaviors. Since drowsiness or slow-wave stage in non-REM sleep is strongly correlated with δ rhythm (Crunelli et al., 2006; Zhu et al., 2006; Destexhe et al., 2007), it is plausible that α1G ko mice might get into these stages faster after KA injection compared to wild type mice.
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Fig. 5 – Latency, duration and frequency of the KA-induced acute hippocampal (A–D) and cortical (E–H) EEG seizures in wild type and αlG ko mice. þ/þ, Open circles, n ¼7 animals; / , filled circles, n ¼ 7 animals. p values at two-tailed t-test. (A, E) Plots of duration of each seizure along with its latency. (B, F) Plots of latency and duration of the first EEG seizures. In hippocampal EEGs, latency (þ/þ, 38.5710.8 min, n¼ 7 seizures; / , 35.7714.1 min, n ¼ 7 seizures, p¼ 0.87); duration (þ/þ, 2.4871.40 min, n¼ 7; / , 0.8570.33 min, p¼ 0.28). In cortical EEGs, latency (þ/þ, 40.0711.5 min, n ¼ 7 seizures; / , 34.6713.6 min, n ¼7 seizures, p¼ 0.77); duration (þ/þ, 0.9270.43 min, n ¼7; / , 0.5270.14 min, p ¼0.40). (C, G) Plots of latency, duration and frequency of seizures during SE. In hippocampal EEGs, latency (þ/þ, 114.275.4 min, total 146 seizures; / , 116.075.5 min, total 213 seizures, p¼ 0.82); duration (þ/þ, 4.7870.88 min; / , 2.0270.49 min, 15 recordings, p¼ 0.007); frequency (þ/þ, 11.071.9/hr; / , 18.172.5/hr, 19 recordings, p¼ 0.038); total seizure duration (þ/þ, 36.0573.07 min/hr recording; / , 25.8073.29 min/hr recording, p¼ 0.033). Total seizure number/total recording time (þ/þ, 167/15.48hr, / , 213/15.64 hr). In cortical EEG, latency (þ/þ, 102.576.1 min, total 144 seizures; /, 113.175.9 min, total 200 seizures, p¼ 0.220); duration (þ/þ, 3.7470.69 min; / , 3.0470.70 min, 15 recordings, p¼ 0.49); frequency (þ/þ, 11.071.2/hr; /, 16.372.4/hr, 19 recordings, p¼ 0.081); total seizure duration (þ/þ, 33.1073.67 min/hr recording; /, 31.4873.63 min/hr recording, p¼ 2.037). Total seizure number/total recording time (þ/þ, 144/15.48 hr, /, 200/15.64 hr). (D, H) Cumulative probability plots of seizure latency, duration and frequency. # indicates a significant difference of Kolmogorov-Smirnov test in the distribution of duration of hippocampal EEG (K-S, two-tailed, D ¼ 0.012).
3.3. Relationship between EEG discharge of TLE models and mossy fiber sprouting Hippocampal structural reorganization was observed in small fraction of mice tested (o20% in either type) with timm and nissl staining in this study. One reason might be too short a latency period to observe the histological phenotypes. A second possibility is that this study shows a typical pathological feature of TLE suggested previously, such that the high variability in the structural organization either even within one structure or across the limbic system (Bertram, 2003), or
little correlation between mossy fiber sprouting and EEG discharge (Bragin et al., 1999; Zhang et al., 2002b; Hunt et al., 2013). A third possibility is that the mouse strain, C57BL/6J, one of parental strain of our null mouse, has been shown to be resistant to cell death and synaptic reorganization despite seizure activity after KA injection (McKhann et al., 2003). Therefore, it remains to be seen how α1G T-type channel is exactly involved in the cellular hippocampal organization and mossy fiber growth. Since this study focuses mainly on the EEG phenotype analysis from the conventional ko mice, the conclusions of
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Fig. 6 – Duration and frequency of the KA-induced chronic hippocampal (A, B) and cortical (C, D) EEG seizures in wild type and αlG ko mice. EEGs fromZ 4 days after the KA-injection were analyzed. Both ## and # indicates where K-S test results on the cumulative distribution shape were shown significantly different, Do0.01 and Do0.05, respectively. A) Left, Bar graph of duration of hippocampal seizures per recording (þ/þ, 0.6470.14 min, n ¼76 recordings from 17 animals, total 7630 seizures; /, 0.3870.09 min, n ¼94 recordings from 17 animals, total 10555 seizures, two-tailed t-test, p¼ 0.114). Right, Cumulative probability plot of hippocampal seizure duration. K-S test shows significant difference (Do0.05). B) Left, Bar graph of duration of cortical seizures per recording (þ/þ, 1.2270.25 min, n¼ 78 recordings from 17 animals, total 9181 seizures; / , 0.6370.05 min, n ¼96 recordings from 17 animals, total 13268 seizures, two-tailed t-test, p¼ 0.0117). * indicates a significant ttest (po0.5). Right, Cumulative probability plot of cortical seizure duration. K-S test shows significant difference (Do0.05). C) Left, Bar graph of frequency of hippocampal seizures per recording (þ/þ, 14.671.8/hr; / , 15.671.9/hr, two-tailed t-test, p¼ 0.648). Right, Cumulative probability plot of hippocampal seizure frequency. K-S test shows significant difference (Do0.01). D) Left, Bar graph of frequency of cortical seizures per recording (þ/þ, 25.972.2/hr; / , 29.572.0/hr, two-tailed t-test, p¼ 0.231). Right, Cumulative probability plot of cortical seizure frequency. K-S test shows significant difference (Do0.05).
this study have to be considered more carefully. First, because EEG recording electrode does get signals from multiple regions of brain, it cannot exclude the possibility that the differential hippocampal EEG effects observed might contain signals generated from other areas such as thalamus (Anderson et al., 2005; Crunelli et al., 2006). Moreover, since conventional α1G calcium channel ko mice might establish compensatory phenotypes during development, more study is needed to make a direct causal relationship between α1G calcium channel and the phenotypes observed in this study, i.e. on the possible compensatory role of Cav3.2 expression, which has many diverse functions in addition to seizure (Chemin et al., 2002; Becker et al., 2008; Gangarossa et al., 2014). More refined methods for hippocampal-specific and timely-deletion or expression of α1G calcium channel should be adopted for directly identifying the role of α1G calcium channel in rodent TLE models.
3.4.
Conclusions
It is shown that α1G is mainly expressed in hippocampal interneurons (Vinet and Sik, 2006) and calretinin-containing hippocampal interneurons are involved in TLE by
synchronizing dendritic inhibitory interneurons (Toth and Magloczky, 2014). Therefore, it will be interesting whether α1G are expressed in calretining-containing hippocampal interneurons and what changes are made at both cellular physiology and neural circuit activity level after α1G is deleted in specific, i.e. calretinin-expressing, interneurons. Furthermore, it is also interesting to compare role of α1G with α1H (Cav3.2), which is known to be involved in pilocarpineinduced TLE. Since the increase of intrinsic neuronal bursting is correlated with the appearance of TLE (Sanabria et al., 2001), possible functions of diverse ion channels in TLE models have been pursued (Sheffer and Berkovic, 2003; Nelson et al., 2006). This study asks whether α1G T-type calcium channel plays a critical role in KA-induced TLE model using α1G ko mice since Cav3.1 calcium channel is known to have a critical role in seizure generation in a GABAB-receptor agonist-induced absence seizure model (Song et al., 2004; Kim et al., 2001) and in BAC transgenic mouse induced pure absence epilepsy (Ernst et al., 2009). The result suggests, first, that α1G calcium channel contributes for the maintenance of individual hippocampal seizures as well as for the epileptogenicity during KAinduced SE without affecting the latency of seizures. Secondly,
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it shows that deletion of α1G calcium channel causes an increase of the seizure frequency via an unknown mechanism. Shorter but more frequent hippocampal seizures are generated in α1G ko mice though 10epileptogenicity is reduced. Thirdly, these effects of α1G calcium channel on KA-induced seizures during SE are specific for hippocampal seizures with little effects on cortical seizure properties. In summary this study suggests that α1G calcium channel is involved in the regulation of individual hippocampal seizure duration, the seizure frequency and PSD of EEG rhythms, especially δ rhythm. The result supports the idea that α1G T-type calcium channel play a role in the modulation of epileptogenicity and seizure properties in KA-induced mouse TLE model and will contribute to the efforts of finding the appropriate clinical targets for the treatment and prevention of TLE seizure in the future, though it needs to show direct causal links between α1G and TLE models with more refined methods such as cell type-specific siRNA or conditional knock-down of α1G.
4.
Experimental procedure
4.1.
Animals and drug administration
The generation of α1G T-type calcium channel ko mice was previously described (Kim et al., 2001). α1G null mutation was maintained in two genetic backgrounds, either 129S4/svJae or C57BL/6J. Heterozygote mutants obtained from the chimera were backcrossed into each genetic background more than 10 times ( Z N10). The hybrid F1 α1G/ and wild-type littermate control mice were generated by mating heterozygotes from each of the two genetic backgrounds. These hybrid F1 mice were used for the experiment and were maintained with free access to food and water under a 12 h light/dark cycle, with the light cycle beginning at 8:00 AM. Experiments were done on 3–4 month old adult mice. All animal procedures were in accordance with the regulation of the institutional guideline (KIST, Seoul, Korea). Single i.p. injection of KA (10 mg/kg bw, Tocris, in 0.9% NaCl) has been used for acute and chronic recordings, but this dose sometimes caused fatality. For chronic observation and to prolong the SE while minimizing mortality, lower dose of KA (7 mg/kg bw) injection was made three times (1 /day), to both genotypes (n ¼7) (Hellier et al., 1998). For intrahippocampal injection for chronic recording only, mice were stereotaxically
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positioned and KA (50 mg/kg, 140 Nl) was applied into the right CA1 area of the dorsal hippocampus using a micropump (AP¼ 1.8 mm, ML¼ 1.6 mm, DV¼ 1.9 mm) under general anesthesia (0.2% avertin, 20 ml/kg i.p., Sigma, USA) over 10 min period or the same amount of saline as control. All of the acute recording data were from i.p. injected mice since the intrahippocampal injection after surgery does not allow the immediate acute recording. In case of the chronic recording data, most data were from i.p. injected mice (in WT, 10/10 (100%) from i.p.; in KO, 6/8 from i.p.) and from male mice (in WT, 8/10 from male; in KO, 7/8 from male). Wild type control mice without any treatment were also recorded (n¼ 2, one male and one female mouse).
4.2.
Electrode implantation
For EEG recordings, total three electrodes were implanted on to the mouse brain. One monopolar tungsten teflon-coated electrode (150 μm; WPI, USA) was implanted at the injected ipsilateral hippocampus and the screw electrode (Φ ¼ 1 mm) into the contralateral side of cortical area. A reference screw electrode was also inserted over the cerebellum. All electrodes were fixed to the skull with cyanoacrylate and dental acrylic cement. The mice were returned to the cage after recovery from the anesthesia. The position of the tungsten electrode was verified with Nissl or Timm staining of the brain sections (See Supplementary methods).
4.3.
EEG acquisition and analysis
After recovery from the surgery, EEG was recorded from the awakening mouse in a cage or 500 ml beaker, where thin wires were connected from the electrodes of mouse head to the amplifier. Grass quad AC amplifier (model QP511; Grass technologies, West Warwick, RI, USA), Digidata 1322 Digitizer and pClamp 9.2 software (Molecular Devices, Sunnyvale, CA, USA) were used for EEG acquisition. Raw EEG data was amplified by 5000 and, to remove drift signal and low frequency noise, band-pass filtered at 0.3–100 Hz, digitized at 200 Hz sampling rate, and stored on PC for off-line analysis. Mouse behaviors during acute EEG experiments were digitally recorded with a webcam (Microsoft LifeCam VX-3000). All data were analyzed single-blind test. PSD was calculated by using Fast Fourier Transformation and pwelch method (hanning window, window length: 512,
Fig. 7 – Plots showing the acute (A, B, E, and F) and chronic (C, D, G, and H) effects of KA i.p. injection on the peak frequency of PSD of each EEG recording. t¼ 0, the time or day of KA injection. (A, B) Peak frequency of PSD from each acute hippocampal EEG recording done before and after KA injection (þ/þ, n¼ 56 recordings from 11 animals; / , n ¼45 from 8 animals). K-S test on the cumulative distribution of peak PSD frequency for the early phase (o20 min) between þ/þ and / , D¼ 0.5809; for the late phase ( Z20 min), D ¼0.0066, suggesting significant difference in420 min period (C, D) Peak frequency of PSD from each chronic hippocampal recording (þ/þ, n¼ 95 from 17 animals; / , n ¼ 106 from 17 animals). K-S test between þ/þ and / for the early phase (o25days), D ¼0.1136; for the late phase ( Z25days), D ¼0.6045, suggesting non-significant difference for all chronic periods (E, F) Peak frequency of PSD from each acute cortical EEG recording measured before and after KA injection (þ/þ, n ¼ 58 from 11 animals; / , n ¼ 45 from 8 animals). K-S test between þ/þ and / for the early phase (o20 min), D ¼0.0082; for the late phase (Z 20 min), D ¼0.03386, suggesting clear significant difference in o20 min period, (G, H) Peak frequency of PSD of each chronic cortical EEG recording (þ/þ, n¼ 93 from 17 animals; / , n ¼ 108 from 17 animals). K-S test between þ/þ and / for the early phase (o25days), D¼ 0.0108; for the late phase ( Z25days), D ¼0.1037, suggesting marginal significant difference for o25days period.
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overlap: 256, nfft: 1024) in Clampfit 9.2. PSD of raw EEG data was initially normalized by subtracting its PSD at 50 Hz. The frequency bands were categorized by five rhythmic band ranges: 0.5–4 Hz(δ), 4–8 Hz(θ), 8–13 Hz(α), 13 30 Hz(β) and430 Hz(γ). The time–frequency analysis of EEG data from i.p. injection experiments was made to observe any change of the PSD amplitude at each specified band with short-time Fourier transform (window: hanning, window length: 200, overlap: 50, nfft: 1024) (Supplementary Fig. 1). To see the effect of KA, the PSD of total or each frequency band EEG data was secondly normalized to that of the control by subtracting the average PSD value of total or each frequency band of control EEG, respectively. Therefore, zero PSD indicates no change of PSD after KA injection compared to that of control EEG. Seizure was defined when two criteria were met: 1) the average of the total PSD is larger than that of control EEG, i.e. 40 value after the second PSD normalization step, and 2) the period fulfilling the first condition is 410 s. For chronic EEG data from intrahippocampal injection experiments, the analysis criteria are the same as ones for i.p. EEG data above, except adopting the average of all i.p. control data as the control. The chronic period was counted as the days after KA administration (day¼ 0).
4.4.
Statistical analysis
Group comparisons of latency, frequency and duration of seizure, the change of PSD peak for acute and chronic recording data were performed by two-tailed Student's t-test at α¼ 0.05 for independent samples and by the KolmogorovSmirnov test.
Competing interests The author declares no competing financial interests.
Authors' contributions CHK designed the experiments and acquired, analyzed, interpreted the data. CHK wrote the manuscript and has been accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Acknowledgments This work was supported by the grant from the KIST Institutional Program (Project no. 2E25210) to CHK. The author thanks Jahyun Kim, Dr. Eunjin Hwang and Dr. Jihyun Choi for the help on the programming for data analysis, and Soyeon Um for the help on the staining experiment, and Dr. Hee-Sup Shin for the helpful discussion.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.06.015.
r e f e r e n c e s
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