The Journal of Neuroscience, June 2, 2004, 24(22):5249-5257; doi:10.1523/JNEUROSCI.5546-03.2004
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Neurobiology of Disease
Role of the 
1G T-Type Calcium Channel in Spontaneous Absence Seizures in Mutant Mice
Inseon Song,1,2
Daesoo Kim,1
Soonwook Choi,1
Minjeong Sun,1,2
Yeongin Kim,3 and
Hee-Sup Shin1
1Center for Calcium and Learning, Korea Institute of Science and Technology, Cheongryang, Seoul, 136-791, Korea, 2Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea, and 3Department of Neurology, Kangnam St. Mary's Hospital, Catholic University of Korea, Seocho-gu, Seoul, 137-701, Korea
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Abstract
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Alterations in thalamic T-type Ca2+ channels are thought to contribute to the pathogenesis of absence seizures. Here, we found that mice with a null mutation for the pore-forming 
1A subunits of P/Q-type channels (1A–/– mice) were prone to absence seizures characterized by typical spike-and-wave discharges (SWDs) and behavioral arrests. Isolated thalamocortical relay (TC) neurons from these mice showed increased T-type Ca2+ currents in vitro. To examine the role of increased T-currents in 1A–/– TC neurons, we cross-bred 1A–/– mice with mice harboring a null mutation for the gene encoding 1G, a major isotype of T-type Ca2+ channels in TC neurons. 1A–/–/1G–/– mice showed a complete loss of T-type Ca2+ currents in TC neurons and displayed no SWDs. Interestingly, 1A–/–/1G+/– mice had 75% of the T-type Ca2+ currents in TC neurons observed in 1A+/+/1G+/+ mice and showed SWD activity that was quantitatively similar to that in 1A–/–/1G+/+ mice. Similar results were obtained using double-mutant mice harboring the 1G mutation plus another mutation also used as a model for absence seizures, i.e., lethargic (
4lh/lh), tottering (1Atg/tg), or stargazer (
2stg/stg). The present results reveal that 1G T-type Ca2+ channels play a critical role in the genesis of spontaneous absence seizures resulting from hypofunctioning P/Q-type channels, but that the augmentation of thalamic T-type Ca2+ currents is not an essential step in the genesis of absence seizures.
Key words: calcium; Ca; EEG; electroencephalogram; epilepsy; gene; mutant; thalamus
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Introduction
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Pharmacological studies suggest that low-voltage-activated T-type Ca2+ channels are involved in the genesis of absence seizures, which are characterized by spike-and-wave discharges (SWDs) (van Luijtelaar et al., 2000
; Porcello et al., 2003). Antagonists of T-type Ca2+ channels suppress both slow intrathalamic rhythms in vitro (Porcello et al., 2003) and SWDs in human absence seizure patients and in rodent models of absence seizures (Heller et al., 1983; Hosford et al., 1992; van Luijtelaar et al., 2000). Previous genetic studies indicate that of the three 1 subunits of T-type Ca2+ channels (G, H, and I), the 1G subunit is critically involved in SWD genesis. Mice with a null mutation of the 1G gene lacked low-threshold burst firing in thalamocortical relay (TC) neurons in vitro (Kim et al., 2001) and in vivo (Kim et al., 2003) and were resistant to SWDs induced by GABAB-receptor agonists (Kim et al., 2001).
T-type Ca2+ channel activity has been studied in rodent models of absence seizures to determine the role of this channel in SWD genesis. The augmentation of T-type Ca2+ currents in the thalamus was first reported in studies using genetic absence epilepsy rats from Strasbourg (Tsakiridou et al., 1995; Talley et al., 2000). Computational modeling studies support the concept that augmented T-type Ca2+ currents increase the number of burst spikes and thereby enhance thalamic synchrony (Destexhe et al., 1996, 1998; Hughes et al., 1999; Thomas and Grisar, 2000). These studies imply that functional enhancement of the T-type Ca2+ channel can contribute to the development of absence seizures by enhancing the probability of thalamocortical hypersynchronization. Mice with mutations in various subunits of the high-voltage-activated (HVA) Ca2+ channels, namely tottering (1Atg/tg), lethargic (4lh/lh), and stargazer (2stg/stg) mice, display SWDs (Noebels and Sidman, 1979; Hosford et al., 1992; Qiao and Noebels, 1993). Recently, these mutant mice were shown to have higher T-type Ca2+ current levels in their TC neurons compared with normal mice, suggesting a possibility that the enhancement of T-currents might underlie the pathogenesis of absence seizures in those mutants (Tsakiridou et al., 1995; Zhang et al., 2002).
To address these issues, we examined absence seizures in mice with a null mutation in the gene coding for pore-forming 1A subunits (1A–/– mice), which therefore lack the P/Q-type Ca2+ currents. We then explored the role of T-type Ca2+ channels in the genesis of absence seizures in these null mice and other spontaneous mutant mice. Our results provide in vivo evidence that baseline T-type Ca2+ currents but not their augmentation in TCs are necessary and sufficient to support absence seizures in various genetic mouse models.
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Materials and Methods
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Animals. The 1A mice used for EEG recording and patch-clamp analyses were F2 progeny derived from intercrossing heterozygotes of the F1 (129/svxC57BL/6J) genetic background. Mice heterozygous for the 1A null mutation (Jun et al., 1999), lethargic (4lh/lh), tottering (1Atg/tg), or stargazer (2stg/stg) were mated with 1G–/– mice (Kim et al., 2001) to obtain 1A+/–/1G+/–, 1Atg/+/1G+/–, 4lh/+/1G+/–, 2stg/+/1G+/– offspring. Double-heterozygous mice for the two mutations were intercrossed to obtain 1A–/–, 4lh/lh, 1Atg/tg, 2stg/stg mice with different numbers of 1G gene alleles. These mice allowed examination of the effect of deleting 1G on the background of absence seizures in 1A–/– mice. In addition, the mice allowed investigation of the pathological role of T-current enhancement in absence seizures. The animals were housed at room temperature (22°C), fed ad libitum, and submitted to a 12 hr light/dark cycle. All handling of mice was in accordance with the regulations of the institute.
Electrode implantation and cortical EEG recording. Differential EEG recording was performed as described previously (Kim et al., 2001). Mice were anesthetized with avertin (tribromoethyl alcohol/tertiary amyl alcohol; Aldrich, Milwaukee, WI). Subdural tungsten electrodes (A-M Systems, Carlsborg, WA) were bilaterally or unilaterally implanted in the temporal lobe region, and a ground electrode was implanted in the occipital region of the brain (Schridde and van Luijtelaar, 2004). The head mount was secured using dental cement, and mice were allowed to recover for at least 24 hr before EEG recordings. EEG activity (sampling frequency, 200 µsec) was recorded during 0.5–1 hr samples for 1–2 hr using a pCLAMP8.0 program (Axon Instruments, Foster City, CA). Only SWDs with a minimum voltage amplitude of twice the background EEG and a minimum duration of 0.7 sec were included in analysis, and SWDs separated by <1 sec were regarded as a single SWD event.
To test the effects of drugs on absence seizures, EEGs of 1A–/– mice (3–4 weeks old) were recorded for 2 hr, starting 1 hr before drug administration. Valproic acid (Sigma, St. Louis, MO) or ethoxusimide (Sigma) was diluted in physiological saline (0.85% NaCl) and injected intraperitoneally. The selection of the drug dose was based on published data (Heller et al., 1983; Aizawa et al., 1997) and preliminary experiments.
Data analysis. EEG signals were amplified, filtered, and recorded using pCLAMP8 software (Axon Instruments). To assess the difference in EEG activity between each group of mice, we used the linear spectra of consecutive EEG data sections (duration over 1 min; range, 1–15 Hz) computed using the pCLAMP8 program using the fast Fourier transform.
Northern blot. Total RNA was isolated from the thalamic region of 3- to 4-week-old mice. RNA (20 µg per lane) was separated on 1.0% agarose gels containing 2.2 M formaldehyde and then transferred to nylon membranes by capillary blot. The hybridization solution comprised 7% SDS, 1% BSA, 0.5 M NaHPO4, 1 mM EDTA, and a random-primed rat cDNA probe corresponding to nucleotides 4699–6174 bp of the 1G clone. A glyceraldehyde 3-phosphate dehydrogenase probe was also hybridized to blots, and this signal was used to normalize for RNA loading. Signal detection and normalization were performed using the ImageQuant Image Analysis system (Amersham Biosciences, Arlington Heights, IL). Concentrations are expressed as "percentage of wild-type control" analyzed on the same blots.
Whole-cell voltage-clamp analysis. Patch-clamp analysis of thalamic relay neurons was performed as described previously (Kim et al., 2001). Thalamic relay neurons were acutely dissociated as described previously (Tsakiridou et al., 1995; Raman and Bean, 1999). Briefly, brains were cooled rapidly in ice-chilled slicing solution consisting of (in mM): 122 NaCl, 26 NaHCO3, 1.2 NaH2PO4, 2 MgCl2, 2 CaCl2, 3 KCl and 10 glucose, after which 300 µm sections were cut in the coronal plane using a Vibratome (Ted Pella, Redding, CA). Slices containing the ventrobasal complex were dissected with a scalpel to isolate the thalamus. Thalamic slices were incubated at 35°C for 6 min with protease XXIII (3 mg/ml; Sigma) in an oxygenated HEPES-buffered solution consisting of (in mM): 82 Na2SO4, 30KSO4, 5 MgCl2, 10 HEPES, 10 glucose, 0.01% phenol red, and adjusted to pH 7.4 with NaOH. The enzymatic reaction was stopped by adding BSA (1 mg/ml; Sigma) and trypsin inhibitor (1 mg/ml; Sigma). Each thalamic slice was triturated with fire-polished Pasteur pipettes and plated onto a recording chamber. Healthy-looking neurons of triangular or multipolar shapes with processed dendrites were used for patch-clamp recordings. Recordings were performed using electrodes (3.5–6.5 M
) fabricated from borosilicate glass (Warner Instruments, Hamden, CT) in an extracellular solution consisting of (in mM): 55 TEA-Cl, 3 CaCl2, 10 HEPES, adjusted to pH 7.4 with TEA-OH. Patch pipettes were filled with a solution containing (in mM): 110 TrisPO4 dibasic, 28 Tris-base, 11 EGTA, 2 MaCl2, 0.5 CaCl2, 4 Na2ATP, 0.3 GTP-Na, 0.001 TTX, pH 7.3. The series resistance compensation (>60%) was used routinely, and patch-recording data with access resistance (>20 M) were discarded. The currents were leak-subtracted using a P/4 protocol. Signals were digitized using an Axopatch 200-B amplifier (Axon Instruments) and analyzed using pCLAMP8 software (Axon Instruments).
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Results
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Absence seizures with 3 Hz SWDs in 1A–/– mice
1A–/– mice develop progressive neurological symptoms characterized specifically by ataxia and dystonia, before dying 
4 weeks after birth (Jun et al., 1999). We recorded cortical EEG activities in 1A–/– and wild-type mice at 3–4 weeks of age. We found that 1A–/– mice (n = 10) exhibited spontaneous 3–5 Hz SWDs (Fig. 1A) and that each episode was accompanied by behavioral arrest, often with twitching of the vibrissa. These abnormal cortical activities were not observed in wild-type littermates (Fig. 1A). SWDs occurred 160 times per hour in 1A–/– mice (Table 1). We examined the effect of the anti-epileptic drugs ethoxusimide (150 mg/kg; n = 6) and valproic acid (10 mg/kg; n = 5) on the incidence of SWDs in 1A–/– mice. These drugs are documented to suppress absence seizure SWDs in humans and rodents (Heller et al., 1983; Hosford et al., 1992). Compared with vehicle-treated 1A–/– mice, we found that ethoxusimide decreased the incidence of SWDs from 161.7 ± 40.5 to 40.8 ± 18.6 per hour, whereas valproic acid reduced the incidence from 181.6 ± 28.9 to 64.4 ± 26.6 per hour (Fig. 1B). Taken together, the behavioral, electrographic, and pharmacological characters of seizures in 1A–/– mice are similar to those of mice absence seizures(Hosford et al., 1992; Aizawa et al., 1997).
Generation of 1A–/–mice with differing numbers of 1G alleles
1G is one of the T-type Ca2+ channels highly expressed in thalamic relay neurons and is selectively involved in SWD seizures induced by GABAB receptor agonists (Kim et al., 2001). We sought to determine whether 1G T-type Ca2+ channels are pathophysiologically involved in the generation and expression of absence seizures in 1A–/– mice. 1G+/– and 1A+/– mice were cross-bred to generate double heterozygotes, 1A+/–/1G+/– mice. A result of double heterozygote matings was 1A–/– mice with different numbers of 1G gene alleles, i.e., 1A–/–/1G+/+, 1A–/–/1G+/–, and 1A–/–/1G–/–. On visual inspection, 1A–/–/1G+/– and 1A–/–/1G–/– mice exhibited severe ataxia and weakness, similar to that observed in 1A–/– single knock-out mice (Jun et al., 1999). In addition, 1A–/–/1G+/– and 1A–/–/1G–/– mice did not survive past weaning.
Decreased HVA Ca2+ currents in 1A–/– thalamic relay neurons
We performed whole-cell voltage-clamp experiments to examine the effect of 1A genetic deletion on HVA Ca2+ currents in acutely dissociated TC neurons, which are characterized by their large size and triangular or multipolar shape with truncated dendrites (Huguenard and Prince, 1992; Pape et al., 1994; Kim et al., 2001). HVA Ca2+ currents, supported by 3 mM Ca2+ as a charge carrier, were activated by step depolarization from a holding potential of –60 mV. As a result, large sustained voltage-dependent Ca2+ inward currents were evoked. Figure 2A shows a typical trace of total Ca2+ currents with a slowly inactivating component recorded from TC neurons. A significant difference was observed between neurons from 1A–/–/1G+/+ mice compared with neurons of control (1A+/+/1G+/+) mice in terms of the amplitude of Ca2+ currents (Fig. 2A). The amplitudes of HVA Ca2+ currents at all command membrane potentials were smaller in 1A–/–/1G+/+ (n = 12) and 1A–/–/1G–/– (n = 11) TC neurons compared with controls (n = 10). This decrease in HVA Ca2+ current can be accounted for by the loss of P/Q-type Ca2+ currents in 1A–/– mice, consistent with previous results in which P/Q-type Ca2+ currents were shown to be a component of HVA Ca2+ currents in TC neurons (Pfrieger et al., 1992; Kammermeier and Jones, 1997).
To determine the current–voltage (I–V) relationship of total HVA Ca2+ currents in neurons from 1A–/–/1G+/+ (n = 10) and control (n = 8) mice, a series of voltage steps from –70 to +20 mV with +10 mV increments was delivered from a holding potential at –70 mV. The I–V relationship curve in control mice revealed a prominent shoulder at negative potentials, indicating channel activation at low voltages, with the peak of the I–V curve occurring at –10 mV (Fig. 2B). In contrast to these findings using control neurons, in neurons from 1A–/–/1G+/+ mice, the I–V relationships showed a significant decrease in the amplitude of the HVA Ca2+ current in voltage steps from –30 to +20 mV, peaking near –10 mV without a change in the shape of the I–V curves (Fig. 2B). We examined the profile of HVA Ca2+ currents in TC cells from neurons of 1A–/–/1G–/– and 1A–/–/1G+/– (data not shown) mice and found no difference in HVA Ca2+ currents between these two genotypes and 1A–/–/1G+/+ mice (Fig. 2A). These findings indicate that genetic reduction of T-type Ca2+ currents does not strongly modulate HVA Ca2+ currents for compensation. We quantitatively compared the peak amplitude of inward Ca2+ currents evoked by depolarization from –60 to –10 mV in TC neurons from control and 1A–/–/1G+/+ mice. Pooled data showed that the total HVA Ca2+ current was 312.3 ± 14.5 pA in neurons of controls (n = 10) (Fig. 2C, black bars) and 149.8 ± 18 pA in neurons of 1A–/–/1G+/+ (n = 12) (Fig. 2C, gray bars) mice (p < 0.001); however, the averaged peak amplitude of the HVA Ca2+ current in neurons of 1A–/–/1G–/– mice at –10 mV (139.2 ± 12.9 pA; n = 11) (Fig. 2C, white bars) was similar to that of neurons of 1A–/–/1G+/+ mice (149.8 ± 18 pA; n = 12) (Fig. 2C, gray bars) (p > 0.05). The differences in the peak amplitudes of HVA Ca2+ currents between neurons of 1A–/–/1G+/+ and control mice were not caused by differences in the surface area of cells because the value of the whole-cell capacitance was not different between 1A–/–/1G+/+ (11.5 ± 0.6 pF; n = 12) and control (11.5 ± 0.9 pF; n = 10) cells.
Increased T-type calcium currents in 1A–/– thalamic relay neurons
To examine T-type Ca2+ currents, we used a voltage protocol in which a voltage step from –110 to –45 mV activates transient T-currents (Huguenard and Prince, 1992) (Fig. 3A). This T-current reached peak amplitude 10 msec after onset of the 100 msec voltage step and then inactivated rapidly. The TC neurons from 1A–/–/1G–/– mice displayed a near complete loss of T-type Ca2+ currents (n = 8) (Fig. 3A), consistent with previous data (Kim et al., 2001). In contrast, transient Ca2+ currents of larger amplitude were evoked from 1A–/–/1G+/+ cells compared with control (1A+/+/1G+/+) cells (Fig. 3A). The data presented in Figure 3B show that the averaged peak value of T-type Ca2+ currents at –45 mV was significantly larger in 1A–/–/1G+/+ neurons (264.4 ± 24.6 pA; n = 10) (Fig. 3B, gray bars) than in controls (165.2 ± 10.5 pA; n = 13) (black bars) (p < 0.0005). In contrast, the peak amplitude of T-currents from 1A–/–/1G+/– neurons was 125.3 ± 10.0 pA (n = 18) (Fig. 3B, dark gray bars), which is 50% of that in 1A–/–/1G+/+ and 75% of that in control TC cells (p < 0.005). These properties of T-type Ca2+ currents from each genotype were consistent with the current density histogram of peak T-currents (Fig. 3C). These data indicate that the enhancement of T-type Ca2+ currents was not caused by an increase in TC neuron membrane size. No significant difference in time-to-peak was observed between groups [8.2 ± 0.4 msec for control (n = 11), 8.4 ± 0.6 msec for 1A–/–/1G+/+ (n = 11), and 8.1 ± 0.5 msec for 1A–/–/1G+/– (n = 18) TC cells].
One explanation for augmented T-type Ca2+ currents in 1A–/– TC cells is a change in voltage dependence, such as steady-state inactivation to a more depolarized level. To obtain steady-state inactivation curves, we delivered a prepulse to various membrane potentials before a –40 mV test stimulus. Current traces of steady-state inactivation of T-currents are shown in Figure 4A. The data presented in Figure 4B show that despite increased T-type Ca2+ current amplitude, steady-state inactivation was similar for both groups when peak current values from the test pulses were normalized to the maximal current amplitude in each cell.
We examined the possibility that the alteration in T-current amplitudes was caused by an increase in the amount of T-type Ca2+ channels. Using rat 1G cDNA [nucleotide (nt) 4699–6174] as a probe in Northern blot analysis, we examined 1G gene expression in thalamus tissue isolated from 1A–/– and 1A+/+ mice. Although no signal was detected in 1G–/– thalamic tissues, visual examination of autoradiographs indicated similar 1G mRNA expression in thalamic tissues from both 1A–/– (n = 5) and 1A+/+ (n = 4) mice (Fig. 5). Quantitative image analysis confirmed that there was no significant difference in expression (94 ± 9 for 1A–/– and 100 ± 5% for 1A+/+; p > 0.5).
Effect of 1G allele number on SWD generation in 1A–/– mice
We investigated whether 1G genes were necessary for spontaneous absence seizures in 1A–/– mice, because they are functionally involved in drug-induced SWD seizures (Kim et al., 2001). Subdural EEG measurements were conducted on 3-week-old 1A–/–/1G+/+ (n = 5) and 1A–/–/1G–/– (n = 4) mice. We found that 1A–/–/1G–/– mice did not exhibit the typical 3–4 Hz SWDs that were observed in 1A–/–/1G+/+ littermates (Fig. 6A) (157.5 ± 43.3 per hour for 1A–/–/1G+/+ and 0 ± 0 per hour for 1A–/–/1G–/– mice; p < 0.005) (Table 1). A power spectrum analysis confirmed this alteration in cortical paroxysmal activity, i.e., disappearance of the 3 Hz peak frequency in 1A–/–/1G–/– compared with 1A–/–/1G+/+ mice (Fig. 7A). It appears that complete genetic deletion of 1G genes functionally abolishes generation of the spontaneous cortical SWD activity observed in 1A–/–/1G+/+ mice.
We investigated whether augmentation of thalamic T-currents was necessary for absence seizures. We examined SWDs in 1G heterozygote mice on a 1A–/– background. These mice were shown previously to exhibit 75% of the T-currents observed in wild-type mice (Fig. 3). Such experiments present an opportunity to investigate the functional consequence of reduced T-current on absence seizures caused by lack of 1A subunits. If increased TC neuron T-current (compared with wild type) is a causative factor in SWD genesis in 1A–/– mice, mice with reduced or the same level of T-currents compared with nonepileptic mice should express no or altered SWD cortical activity. Contrary to our expectations, we found no significant difference between 1A–/–/1G+/– and 1A–/–/1G+/+ mice in terms of SWD duration or frequency (Table 1).
Role of 1G in SWD generation in various absence seizure mice models
Having found that SWDs in 1A/G double-mutant mice differed according to 1G allele "dose," we examined the role of 1G in the genesis of SWDs in other mouse models of absence seizures. We again cross-bred heterozygote mice to produce double mutants containing 1G–/– and 1Atg/tg (Noebels and Sidman, 1979), 4lh/lh (Hosford et al., 1992), or 2stg/stg (Noebels et al., 1990). Our study was on the basis of findings that compared with neurons of control mice, T-type Ca2+ currents are greater in thalamic relay neurons from 1Atg/tg, 4lh/lh, and 2stg/stg mice (146, 151, and 145% of control, respectively) (Zhang et al., 2002). We performed EEG recordings in freely moving young (3–4 week) 1Atg/tg/1G+/+ (n = 3), 4lh/lh/1G+/+(n = 4), and 2stg/stg/1G+/+ (n = 3) mice on a mixed genetic background (129/svxC57BL/6J). These mice exhibited 5–7 Hz SWDs with high amplitude, with the morphology and dominant frequency of the SWDs being slightly different among groups (Fig. 6B–D, Table 1). During a period of robust cortical paroxysmal activity, all mice exhibited a sudden behavioral arrest of movement and a fixed posture, indicating behavioral absence seizures. The mean seizure durations were 2.2 ± 0.2 sec (range, 0.7–6 sec) for 1Atg/tg/1G+/+ mice, 1.5 ± 0.1 sec (0.7–5 sec) for 4lh/lh/1G+/+ mice, and 1.9 ± 0.2 sec (0.7–10 sec) for 2stg/stg/1G+/+ mice (Table 1). The mean number of SWD events per hour were 98.3 ± 12.2 in 1Atg/tg/1G+/+ (n = 3), 164 ± 25.7 in 4lh/lh/1G+/+ (n = 4), and 132.0 ± 8.7 in 2stg/stg/1G+/+ (n = 3) mice (Table 1). The SWD patterns in these mutants were not different from those described previously (Noebels, 1984; Hosford et al., 1992; Qiao and Noebels, 1993). We took EEG measurements in double mutants completely lacking 1G genes. We found a complete suppression of SWDs in 1Atg/tg/1G–/– mice (n = 5). Indeed, we did not observe any 6–7 Hz SWDs with minimum voltage amplitude of twice the EEG background and a minimum duration of 0.7 sec (Figs. 6B, 7B; Table 1). For both 4lh/lh/1G–/– (n = 4) and 2stg/stg/1G–/– (n = 4) mice, cortical SWD paroxysmal activities were strongly suppressed (Fig. 6C,D; Table 1). The 5–7 Hz SWDs with very short duration (0.7–2 sec) were rare in 2stg/stg/1G–/– mice (9.4 ± 3.4 per hour; n = 4), and very few 5–6 Hz SWDs with a duration similar to those observed in 4lh/lh/1G+/+ mice were detected on EEG recordings from 4lh/lh/1G–/– mice (7.1 ± 4.7 per hour; n = 4) (Table 1). Interestingly, the results show that contrary to genetic ablation of 1G, which abolished cortical SWDs in mice harboring genetic dysfunction, in the 1A gene that encodes the main subunit of P/Q-type Ca2+ channel there was some paroxysmal cortical activity in 1G–/– mice, with a mutation in the regulatory subunit of the HVA Ca2+ channel (i.e., 4 and 2). We used power spectrum analysis to simplify our data regarding changes in the dominant peak frequency. From this analysis, we conclude that there are few or no SWDs in any of these mutants as a result of homologous deletion of 1G genes (Fig. 7). Additionally, EEG analysis showed that SWDs were present in all double mutants that were heterozygous for 1G (i.e., 1G+/–) (Fig. 6B–D). Indeed, there was no difference in SWDs between these mutants and those with 1G+/+ (Table 1).
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Discussion
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P/Q-type Ca2+ channels and absence seizures
HVA Ca2+ channels (subdivided into L, N, P/Q, and R types) play critical roles in neuron function, such as neurotransmitter release (Wheeler et al., 1994), patterning of cell excitability (Cavelier et al., 2002; Park et al., 2003), and gene expression (Sutton et al., 1999). Spontaneous absence seizures are reported in mice with Ca2+ channelopathy caused by various mutations in the subunits of P/Q-type Ca2+ channels, which show a partial reduction in P/Q-type currents with no difference in mRNA and protein levels of the channels (Fletcher et al., 1996; Mori et al., 1996; Wakamori et al., 1998; Zwingman et al., 2001). Consistent with these findings, we found that 1A–/– mice, which lack P/Q-type channels, had SWD activity similar to that reported in mice with other 1A point mutations (Fletcher et al., 1996; Mori et al., 1996; Wakamori et al., 1998; Zwingman et al., 2001); however, in contrast to the reports showing that mutations in 1Atg/tg, 4lh/lh, and 2stg/stg mice result in increased total HVA Ca2+ currents in TC neurons (Zhang et al., 2002), 1A null TC neurons had reduced total HVA Ca2+ currents, possibly because of the absence of P/Q-type Ca2+ currents. Thus, the present results suggest that the loss of P/Q-type current function contributes to the genesis of SWDs without any indirect changes to other HVA Ca2+ current types, at least in TC neurons. Consistent with this proposal, expression studies using a cloned 1A gene from a child patient with absence seizure and ataxia revealed a near-complete deletion of P/Q-type currents (Jouvenceau et al., 2001), similar to the findings obtained from 1A–/– mice (Kammermeier and Jones, 1997; Jun et al., 1999).
Heterogeneity of absence seizures: is 1G a common mediator for absence seizures?
Data from mouse models displaying various and nonoverlapping neurological abnormalities indicate the involvement of multiple mechanisms in the genesis of SWDs (Hosford et al., 1992; Di Pasquale et al., 1997; Zhang et al., 2002). Pharmacological studies support the complexity of SWDs. Although GABAB receptor-induced SWDs are associated with thalamocortical pathways (Caddick and Hosford, 1996; Kim et al., 2001), systemic administration of GABAA antagonists can induce SWDs in athalamic cats (Steriade and Contreras, 1998; Kim et al., 2001). Previously, we revealed that there were different mechanisms involved in GABAB receptor-mediated and GABAA antagonist-induced SWDs by showing that they are either dependent or independent of 1G T-type channels, respectively, because 1G–/– mice that lack thalamic burst firings are exclusively resistant to GABAB antagonist-induced SWDs (Kim et al., 2001). Thus, an aim of the present study was to characterize various mouse models of spontaneous absence seizures according to their dependence on 1G gene function. The present results show that 1G null mutation abolished SWDs of 1A–/– and 1Atg/tg mice and drastically reduced SWDs in 4lh/lh and 2stg/stg mice. It is interesting to note that there were residual SWDs in double mutants with 1G–/– and 2stg/stg or 4lh/lh. Considering that both 2 and 4 are auxiliary subunits of 1 subunits, which have been known to modulate voltage dependence, kinetics, and amplitude of other types of Ca2+ channels as well as the P/Q-type (Kang et al., 2001; Schjott et al., 2003), the residual SWDs in 2stg/stg/lG–/– and 4lh/lh/lG–/– mice appear independent of pathological interactions between 1A and dysfunctional 2 and 4 subunits. This concept is supported by pharmacological studies using these mice in which absence seizures in 2stg/stg mice were sensitive to MK-801, which is ineffective in the treatment of absence seizures in other mice (Heller et al., 1983; Aizawa et al., 1997); however, a common thread that weaves through the generation and propagation of absence seizures in 1A–/– mice, as well as other mutant mice, is critical dependence on the 1G gene.
Functional significance of T-currents in TC neurons
Many studies on Ca2+ channelopathy have reported altered Ca2+ current profiles as a result of Ca2+ channel mutations, indicating a strong correlation between a disease symptom and alteration in Ca2+ currents. For example, altered expression of the N-type Ca2+ channel 1B in 4lh/lh mice is suggested as a possible mechanism underlying absence seizures (McEnery et al., 1998), and increased expression of the 1C Ca2+ channel in cerebellar Purkinje cells in 1Atg/tg mice is associated with the dystonia in these mutant mice (Campbell and Hess, 1999). Similarly, the importance of T-currents in the development of absence seizures is underlined by recent studies using animal models of absence seizures (Tsakiridou et al., 1995; Zhang et al., 2002). These findings have motivated computational modeling studies to describe how augmented T-currents in thalamic neurons contribute to either physiological or pathophysiological synchrony in thalamocortical networks (Destexhe et al., 1996, 1998; Hughes et al., 1999; Thomas and Grisar, 2000). Somewhat unexpectedly, however, the present study using double-mutant mice (epileptic mice on a 1G–/– background) revealed that the basal level of T-currents in TC neurons was enough to support SWD generation. There was no quantitative difference in the severity of SWDs between 75 and 150% of wild-type dosage of T-currents in TC neurons isolated from the double mutants, suggesting that an increase in thalamic T-currents might not contribute to SWD genesis in vivo. At this point it cannot be ruled out that the increase of T-currents in other brain regions may support absence seizure development in these mutants, considering that the expression of 1G is also detected in other regions of brain, including cortex, olfactory bulb, and cerebellum (Talley et al., 1999). The question arises as to how normal levels of T-type Ca2+ currents in TC neurons contribute to SWDs generation. It is interesting to note that the hyperpolarizing shift in the resting membrane potential of hyperpolarization-activated cation channel 2-deficient thalamic relay neurons removes inactivation of T-type Ca2+ channels and thereby promotes burst rather than tonic firing in response to depolarizing inputs resulting in increased susceptibility to oscillations (Ludwig et al., 2003). Reduced excitatory but normal inhibitory synaptic transmission in 4lh/lh and 1Atg/tg mice thalami (Caddick et al., 1999) and enhanced GABAB receptor expression (Hosford et al., 1992) would result in relatively enhanced GABAergic input in 4lh/lh and 1Atg/tg thalamic neurons. Thus, more effective hyperpolarization per se could increase the likelihood of a T-type Ca2+ channel opening in TC neurons, enough to support SWDs without an increase in T-currents.
Finally, the present results suggest that a shift in research direction is required to determine the mechanisms underlying absence seizures. Beyond the issue of augmentation of T-type Ca2+ channels in TC neurons, studies are required to elucidate how hyperpolarizing inputs are overloaded in these neurons. The relationship between hypofunctioning P/Q-type channels and hyperpolarization of TC neurons sheds light on a possible novel therapeutic strategy for absence seizures.
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Footnotes
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Received Dec 17, 2003;
revised April 21, 2004;
accepted April 22, 2004.
This work was supported by a grant from the Chemoinformatics Program of the Korea Institute of Science and Technology and by a Creative Research Initiative grant from the Ministry of Science and Technology, Korea. We thank Dr. Donghyun Park for advice on analysis of EEG data and Heekyung Lee for reading this manuscript.
Correspondence should be addressed to Hee-Sup Shin, Center for Calcium and Learning, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul, 136-791, Korea. E-mail: shin{at}kist.re.kr.
DOI:10.1523/JNEUROSCI.5546-03.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/245249-09$15.00/0
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References
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Abstract
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