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Neurobiology of Disease

Elevated Thalamic Low-Voltage-Activated Currents Precede the Onset of Absence Epilepsy in the SNAP25-Deficient Mouse Mutant Coloboma

Yi Zhang, Alexander P. Vilaythong, Daniel Yoshor and Jeffrey L. Noebels
Journal of Neuroscience 2 June 2004, 24 (22) 5239-5248; DOI: https://doi.org/10.1523/JNEUROSCI.0992-04.2004
Yi Zhang
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Alexander P. Vilaythong
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Daniel Yoshor
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Jeffrey L. Noebels
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This article has a correction. Please see:

  • Erratum - June 23, 2004

Abstract

Recessive mutations in genes encoding voltage-gated Ca2+ channel subunits alter high-voltage-activated (HVA) calcium currents, impair neurotransmitter release, and stimulate thalamic low-voltage-activated (LVA) currents that contribute to a cortical spike-wave epilepsy phenotype in mice. We now report thalamic LVA current elevations in a non-Ca2+ channel mutant. EEG analysis of Coloboma (Cm/+), an autosomal dominant mutant mouse lacking one copy of the gene for a synaptosomal-associated protein (SNAP25) that interacts with HVA channels, reveals abnormal spike-wave discharges (SWDs) in the behaving animal. We compared the biophysical properties of both LVA and HVA currents in Cm/+ and wild-type thalamic neurons and observed a 54% increase in peak current density of LVA currents evoked at –50 mV from –110 mV in Cm/+ before the developmental onset of seizures relative to control. The midpoint voltage for steady-state inactivation of LVA currents in Cm/+ was shifted in a depolarized direction by 8 mV before epilepsy onset, and the mean time constant for decay of LVA Ca2+ currents at –50 mV was also prolonged. No significant differences were found in recovery from inactivation of LVA currents or in HVA current densities and kinetics. Our data demonstrate that a non-Ca2+ channel subunit gene mutation leads to potentiated thalamic LVA currents that precede the appearance of SWDs and that altered somatodendritic HVA currents are not required for abnormal thalamocortical oscillations. We suggest that presynaptic release defects shared by these mutants lead to postsynaptic LVA excitability increases in thalamic pacemaker neurons that favor rebound bursting and absence epilepsy.

  • T-type calcium channels
  • thalamocortical relay cells
  • calcium channelopathy
  • stargazer
  • tottering
  • lethargic

Introduction

Ca2+ currents, especially the low-voltage-activated (LVA) currents within thalamic circuitry, play a critical role in the development of thalamocortical network oscillations underlying absence epilepsy (Huguenard, 1996; McCormick and Contreras, 2001; Crunelli and Leresche, 2002). Mutant alleles of high-voltage-activated (HVA) subunit genes Cav 2.1/α1A (tottering/tg; leaner/tgla; rocker/tgrkr), β4 (lethargic/lh), γ2 (stargazer/stg), and α2δ2 (ducky/du) have been associated with cortical spike-wave discharges (SWDs) in mice resembling those in human idiopathic generalized absence epilepsy (Noebels, 1984; Noebels et al., 1990; Fletcher et al., 1996; Burgess et al., 1997; Letts et al., 1998; Barclay et al., 2001; Zwingman et al., 2001). Human α1A mutations associated with SWDs have also been described (Jouvenceau et al., 2001). Several of these mutations have been shown to alter membrane excitability and impair presynaptic neurotransmitter release by reducing P/Q-type Ca2+ currents (Dove et al., 1998; Wakamori et al., 1998; Qian and Noebels, 2000).

Although it was initially unclear how decreased P/Q Ca2+ current could lead to membrane hyperexcitability and seizures, recent analysis of thalamic neurons in tg, lh, and stg mutants reveals not only altered HVA current densities and kinetics, but striking increases in LVA peak currents and channel availability (Zhang et al., 2002). The elevated thalamic LVA currents provide a common downstream excitability defect that favors seizure generation by promoting rebound bursting. The molecular link between the mutated channel subunit and the potentiated LVA current is unclear. There is no evidence that α1A, β4, or γ2 subunits directly interact with the α1G-I subunits that mediate thalamic LVA currents. Furthermore, no increases in α1G-I mRNA expression or altered modulation of LVA currents were found. One possibility remaining to be explored is whether the LVA potentiation specifically depends on impaired calcium entry through the mutant P/Q-type channels or whether it can arise independently of the primary calcium channelopathy. A second is whether LVA increases might be induced by seizures themselves.

The neurological mouse mutant Coloboma (Cm/+; homozygous lethal) is an autosomal dominant mutation showing hyper-activity and learning deficits that maps to a small deleted region on chromosome 2 (Hess et al., 1992, 1994). The Coloboma locus spans the target membrane receptor (t-SNARE) gene encoding soluble N-ethyl-maleimide-sensitive factor attachment protein (SNAP25), a core complex protein that interacts with Ca2+ channel α subunits and participates in Ca2+-dependent neurotransmitter exocytosis (Rettig et al., 1996; Washbourne et al., 2002; Sorensen et al., 2003). Haploinsufficency of the SNAP25 gene in Cm/+ mice reduces 50% of the SNAP25 mRNA and protein content in brain and impairs evoked neurotransmitter release (Raber et al., 1997; Wilson, 2000). No genes for voltage-gated ion channels are contained within or flank this region, and the neurobehavioral phenotype is rescued by SNAP25 transgene expression (Hess et al., 1996). On the basis of the functional overlap of altered transmitter release shared with the Ca2+ channelopathy models, we examined Cm/+ mice for abnormal cortical synchronization and thalamic excitability changes. We discovered that Cm/+ mice show robust cortical SWDs and thalamic T-type current increases closely resembling those described in Ca2+ channel mutants. In addition, the T-type current increases clearly precede the developmental onset of cortical discharges.

Materials and Methods

Chronic electroencephalographic recordings

Cm/+ mice and their wild-type littermates (C3H/HESNJ) from the age of postnatal day (P) 10–30 were implanted for chronic EEG recordings as described previously (Qiao and Noebels, 1993). Mice were anesthetized with Avertin (1.25% tribromoethanol/amyl alcohol solution, i.p.) using a dose of 0.02 ml/gm. Teflon-coated silver wire electrodes (0.005 inch diameter) soldered to a microminiature connector were implanted bilaterally into the subdural space over frontal and parietal cortices. Digital EEG activity was recorded daily during random 2 hr samples (Stellate Systems; Harmonie software version 5.0b). A digital video camera was used to simultaneously monitor behavior during the EEG recording periods. All recordings were performed at least 24 hr after surgery on mice moving freely in the test cage. After the recordings, the EEG and digital video from mice were analyzed for the incidence and duration of spontaneous cortical spike activity. Abnormal synchronous discharges with a minimum amplitude of twice the background EEG voltage and a minimum duration of 1 sec were included for analysis, and spike bursts separated by <1 sec were counted as a single discharge. To developmentally compare the EEG results of Cm/+ with those from tg and stg mutants, similar recordings were conducted in tottering (C57BL/6J-Cacna1atg/tg), stargazer (C57BL/6J-Cacnγ2stg/stg), and their wild-type control (C57BL/6J, +/+) mice over a similar age range.

Preparation of brain slices

Coronal brain slices (350 μm thick) were prepared from P13–16 heterozygous Coloboma (C3H/HESNJ, Cm/+) and homozygous wild-type (C3H/HESNJ, +/+) littermate mice. Slices were obtained at the level of the lateral dorsal nucleus (LDN) of the thalamus as described previously (Zhang et al., 2002). Tissue was sectioned by vibratome in a solution containing (in mm): 125 choline-Cl, 3.0 KCl, 1.25 NaH2PO4, 25 NaHCO3, 1.0 CaCl2, 7.0 MgCl2, 10 dextrose, 1.3 ascorbate acid, and 3.0 pyruvate, bubbled with 95% O2–5% CO2 (at 4°C) (Kapur et al., 1998). Slices were then incubated in an artificial CSF solution for 40 min at 37°C and then maintained at room temperature (22–25°C). The artificial CSF was gassed with 95% O2–5% CO2 and contained (in mm): 130 NaCl, 3.0 KCl, 2.0 MgCl2, 2.0 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose.

Electrophysiological recording

Macroscopic Ca2+ currents from thalamocortical (TC) cells in LDN were recorded using the whole-cell configuration of the patch-clamp technique. An upright microscope (Axioskop, Zeiss Instruments) fitted with a 40× water-immersion objective and differential interference contrast optics was used to view the slices and identify neurons for analysis. Voltage command pulses were generated by a computer using pCLAMP 8.02 software. Currents were recorded with an Axopatch-1D amplifier, filtered at 10 kHz (–3 dB), and compensated for series resistance (∼70%). Patch electrodes were drawn from borosilicate glass and coated with Sylgard. Ca2+ currents were corrected for leak and capacitive currents by subtracting a scaled current elicited by a 10 mV hyperpolarization from the standard holding potential of –70 mV. All recordings were performed at room temperature (22–25°C).

Solutions. The recording bath solution consisted of (in mm): 115 NaCl, 3.0 KCl, 10 sucrose, 10 glucose, 26 NaHCO3, 2 MgCl2, 2.5 CaCl2, 0.5 4-aminopyridine, 5 CsCl, 10 TEA-Cl, and 0.001 TTX, pH 7.4 (gassed with 95% O2 and 5% CO2). The intracellular pipette solution contained (in mm): 78 Cs-gluconate, 20 HEPES, 10 BAPTA-Cs4 (cell impermeant), 0.5 CaCl2, 1.0 MgCl2, 4 Mg-ATP, 0.3 GTP-Tris, 6 phosphocreatine (Di-Tris salt), 4.0 NaCl, and 20 TEA-Cl, pH 7.3 (titrated with CsOH).

Voltage protocols. To generate Ca2+ channel current–voltage (I–V) curves, currents were elicited by applying voltage step commands (200 msec) to varying potentials from a 3 sec prepulse potential at –60 or –110 mV. The I–V protocol for HVA Ca2+ currents consisted of voltage steps from –80 to +60 mV in 5 mV increments triggered from a 3 sec prepulse potential at –60 mV. To define LVA Ca2+ currents, difference currents obtained by digital subtraction of the currents elicited during depolarizing voltage steps from –60 and –110 mV were used. Standard voltage protocols for steady-state activation (SSA) of HVA Ca2+ currents, as well as the steady-state inactivation (SSI) and recovery from inactivation (RFI) of LVA currents, respectively, were applied and are explained in further detail in the Figure legends. In our study, we did not find any significant time-dependent ICa2+ rundown within 40 min after membrane rupture, and all data included in this study were obtained within this time interval. Statistical data analysis was performed using the Student's t test or one-way ANOVA with the post hoc test. Differences where p < 0.05 were accepted as statistically significant. The data shown represent means ± SE.

Results

Cortical spike-wave discharges in the Cm/+ mutant are associated with elevated LVA Ca2+ currents in thalamic neurons

Similar to mice with recessive mutations in calcium channel subunit genes, we observed frequent spontaneous bursts of 5–6 Hz bilateral cortical SWDs in all Cm/+ mutants. These stereotyped discharges appeared in the awake animal and were always accompanied by simultaneous behavioral arrest. Representative bilateral EEG traces recorded in adult Cm/+ mice are shown in Figure 1A (top). Intraperitoneal injection of 5 mmol/kg ethosuximide (ETX) completely blocked SWDs within 2–4 min (Fig. 1A, middle). The mean number of SWDs in adult Cm/+ mice was reduced from 29.3 ± 4.2 per hour before injection to 0 per hour for 2–4 hr after injection (Fig. 1A, bottom), followed by full recovery of the SWDs and absence-type seizures. The sensitivity of intraperitoneal injection of ETX in Cm/+ mutants was reduced significantly compared with the effective doses used in mice absence models: tg, lh, and stg models (1–1.4 mmol/kg) (Heller et al., 1983; Aizawa et al., 1997). Spike wave discharges of this general pattern and behavioral phenotype are seen in human absence epilepsy and Ca2+ channel mouse mutants (Noebels and Sidman, 1979; Hosford et al., 1992; Qiao and Noebels, 1993), as well as in other genetic models of absence in rat (Marescaux et al., 1992; Coenen and Van Luijtelaar, 2003).

Figure 1.
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Figure 1.

Spike-wave discharges coexist with elevated low-voltage-activated Ca2+ peak current in thalamic neurons of Coloboma mutant mice. A, Representative bilateral EEG traces showing 5∼6 Hz spike-wave discharges (top) and pharmacological sensitivity to 5 mmol/kg ethosuximide (ETX, i.p.) in Cm/+ mutants (n = 5; at P30) (middle). L and R designate left and right frontal cortex recording sites. The bottom panel shows the mean incidence of spike-wave discharges in Cm/+ mutant mice before and after ETX injection. B, Increased thalamic LVACa2+ peak current in Coloboma. Top panel, Representative LVA current traces from thalamocortical relay cells (TCs) of the LDN in control (C3H) and Cm/+ mice at age P15. The cell capacitance values of the two neurons were 100 and 101 pF in C3H and Cm/+, respectively; holding potential =–70 mV. The membrane potential was prepulsed to –110 mV for 3 sec before stepping to –50 mV for 200 msec. Decay of the current was fitted by a single-exponential function (superimposed dotted line). A significant alteration in the macroscopic current decay was found in Cm/+ neurons. The time constants (τ) for decay of the representative currents are 24.6 msec in C3H and 29.8 msec in Cm/+, respectively. Bottom panel, Elevated LVACa2+ current amplitude and peak current density fromCm/+ TCs. LVA currents were evoked at the same membrane potential as described in top panel. **p<0.001 versus control at corresponding age.

We then analyzed low-voltage-activated Ca2+ current amplitudes and kinetics in TC neurons to determine whether any alterations coexist with the expression of absence epilepsy in the Cm/+ mutant. In Figure 1B, the top panel shows representative traces of LVA Ca2+ currents in response to a test pulse to –50 mV arising from a 3 sec prepulse to –110 mV in TC neurons from wild-type and Cm/+ mice. At a membrane potential of –50 mV, all LVA Ca2+ currents have recovered from inactivation and are thus available for opening in both wild-type and mutant neurons (see Figs. 4B, 5B), whereas HVA Ca2+ currents in these cells have not yet started to activate (see Fig. 6B). The current traces of the LVA calcium channels show fast activation and inactivation, similar to that found in vitro by expression of Cav3.1(α1G) and Cav3.2(α1H) T-type calcium channels (Lee et al., 1999; Delisle and Satin, 2000; Zhang et al., 2000) as well as native LVA currents from dissociated rat TC neurons (Destexhe et al., 1998) and TC neurons of tg, lh, and stg brain slices (Zhang et al., 2002). The peak current densities (normalized by cell capacitance) of LVA currents at a membrane potential of –50 mV were increased by 60% compared with the corresponding value in control neurons at this age (P14–16) (Fig. 1B, right bottom panel). The mean peak current amplitudes and peak current densities were –1142.3 ± 131.9 (pA) and 12.4 ± 1.1 (pA/pF) in control mice and –1916.2 ± 54.3** (pA) and 19.8 ± 0.4** (pA/pF) in Cm/+ (**p < 0.001 vs control).

Figure 4.
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Figure 4.

Depolarized shift of the voltage dependence of low-voltage-activated calcium channel availability (steady-state inactivation) in Coloboma mutants. A, Representative current traces for SSI of LVA Ca2+ currents. A standard double-pulse protocol for steady-state inactivation was given from the holding potential of –70 mV. A 4 sec prepulse at potentials ranging from –120 to –40 mV preceded each depolarization, followed by a subsequent voltage step to –50 mV for 200 msec. The interpulse interval was 10 sec. B, Normalized current–voltage curves for SSI of LVA Ca2+ currents. Current amplitude from the inactivation protocol, normalized to maximum, was plotted as a function of prepulse membrane potentials and best fitted with a Boltzmann function: I/Imax = {1 + exp(V – V1/2)/k}–1.

Figure 5.
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Figure 5.

Recovery from inactivation of LVA Ca2+ currents. A, Representative current traces for recovery from inactivation of LVA currents in control and Cm/+. The holding potential was set to –50 mV, and 50 mV hyperpolarizations of incremental duration were applied. LVA peak amplitude was measured after returning to –50 mV. B, Recovery from inactivation curves. Recovery curves were established by plotting the normalized peak amplitude versus duration. The recovery curves followed a two-exponential time course, and the fast time constant (τ1) and slow time constant (τ2) were derived from curve fitting.

Figure 6.
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Figure 6.

HVA Ca2+ currents in Cm/+ and wild-type mice. A, Representative superimposed HVA Ca2+ current traces from thalamic cells in control and Cm/+ mice. The I–V protocol consisted of a 3 sec prepulse potential at –60 mV followed by voltage steps (200 msec) ranging from –80 to +60 mV in 5 mV increments, with the holding potential maintained at –70 mV. B, Current density–voltage curves for HVA Ca2+ currents, constructed by plotting the normalized current amplitude at various membrane potentials. The voltage protocol used was identical to that described in A. C, Peak current density and current amplitude from B. D, SSA of HVA Ca2+ currents in control and Cm/+. The steady-state conductance (G) and voltage (V) data were transformed from I–V data shown in B. The solid and dotted curves are fits of the data to the Boltzmann equation of the form: G/Gmax = 1/(1 + exp(V1/2 – V)/k), where Gmax is maximum conductance, V1/2 is half-maximal voltage, and k is the slope.

Age of onset and mean incidence of spontaneous cortical SWDs in Cm/+ mutants

The onset of cortical SWDs in Cm/+ mice, like those in mice bearing Ca2+ channelopathies, displayed a highly reproducible developmental profile. Representative bilateral EEG traces of both Cm/+ mutants and C3H control mice recorded at various postnatal ages from P13 through adulthood are shown in Figure 2A and are compared with EEG traces at the same ages from tg and stg mutants as well as their control genotypes. In our study, no synchronous discharges were ever recorded from Cm/+ mutants at ages P11–13. SWDs emerged at P14–15 and were always present by P20 in Cm/+ mice. As shown in Figure 2A, most of the spike-wave discharges in the immature (P14–18) Cm/+ mouse brain display a pattern of prolonged and frequent 3–4 Hz synchronous discharges, intermixed with occasional shorter 5–6Hz bursts. From P20 onward, a 5–6 Hz spike-wave pattern becomes the predominant pattern in Cm/+ mutants. In comparison, cortical SWDs were also first visible at P13 and P14, respectively, in stg and tg mutants. Similar to Cm/+, SWDs exhibited a variable spike frequency pattern of 3∼6 Hz during the period from P13 to P18 and then stabilized at 5–6 Hz from P20 onward in both tg and stg mice. No SWDs were ever observed in C3H (wild-type littermates of Cm/+) and C57/BL6 (+/+ control for tg and stg) mice within the age range of P11 to P25. Group data showing the mean incidence of cortical discharges in Cm/+ mutants and C3H +/+ control mice during development are demonstrated in Figure 2B. As shown, the onset of synchronous cortical discharges began with rare bursts (1–30 sec duration; some as long as 150–300 sec) in Cm/+ mutant mice at the age of P14 and P15 (two to five bursts per hour) and gradually increased to a stable rate exceeding 20 per hour. The mean incidence of spike-wave discharges in Cm/+ mice at P20 was ∼15 bursts per hour and then reached 25–45 bursts per hour between P21 and P25. Behavioral video image data obtained simultaneously with EEG recordings showed that except for very brief duration discharges, the SWDs were seen when Cm/+ mutants were motionless in a state of quiet wakefulness, similar to the correlation with behavioral arrest in other absence seizure models. The patterns of onset and incidence of spike-wave discharges in Cm/+ mutants are essentially identical to those exhibited in Ca2+ channel mutants (Noebels and Sidman, 1979; Noebels, 1984; Noebels et al., 1990). No synchronous discharges were ever recorded in adolescent C3H control mice; however, rare isolated SWDs were noted in recordings from some +/+ C3H aged ≥5 weeks, suggesting that this genetic background may express additional permissive susceptibility genes for this trait.

Figure 2.
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Figure 2.

Onset and mean incidence of spike-wave discharges in Cm/+ mutant mice during development. A, Representative bilateral EEG traces recorded in Cm/+, compared with those in tg and stg mutants at the same age. B, Group data showing the mean incidence (i.e., number of SWDs per hour) of cortical discharges in Cm/+ and C3H control mice at ages ranging from P11 to P25. In C3H (n = 8), no SWDs were ever observed throughout the recording period. For Cm/+ mice, each age point includes mean data from two to seven mice ± SE; #p < 0.01; *p < 0.001 versus corresponding values of Cm/+ at P14–17, respectively.

The increased peak current density and prolonged current decay of thalamic LVA calcium currents precede seizure onset

We examined thalamic LVA calcium currents in brain slices obtained at age P13, at least 24 hr and typically several days before the earliest evidence of cortical SWDs in Cm/+ mutants. The data collected at P13 are compared with those obtained at P14–16 when seizures began to emerge. Figure 3A shows representative traces of LVA Ca2+ currents in response to a test pulse to –50 mV from a 3 sec prepulse to –110 mV in TC neurons from wild-type and Cm/+ mice. The current traces of the LVA calcium channels at both P13 (before the onset of SWDs) and P15 (after the emergence of SWDs) display fast activation and inactivation kinetics as well as activation at low voltages, which together define the characteristic properties of T-type Ca2+ channels (Destexhe et al., 1998; Lee et al., 1999; Zhang et al., 2000, 2002). The peak current densities of LVA currents at a membrane potential of –50 mV increased by 54% in Cm/+ at P13 and 60% in Cm/+ at P14–16 compared with corresponding values in control +/+ neurons at same age (Fig. 3B). The mean peak current amplitude and peak current density were –1418.0 ± 132.9 (pA) and 12.7 ± 0.5 (pA/pF) in control and –2015.8 ± 117.9* (pA) and 19.5 ± 1.2** (pA/pF) in Cm/+ at age P13, –1142.3 ± 131.9 (pA) and 12.4 ± 1.1 (pA/pF) in control, and –1916.2 ± 54.3** (pA) and 19.8 ± 0.4** (pA/pF) in Cm/+ aged P14–16 (**p < 0.001; *p < 0.01 vs control). The amplitude and peak current density of control and Cm/+ at P13 were not significantly different from corresponding data at P14–16, respectively. Because synchronous discharges in Cm/+ mutant mice are never seen until P14–16 (Fig. 2B), this result demonstrates that thalamic LVA Ca2+ currents are elevated before the onset of discharges and therefore are not induced by the seizure itself.

Figure 3.
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Figure 3.

Increased low-voltage-activated Ca2+ peak current in Coloboma. A, Representative LVA current traces from thalamocortical relay cells (TCs) in control (C3H) and Cm/+ mice at ages P13 and P15. The cell capacitance values of these neurons were 104 and 105 pF in C3H and Cm/+ at P13, and 100 and 101 pF in C3H and Cm/+ at P15, respectively. The holding potential and voltage protocol used were the same as described in Figure 1 B. Decay of the current was fitted by a single-exponential function (superimposed dotted line). A significant alteration in macroscopic current decay was found in Cm/+. The time constants τ for decay of representative current traces at P13 and P15 are 22.53 and 24.6 msec in C3H and 29.4 and 29.8 msec in Cm/+, respectively. B, Elevated LVA Ca2+ current amplitude and peak current density from Cm/+ TC neurons. LVA currents were evoked at the same membrane potential as described in A. **p < 0.001; *p < 0.01 versus control at corresponding age.

The duration of macroscopic inactivation of LVA Ca2+ currents in both control and mutant mice is closer to the time scale observed in Cav3.1 but not Cav3.2 T-type calcium channels expressed in mammalian cells (Lee et al., 1999; Zhang et al., 2000). The decay of macroscopic LVA currents evoked at –50 mV was fitted by a single-exponential function (Fig. 3A); however, unlike what we observed in tg, lh, and stg neurons, we found significant prolongation of the time constant for decay of macroscopic LVA currents in Cm/+ (28.1 ± 0.69** msec; P13–16) compared with that in wild-type neurons at the same age range (23.6 ± 0.86 msec; **p < 0.001). We here combined data derived from mice at P13 and at ages ranging from P14 to P16 in both the control group and the Cm/+ group, because theτ for decay at P13 is not significantly different from that at P14–16 in either control or mutant mice.

Depolarizing shifts in voltage dependence of steady-state inactivation and unaltered kinetics for recovery from inactivation of LVA currents in Cm/+ mice

We next examined the voltage-dependent inactivation and recovery from inactivation of LVA Ca2+ currents in both wild-type and Cm/+ neurons. The current traces of SSI of LVA are shown in Figure 4A. For the SSI protocol, we used a 4 sec prepulse to various membrane potentials before delivering a second test stimulus to –50 mV. The 4 sec prepulse was long enough to bring channels to a steady-state condition, because all LVA Ca2+ channels in TC neurons recover from inactivation within 3 sec (Fig. 5B). As demonstrated in Figure 4A, LVA currents elicited at –50 mV from different premembrane potentials in both control and Cm/+ mutant show fast inactivation and decay completely within 200 msec. We found a significant depolarizing shift of the steady-state inactivation curves of LVA currents in the mutant both before the onset of seizure (P13) and after seizure generation (P14–16) in contrast to wild-type neurons (Fig. 4B). The mean half-maximal voltages (V1/2) for SSI curves were –84.0 ± 0.7 mV in control (P13–16), –76.0 ± 2.1* mV in Cm/+ at P13, –75.5 ± 3.1# mV in Cm/+ at P14–16, respectively (*p < 0.01; #p < 0.05 vs control). The 8–8.5 mV depolarizing shifts of the voltage dependence for SSI of LVA currents in TC neurons of Cm/+ mice suggest that at physiological membrane potentials varying from –70 to –75 mV, a higher fraction of all LVA calcium channels is available for opening in the mutant relative to control mice. As seen for the LVA peak current density in Cm/+ (Fig. 4), the V1/2 for SSI in mutant mice also shifted significantly in a depolarizing direction at P13, a time preceding the onset of SWDs (Fig. 2B). This depolarized shift of V1/2 for SSI of LVA currents remained after seizure onset (Fig. 4B). Accordingly, in parallel with an increased current density (Fig. 3), the depolarizing shift of V1/2 for SSI of LVA currents in Cm/+ also preceded the emergence of SWDs. The depolarizing shift of the SSI curve for LVA currents in Cm/+ is consistent with our previous data showing 7.5–13.5 mV depolarizing shifts of SSI for thalamic LVA currents in tg, lh, and stg mutants (Zhang et al., 2002). This shift in the voltage dependence of SSI of LVA currents to a more depolarized level in Cm/+ mice will increase membrane excitability and therefore provide an additional biophysical mechanism that may contribute to neuronal burst synchronization in Cm/+ mutants.

Figure 5A displays raw current traces of the RFI of thalamic LVA Ca2+ currents in control and Cm/+ mice. As shown, RFI in both control and mutant cells was complete within 3 sec (Fig. 5B). The recovery from inactivation curve was best fitted with a two-exponential function, and the fast and slow time constants derived from curve fitting did not significantly differ in Cm/+ when compared with control mice, either before or after the onset of SWDs. The values of the fast time constant (τ1) were 260 and 240 msec for control and Cm/+ at P13 and 250 and 210 msec for control and Cm/+ at P14–16, respectively. The values of slow time constant (τ2) were 900 and 860 msec for control and Cm/+ at P13 and 820 and 920 msec for control and Cm/+ at P14–16, respectively.

High-voltage-activated Ca2+ peak currents and voltage dependence of steady-state activation are unaffected in Cm/+ mutants

Finally, we investigated whether thalamic HVA Ca2+ currents are altered by SNAP25 deficiency as seen in Ca2+ channel mutants. HVA Ca2+ currents are mediated by pore-forming α1 subunits, with current amplitude and gating regulated by cytoplasmic β subunits and transmembrane α2δ and γ subunits (Ahlijanian et al., 1990; Chien et al., 1995; Witcher et al., 1995; Gurnett et al., 1996; Walker and De Waard, 1998; Meir et al., 2000; Kang et al., 2001). Our previous study demonstrated increased HVA Ca2+ currents as well as altered channel kinetics in the tg, stg, and lh absence seizure models (Zhang et al., 2002), and there is evidence that SNAP25 directly interacts with HVA Ca2+ channels (Rettig et al., 1996; Sheng et al., 1998).

Figure 6A shows representative HVA Ca2+ current traces from wild-type and Cm/+ mutant neurons. The I–V relationships of Ca2+ currents for control and Cm/+ mutant are shown in Figure 6B. Both control and mutant HVA Ca2+ currents start to activate at around –40 mV and reach a peak between –10 and –15 mV (Fig. 6B). Pooled peak currents and peak current densities are shown in Figure 6C. The mean peak current density was 10.3 ± 1.2 and 11.1 ± 1.4 (pA/pF) in control and Cm/+ mutant neurons at P13 and 10.9 ± 1.2 and 10.9 ± 0.9 (pA/pF) in control and Cm/+ at P14–16, respectively. The mean peak amplitude was –1198.4 ± 219.5 and –1228.6 ± 135.6 (pA) in control and Cm/+ mice at P13 and –1149.9 ± 217.8 and –1122.4 ± 129.6 (pA) in control and Cm/+ mice at P14–16, respectively. These results show that there is no significant alteration in either peak current density or current amplitude in Cm/+ mutants in comparison with those of wild-type mice both before the onset of SWDs and after seizure generation.

Similar to the unaltered peak current in Cm/+, the voltage dependence of SSA for HVA Ca2+ currents in mutant TC neurons also did not shift significantly relative to that of control. The SSA curves of HVA currents in both control and mutant are demonstrated in Figure 6D. The mean values of V1/2 and slope for SSA of HVA channels were –20.1 ± 1.3 mV and 4.3 ± 0.3 in control and –20.4 ± 1.1 mV and 3.8 ± 0.2 in Cm/+ at P13, and –19.1 ± 1.2 mV and 3.7 ± 0.2 in control and –18.6 ± 1.3 mV and 4.0 ± 0.3 in Cm/+ at P14–16. The unchanged peak current density and voltage dependence of SSA differ from what we observed in tg, lh, and stg mutants (Zhang et al., 2002), which demonstrated increased peak current densities in tg and stg as well as a depolarizing shift of SSA curve in lh mice. Thus, the insignificant changes in HVA peak current and voltage dependence of SSA in Cm/+ mice suggest that the Cm/+ mutation does not directly or indirectly affect HVA Ca2+ channel gating.

Discussion

Our results demonstrate that an increased peak current density and a depolarizing shift in the SSI of thalamic LVA Ca2+ currents coexist with frequent cortical spike-wave discharges in the dominant mouse mutant Coloboma. The LVA current elevations precede the developmental onset of the discharges, showing that they are not seizure induced, and thus provide a mechanism for the generation of the absence epilepsy phenotype in Cm/+. The changes in HVA peak current density and voltage dependence of SSA found in Ca2+ channelopathy mutants were not present in Cm/+, supporting the key pathogenic role of thalamic LVA Ca2+ channels in mediating neuronal hyperexcitability and epileptogenesis.

Thalamic Ca2+ currents in Cm/+ compared with calcium channel mutations

LVA currents

Thalamic LVA currents are mediated by α1G-I genes expressed in nonoverlapping thalamic regions (Talley et al., 1999). In a previous study of tg, lh, and stg mice, we found elevated LVA current in LDN, where the α1G gene is expressed (Zhang et al., 2002). The magnitude of the increase (∼50%) in peak LVA currents in Cm/+ is equivalent, signifying that mutations of either Ca2+ channel subunits or non-Ca2+ channel genes can lead to increased thalamic LVA currents in absence models. This finding, taken together with the report that mice lacking the α1A subunit for P/Q-type Ca2+ current also show elevated thalamic LVA currents and spike-wave seizures (Song et al., 2001), excludes the possibility that the potentiated LVA current arises from aberrant interactions between α1G and other mutant calcium channel subunits. It is also supported by the lack of biochemical evidence for direct interactions between wild-type α1G and other poreforming or regulatory Ca2+ channel subunits, except for the finding that functional expression of α1G increases with coexpression of α2δ and β1b subunits (Dolphin et al., 1999). These data demonstrate that mutations of Ca2+ channel subunit genes are not required to induce the elevated LVA Ca2+ currents in thalamic neurons that are associated with SWDs.

HVA currents

In contrast, we did not observe any significant effects of reduced SNAP25 on either the peak current density or voltage-dependent SSA of HVA currents in the Cm/+ mutant. This result differs from tg, lh, and stg mice, in which HVA peak current densities increased by 22% in tg and 45% in stg, and the SSA curve shifted in a depolarizing direction by 5 mV in lh mice (Zhang et al., 2002). In these mutants, the altered HVA currents arise from primary genetic defects of the P/Q-type channel or lost interactions with its associated subunits (De Waard and Campbell, 1995; Letts et al., 1998; Burgess et al., 1999; Kang et al., 2001) and may also reflect the up-regulation of other non-P/Q HVA channel types (Campbell and Hess, 1999). The lack of HVA current change in Cm/+ was unexpected but may be explained by neuronal compartmentation. SNAP25 directly interacts with P/Q-type channels (Rettig et al., 1996) and is reduced in Cm/+ mice; however, the membrane currents reported in these studies are recorded at the cell soma, and it is likely that any alteration of HVA currents in Cm/+ neurons could be detected only at presynaptic terminals where the interaction between SNAP25 and HVA channel proteins occurs (Sheng et al., 1998; Atlas, 2001).

Potentiation of thalamic LVA Ca2+ current precedes the onset of SWDs in Cm/+ mutants

Because potentiation of LVA currents could be activity driven, we examined thalamic LVA currents in Cm/+ and control mice before and after the onset of seizures. At P13, a full day before even the first SWD ever appears, we found a 54% increase in peak current density of LVA Ca2+ currents, as well as an 8 mV depolarizing shift of the inactivation curve, demonstrating that potentiation of thalamic LVA currents precedes the emergence of synchronous discharges and therefore was not seizure induced. In fact, even after several days of seizures occurring with an incidence of two to five bursts per hour, we still observed a comparable (60%) elevation in peak current density of LVA currents together with an 8.5 mV depolarizing shift of the voltage dependence of steady-state inactivation that was changed little from pre-seizure levels. Thus, the onset of SWDs did not add an appreciable supplemental enhancement in either LVA peak current or the shift of inactivation curve in Cm/+. These data correspond well with other evidence that the spike-wave seizure pattern, unlike that induced by convulsants (Nahm and Noebels, 1998), does not significantly induce gene expression patterns.

The increased thalamic LVA currents and channel availability near resting membrane potentials in the Cm/+ mutant favor augmented burst firing and membrane hyperexcitability, because T-type Ca2+ channels begin to activate at relatively hyperpolarized membrane potentials (Huguenard and Prince, 1992; Zhang et al., 2000) attributable in part to rhythmic input from GABAergic nucleus reticularis thalami neurons. Because SNAP25 deficiency reduces transmitter release in Cm/+ synaptosomal preparations (Raber et al., 1997), other complex changes may contribute to the altered synchronization pattern. Although we have not established a unique causal relationship between the potentiated LVA currents and seizure initiation in Cm/+, the evidence for involvement of thalamic T-type channels in spike-wave generation, as well as the exclusion of activity-dependent causes for LVA current increases in Cm/+, suggests an important primary contribution. Interestingly, a comparable depolarizing shift of SSI with prolongation of LVA current decay has just been described in human mutations identified in human childhood absence epilepsy patients (Khosravani et al., 2004).

Mechanisms for enhanced LVA current

Several possibilities may account for elevated LVA currents in Cm/+ mutants. First, T-type channel synthesis might increase because of impaired synaptic transmission caused by deficient SNAP25 expression or phosphorylation (Boschert et al., 1996; Genoud et al., 1999). Presynaptic release defects may lead to developmental differences in the transcriptional regulation of a heterogeneous population of T-type channel isoforms (Bertolesi et al., 2003; Yunker et al., 2003), which in turn have been shown to influence patterns of neuritogenesis during neuronal differentiation (Chemin et al., 2002). Although a minor increase in expression of Cav3.1/α1G and Cav3.2/α1H has been reported in adult GAERS (genetic absence epilepsy rats from Strasbourg) rats, a genetically undefined absence model (Talley et al., 2000), no alteration in the thalamic expression pattern of any of the three Cav3.1–3 genes that closely resemble the Cm/+ model or evidence for abnormal modulation have been detected (Zhang et al., 2002). Second, the currents may be modulated. Modulation pathways potentially responsible for increased LVA currents in Cm/+ include the modification of T-type channels by protein kinase C (Park et al., 2003), CamKII (Welsby et al., 2003), opioid receptors (Schroeder et al., 1991), pH (Delisle and Satin, 2000; Shan et al., 2001), and anandamide (Chemin et al., 2001). It is worth noting that the Coloboma locus also contains the genes for phospholipase C (PLC) β1 and β4; hence Cm/+ mice are also haploinsufficient for these enzyme isoforms. Mice with homozygous deletion of PLC β1 show lethal tonic–clonic seizures in the second postnatal week, and β4 mice show ataxia without seizures; however, heterozygous mice display no neurological abnormalities (Kim et al., 1997). Whether these heterozygotes show SWDs and altered LVA currents that might be related to PLC-β signal transduction abnormalities remains to be determined.

Overlapping mechanisms for absence epilepsy phenotype in Coloboma and calcium channel mutants

The spike-wave phenotype exhibited in developing Cm/+ mice parallels the early onset (P14–16), increasing incidence (reaching >40 per hour in adulthood), and ETX sensitivity of the SWDs found in tg, lh, and stg (Noebels and Sidman, 1979; Noebels et al., 1990; Hosford et al., 1992). Interestingly, in tg and stg mutants, we also observed a 50–60% increase in peak current densities and an 11–12 mV depolarized shift of inactivation over an age range (P8–11) before SWDs appear (data not shown). Significantly, there is additional functional overlap between epileptogenic calcium channelopathies and the SNAP25-deficient Cm/+ mutants, because SNAP25 interacts with HVA channels involved in exocytosis, and both show transmitter release defects. We did find a difference in pharmacological sensitivity: namely, a 3.6- to 5-fold reduction in ETX sensitivity in Cm/+ mice compared with Ca2+ channel mutants. In Cm/+, only a bolus dose of 5 mmol/kg (i.p.) completely blocked the occurrence of SWDs within 2–4 min, and lower doses were ineffective, whereas in Ca2+ channel mutants, 1–1.4 mmol/kg is an effective dose (Heller et al., 1983; Aizawa et al., 1997). The blockade of SWDs by ETX could be attributable to the action of the drug on either thalamic T-type Ca2+ channels (Coulter et al., 1989) or slow inactivated Na+ currents and Ca2+-activated K+ currents (Leresche et al., 1998). Future experiments will address which currents may be less sensitive to ETX in Cm/+ neurons.

In conclusion, our results provide the first demonstration that potentiated thalamic LVA currents precede abnormal neuronal synchronization and, together with the direct effect of the α1G gene on the threshold for thalamocortical SWD generation (Kim et al., 2001), provide the strongest supportive evidence so far for a major role of thalamic LVA currents in murine models of absence epilepsy. The evidence also points to developmental defects in synaptic transmission as a common cellular mechanism for the dysregulation of T-type currents that lead to this epileptic phenotype.

Footnotes

  • This work was supported by National Institute of Neurological Disorders and Stroke Grant NS29709 (J.L.N.) and the Blue Bird Circle Foundation.

  • Correspondence should be addressed to Dr. Jeffrey L. Noebels, Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail: jnoebels{at}bcm.tmc.edu.

  • DOI:10.1523/JNEUROSCI.0992-04.2004

  • Copyright © 2004 Society for Neuroscience 0270-6474/04/245239-10$15.00/0

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The Journal of Neuroscience: 24 (22)
Journal of Neuroscience
Vol. 24, Issue 22
2 Jun 2004
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Elevated Thalamic Low-Voltage-Activated Currents Precede the Onset of Absence Epilepsy in the SNAP25-Deficient Mouse Mutant Coloboma
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Elevated Thalamic Low-Voltage-Activated Currents Precede the Onset of Absence Epilepsy in the SNAP25-Deficient Mouse Mutant Coloboma
Yi Zhang, Alexander P. Vilaythong, Daniel Yoshor, Jeffrey L. Noebels
Journal of Neuroscience 2 June 2004, 24 (22) 5239-5248; DOI: 10.1523/JNEUROSCI.0992-04.2004

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Elevated Thalamic Low-Voltage-Activated Currents Precede the Onset of Absence Epilepsy in the SNAP25-Deficient Mouse Mutant Coloboma
Yi Zhang, Alexander P. Vilaythong, Daniel Yoshor, Jeffrey L. Noebels
Journal of Neuroscience 2 June 2004, 24 (22) 5239-5248; DOI: 10.1523/JNEUROSCI.0992-04.2004
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