Rebound burst firing in the reticular thalamus is not essential for pharmacological absence seizures in mice

Significance

Intrinsic bursts and rhythmic burst discharges are elicited by activation of T-type Ca²⁺ channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to spike-and-wave discharges (SWDs) on the cortical electroencephalogram, which are the hallmarks of absence seizures. Using knockout mice for T-type Ca²⁺ channels that completely lack TRN bursts, however, we show that increased tonic firing in the TRN seems sufficient for drug-induced SWD generation. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.

Abstract

Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca²⁺ channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to the spike-and-wave discharges (SWDs), which are the hallmarks of absence seizures. We observed that the RBDs were completely abolished, whereas tonic firing was significantly increased, in TRN neurons from mice in which the gene for the T-type Ca²⁺ channel, Ca_V3.3, was deleted (Ca_V3.3^−/−). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in Ca_V3.3^−/− mice and in mice in which the Ca_V3.3 gene was silenced predominantly in the TRN. Ca_V3.3^−/− mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both Ca_V3.3 and Ca_V3.2, which showed complete elimination of burst firing and RBDs in TRN neurons, also displayed enhanced drug-induced SWDs and absence seizures. On the other hand, tonic firing in the TRN was increased in these mice, suggesting that increased tonic firing in the TRN may be sufficient for drug-induced SWD generation in the absence of burst firing. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.

Absence seizures are generalized, nonconvulsive seizures characterized by the appearance of bilaterally synchronous spike-and-wave discharges (SWDs) on the electroencephalogram (EEG). The frequency of the SWDs is variable among different models and is usually higher (4–12 Hz) in rodents than in humans (3 Hz) (1). SWDs represent synchronized oscillations of the thalamocortical network (2–4), a network that includes neurons of the cerebral cortex, thalamocortical nucleus (TC), and thalamic reticular nucleus (TRN) (5). This thalamocortical circuitry is a key CNS structure for gating the flow of sensory information from the periphery to the cortex (6, 7). Both thalamocortical and corticothalamic connections are mainly glutamatergic (8). The TRN is a shell-like structure that covers most of the rostral, lateral, and ventral parts of the thalamus (5) and is composed exclusively of GABAergic interneurons that provide massive inhibitory input to TC neurons (9). The most distinctive feature of thalamocortical circuitry is its intrinsic ability to generate oscillations via the reciprocal circuits between TC and TRN neurons (10–12).

Both TC and TRN neurons are able to generate two distinctive patterns of action potential firing: tonic and burst (13, 14). Burst firing is mediated by low-voltage–activated (LVA) T-type Ca²⁺ channels (15). There are three subtypes of T-type Ca²⁺channels, called Ca_V3.1, Ca_V3.2, and Ca_V3.3, each with distinctive expression patterns and kinetic properties (16). Within the thalamocortical circuit, Ca_V3.1 channels are predominantly expressed in TC neurons, whereas Ca_V3.2 and Ca_V3.3 channels are expressed only in TRN neurons (17). Unlike high-voltage–activated Ca²⁺ channels, T-type Ca²⁺ channels are inactivated at membrane potentials around −60 to −50 mV (15). However, when the membrane potential is hyperpolarized for longer than 100 ms, these channels are de-inactivated and can then initiate a burst of action potentials once the membrane potential is repolarized (18). TC neurons with a relatively depolarized resting membrane potential exhibit a tonic mode of firing associated with conventional, Na/K-dependent action potentials upon receiving excitatory inputs. However, when the neurons are hyperpolarized by inhibitory inputs, high-frequency burst firing can be triggered by Ca_V3.1 channels. TRN neurons, on the other hand, have a relatively hyperpolarized resting potential around −70 mV and most of their T-type Ca²⁺ channels are available for activation. These channels, especially Ca_V3.3, enable TRN neurons to generate rhythmic bursts, even in response to excitatory inputs (12, 19). These oscillatory bursts of action potentials in the TRN provide a barrage of hyperpolarizing inhibitory postsynaptic potentials (IPSPs) to the TC neurons, which in turn respond with action potential bursts due to the activation of Ca_V3.1. These TC bursts provide rhythmic excitatory drive to the cortex, eventually resulting in SWDs.

Several models have been proposed to account for the mechanism of SWD generation in the thalamocortical circuit. One well-known model argues that intrinsic cortical oscillations drive the synchronized activity of the entire thalamocortical circuit to cause SWDs (20). The most widely accepted model suggests that rhythmic bursting activity in TRN neurons is a key element for initiation and maintenance of SWDs (4, 10, 12). This model has been indirectly supported by previous observations that lesions or blockade of voltage-gated Ca²⁺ channels in TRN disrupt SWDs (21), leading to the idea that the degree of the synchrony between TC and TRN is regulated by the burst firing of TRN neurons (10). However, there have been no direct experimental tests of the hypothesis that TRN bursts are indeed essential for SWDs in absence seizures. Here we have tested the role of TRN bursts in absence epilepsy and have surprisingly found that mice with complete genetic deletion of TRN burst activity exhibited enhanced SWDs with higher susceptibility to γ-butyrolactone (GBL), a widely used seizure-inducing drug. Apparently that enhanced TRN tonic firing is observed in the absence of bursts is sufficient to support SWDs.

Results

Generation of Ca_V3.3 Ca²⁺ Channel Knockout Mice.

To explore the physiological function of Ca_V3.3, we carried out targeted disruption of the Ca_V3.3 gene in mice as shown in Fig. 1A. The targeting vector was designed to delete exon3 and exon4 that comprise the N terminus of the Ca_V3.3 protein (Amgen Co.; Fig. 1A). Following successful germ-line transmission, mice heterozygous for the targeting event were interbred to obtain homozygous Ca_V3.3 knockouts (KOs), as confirmed by PCR and/or Southern blot (Fig. 1B). No sex bias was observed in the offspring, and the expected Mendelian ratio was observed among wild-type, heterozygous, and homozygous mutant mice (data not shown). To assess whether disruption of the Ca_V3.3 gene was effective, we examined the level of Ca_V3.3 mRNA. RT-PCR analysis showed that no Ca_V3.3 mRNA was produced in the Ca_V3.3 brain, indicating that the gene targeting resulted in a null mutation for this locus (Fig. 1C). We also used in situ hybridization to check the expression and cellular localization of Ca_V3.3 in wild-type and mutant mouse brains (Fig. 1D).

Fig. 1.

Targeted disruption of the mouse Ca_V3.3 gene and decreased T-type current in Ca_V3.3 KO. (A) Schematic representations of the wild-type Ca_V3.3 allele, targeting vector, and mutant allele. Exons were represented by black boxes. Exon3 and exon4 were designed to be deleted to generate the mutant allele. (B) Southern blot analysis of genomic DNA isolated from tails of wild-type (WT) and homozygous KO mice. The restriction enzymes and probes used are shown in A. The 12 kb segment corresponds to the wild allele; the 9 kb, the targeted allele. (C) RT-PCR analysis was performed with mRNA derived from pooled samples of wild-type or the Ca_V3.3 mutant whole brain. PCR primers for RT-PCR were exon3–7. The 690 bp band indicates the PCR product of the wild type, whereas Ca_V3.3 mutant displays no band. (D) Quantitative analysis of the expression of the Ca_V3.3 mRNA with in situ hybridization. There is robust expression of Ca_V3.3 in the TRN of wild-type mouse but no expression in mutant TRN. Probes for in situ hybridization were exon3 and 4. (E) LVA Ca²⁺ currents were evoked by the depolarizing pulses ranging from −100 to −40 mV in wild-type and Ca_V3.3^−/− neurons. (F) Current–voltage relationship displayed a significant reduction in the peak current density in Ca_V3.3^−/− (open circle) compared with wild type (closed circle). Data are represented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed t test.

T-Type Currents and Oscillatory Burst Firing Are Impaired in Ca_V3.3 KO Mice.

The functional loss of Ca_V3.3 was examined by whole-cell voltage clamp analysis of TRN neurons in acute brain slices from ∼3–4-wk-old KO and wild-type mice. LVA inward currents were evoked from a holding potential of –100 mV to depolarizing levels ranging from –90 to –40 mV (Fig. 1E). The peak density of a fast-inactivating current, typical of T-type Ca²⁺ currents, evoked at appropriate test potentials were significantly reduced in Ca_V3.3^−/− TRN neurons compared with wild-type controls (Fig. 1F). These results are consistent with those reported previously (22) and suggest that Ca_V3.3 is responsible for most T-type Ca²⁺ currents in TRN neurons. The small amount of residual current observed in Ca_V3.3^−/− neurons at –40 mV is probably mediated by Ca_V3.2 and as observed previously (22).

To identify GABAergic neurons that project from TRN to TC, we crossed the Ca_V3.3 mutant mice with glutamate decarboxylase 65 (GAD65)-GFP transgenic mice. Injection of a retrograde tracer into the TC region of these mice retrogradely labeled some of the GFP-positive GABAergic neurons in the dorsomedial region of TRN (Fig. 2A), allowing us to identify and characterize GABAergic projection neurons of TRN. In acute brain slices from wild-type mice, a brief hyperpolarizing current pulse that brought the membrane potential to −100 mV evoked subsequent oscillatory rebound bursting in ∼40% of projection neurons, whereas 45% showed only a single low-threshold (LT) burst, and 15% exhibited no LT bursting. In Ca_V3.3^−/− mice, 80% of GFP-positive projection neurons showed no LT burst firing, whereas 20% exhibited a single weak LT burst and oscillatory bursts were never observed (Freeman–Halton extension of the Fisher’s exact probability test, P = 0.0000053) (Fig. 2B). Thus, the ability of TRN neurons to fire bursts of action potentials is severely impaired in Ca_V3.3^−/− mice.

Fig. 2.

Altered firing properties of TRN neurons lacking Ca_V3.3 channels and increased susceptibility to GBL-induced SWD in Ca_V3.3 KO mice. (A) TRN neurons labeled with red retrograde tracer injected into TC of GAD65-GFP mice. Confocal image of TRN neurons colabeled with retrograde beads (red) and GFP (expanded image). (B) Firing characters of Ca_V3.3^+/+ (upper three traces) and Ca_V3.3^−/− (lower two traces) TRN neurons. (C) EEG traces of adult Ca_V3.3^+/+ (Upper) and Ca_V3.3^−/− (Lower) mice for 1 min before and various times after GBL injection. SWDs marked with asterisks are expended at the bottom. (D) SWD density calculated by total duration of SWD per min in Ca_V3.3^+/+ (black circle) and Ca_V3.3^−/− (red circle) mice. (E) Distribution of SWD episode duration after GBL injection. (F) Average EEG power spectrograms of Ca_V3.3^+/+ and Ca_V3.3^−/− mice during a 50-min recording. GBL was injected after 10 min of baseline recording. Data are represented as mean ± SEM; *P < 0.05, **P < 0.01.

Increased GBL-Induced Absence Seizures in Ca_V3.3 KO or Knockdown Mice.

Because Ca_V3.3 is abundantly expressed in TRN but not in TC (17), we could use Ca_V3.3^−/− mice to determine the role of TRN neuron bursting in SWDs. Based on the current model (10), we predicted that SWDs should decrease or disappear in Ca_V3.3^−/− mice. To test this prediction, we examined the susceptibility of adult Ca_V3.3^−/− mice to the seizure-inducing drug GBL (Fig. 2C). Administration of GBL (70 mg/kg body weight, i.p.) to wild-type mice at the age of ∼10 wk induced typical paroxysmal SWDs (Fig. 2C) and behavioral arrest (Fig. S1A). Contrary to our expectations, in Ca_V3.3^−/− mice, SWDs were not reduced; in fact, GBL more effectively induced SWDs both in density (s/min) and total duration in Ca_V3.3^−/− mice than in wild-type mice [repeated-measures ANOVA (rmANOVA), group effect, F₍₁₎ = 15.67, P = 0.0008; TIME effect, F₍₅₇₎ = 14.46, P < 0.0001; Group × Time interaction, F₍₅₇₎ = 4.35, P < 0.0001] (Fig. 2D), with longer SWD episode duration (Kolmogorov–Smirnov test, P = 0.00003) (Fig. 2E). The relative EEG power in the 3–5 Hz range characteristic of SWDs was significantly higher in the Ca_V3.3^−/− mice than wild-type for the entire recording duration after GBL treatment (Fig. 2F). Similar results were found in juvenile (age 3–4 wk) mutant mice [rmANOVA, group effect, F₍₁₎ = 1.94, P = 0.1807; time effect, F₍₅₇₎ = 15.2, P < 0.0001; Group × Time interaction, F₍₅₇₎ = 1.9, P < 0.0001] (Fig. S1 C and D). SWDs were analyzed based on the raw and filtered waveform of EEG recordings as described in detail in SI Materials and Methods (Fig. S1 A and B). The antiabsence drug ethosuximide (ETX) dramatically suppressed GBL-induced SWDs in mutant animals, which confirms that these were typical SWDs (Fig. S1B₁).

Within the thalamocortical pathway, Ca_V3.3 is expressed both in cortex and in TRN (17). To address possible cortical contributions to the seizure phenotype of Ca_V3.3^−/− mice, we used virus-mediated gene silencing to knock down Ca_V3.3 predominantly in TRN. An adeno-associated viral (AAV) vector containing shRNA specific for Ca_V3.3 (Fig. S2 A and B) was injected bilaterally into the TRN of 8-wk-old GAD65-GFP mice (Fig. 3A, Upper). Three weeks later, both scrambled (control) and shRNA for Ca_V3.3 (shCa_V3.3) visualized with red fluorescence (mCherry) colocalized with GAD65-positive (GFP expressing) neurons in TRN. Expression of virus was restricted to TRN neurons (Fig. 3A, Middle and Lower) and significantly reduced Ca_V3.3 mRNA expression by ∼62% in TRN compared with control (Fig. 3B). Such TRN-specific gene silencing of Ca_V3.3 reduced T-type Ca²⁺ currents in TRN neurons (Fig. S2 C and D) and enhanced sensitivity to GBL, similar to what we observed in the Ca_V3.3^−/− mice. Ca_V3.3 knockdown caused a significantly shorter onset time and a higher density of SWD compared with mice injected with control virus [rmANOVA, group effect, F₍₁₎ = 4.52, P = 0.0468; time effect, F₍₅₇₎ = 9.21, P = 0; Group × Time interaction, F₍₅₇₎ = 1.12, P = 0.2514] (Fig. 3 C and D). An EEG power spectrum in the 3–5 Hz range also was significantly higher in Ca_V3.3 knockdown mice than in control mice (Fig. 3E). Therefore, these results suggest that an increased GBL-induced absence seizure in Ca_V3.3 KO was mediated by deletion of Ca_V3.3 at TRN, not cortex.

Fig. 3.

Enhanced susceptibility to GBL-induced SWD after microinjection of AAV-shCa_V3.3 in the TRN of GAD65-GFP mice. (A) Confocal images showing expression of AAV-control and AAV-shCa_V3.3 (yellow circles indicate the TRN region in Top panels). Virus-infected neurons are colabeled with GFP (green, GAD65-GFP–positive neurons; red, AAV-control–infected neurons in Middle panel; and AAV-shCa_V3.3 in Bottom panel; yellow, merged). (B) Representative images of in situ hybridization (Upper, AAV-control; Lower, AAV-shCa_V3.3). (C) EEG traces of AAV-control (Left) and AAV-shCa_V3.3 mice (Right) for 1 min before and various time after GBL injection. (D) SWD density calculated by total duration of SWD per min in AAV-control– (black circle) and AAV-shCa_V3.3– (red circle) injected mice. (E) Average EEG power spectrograms of AAV-control– and AAV-shCa_V3.3–injected mice during a 50-min recording. GBL was injected after 10 min of baseline recording. Data are represented as mean ± SEM, *P < 0.05.

Complete Deletion of Burst Firing in TRN Enhances Absence Seizures in Ca_V3.3^−/− KO and Ca_V3.2^−/−/3.3^−/− Double-KO Mice.

To sidestep the possible effects of residual T-type channel activity present in the TRN of Ca_V3.3 knockdown and Ca_V3.3^−/− mice (Fig. 1F), we made double-KO (DKO) mice lacking both Ca_V3.2 and Ca_V3.3 (Ca_V3.2^−/−/3.3^−/− mice) (Fig. S3A). Although these mice had no bursting activity in their TRN neurons (Fisher’s exact probability, P = 0.0012) (Fig. 4A), the effect of GBL injection was similar to what we observed in Ca_V3.3^−/− and Ca_V3.3 knockdown mice [rmANOVA, group effect, F₍₁₎ = 21.35, P = 0.0004; time effect, F₍₅₇₎ = 13.99, P < 0.0001; Group × Time interaction, F₍₅₇₎ = 5.43, P < 0.0001] (Fig. 4 B–F). The interspike interval within an SWD was not changed. Further, neither Ca_V3.3^−/− KO nor Ca_V3.2^−/−/3.3^−/− DKO mice exhibited spontaneous SWD (data not shown). These findings indicate that SWD can be maintained in the complete absence of TRN burst firing and, in fact, is enhanced when bursts are abolished.

Fig. 4.

Altered firing properties of TRN neurons and increased susceptibility to GBL-induced SWD in Ca_V3.2^−/−/3.3^−/− mice. (A) Firing properties of Ca_V3.2^+/+/3.3^+/+ (Right) and Ca_V3.2^−/−/3.3^−/− (Left) TRN neurons. (B) EEG traces of adult Ca_V3.2^+/+/3.3^+/+ (Left) and Ca_V3.2^−/−/3.3^−/− (Right) mice for 1 min before and various times after GBL injection. (C) SWD density calculated by total duration of SWD per min in Ca_V3.2^+/+/3.3^+/+ (black circle) and Ca_V3.2^−/−/3.3^−/− (red circle) mice. (D) Average EEG power spectrograms of Ca_V3.2^+/+/3.3^+/+ and Ca_V3.2^−/−/3.3^−/− mice during a 50-min recording. GBL was injected after 10 min of baseline recording. (E) Onset time of GBL-induced SWDs in Ca_V3.3^−/− (white) and Ca_V3.2^−/−/3.3^−/− (red) mice and their controls (black and gray, respectively). (F) Total duration of SWD after GBL injection. Data are represented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Tonic Firing Is Increased in Ca_V3.3^−/− KO and Ca_V3.2^−/−/3.3^−/− DKO Mice.

To explain this unexpected observation, we considered the possibility that loss of T-type Ca²⁺ currents could have other effects on TRN neurons that enhance SWDs. In contrast to the loss of TRN bursts, the frequency of tonic firing evoked by depolarizing currents was significantly increased in TRN GFP-positive projection neurons of Ca_V3.3^−/− mice compared with those in wild-type mice [two-way ANOVA, group effect, F_(1,36) = 4.8, P = 0.035] (Fig. 5 A and B). Tonic firing was also increased in Ca_V3.2^−/−/3.3^−/− mice (Fig. S3 B and C). To examine the cause of the increased tonic firing, we analyzed the after-hyperpolarizations (AHPs) that are known to regulate action potential frequency. The amplitude of the fast AHP (fAHP), which plays an important role in repolarization of membrane potential after an action potential, was not significantly different between Ca_V3.3^−/− and wild-type neurons (Fig. 5C). Coupling between T channels and Ca²⁺-activated K⁺ channels is known to be indispensable for oscillatory discharges of TRN neurons (23). Thus, we examined the amplitude of the medium AHP (mAHP) caused by activation of Ca²⁺-activated K⁺ channels and found that mAHPs were significantly reduced in the mutant mice (Fig. 5D). This was also observed in Ca_V3.2^−/−/3.3^−/− mice (Fig. S3 D and E). Therefore, an increased tonic firing of mutant TRN neurons might be at least partially due to a decreased mAHP. In contrast, other intrinsic electrophysiological properties of TC neurons were unaffected in mutant mice (Fig. S4 A–D). These neurons also exhibited no differences in the amplitude or frequency of spontaneous inhibitory postsynaptic currents (IPSCs) resulting from TRN input (Fig. S4 E–G), which is consistent with the lack of spontaneous SWDs in Ca_V3.3^−/− mice.

Fig. 5.

Enhanced inhibitory synaptic inputs to TC neurons during tonic firing in TRN neurons of Ca_V3.3^−/− mice. (A) Representative traces of tonic firings in Ca_V3.3^+/+ (Left) and Ca_V3.3^−/− TRN neurons (Right). (B) Average number of tonic firings with different current pulses in Ca_V3.3^+/+ and Ca_V3.3^−/− TRN neurons. (C) Average fAHPs in Ca_V3.3^+/+ and Ca_V3.3^−/− TRN neurons. (D) Average mAHPs in Ca_V3.3^+/+ and Ca_V3.3^−/− TRN neurons. (E) IPSC evoked by tonic-frequency (upper trace, five stimuli at 100 Hz) or burst-frequency (lower trace, five stimuli at 500 Hz) electrical stimulations in Ca_V3.3^+/+ and Ca_V3.3^−/− TC neurons. (F) Average IPSC area in Ca_V3.3^+/+ and Ca_V3.3^−/− TC neurons. (G) PPR in Ca_V3.3^+/+ and Ca_V3.3^−/− TC neurons. Data are represented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Enhanced TRN–TC Inhibitory Synaptic Connection in the Mutant.

To determine the downstream effect of increased tonic firing in TRN neurons, we next examined the TRN–TC inhibitory synaptic connection. Whole-cell patch clamp recordings from TC neurons of Ca_V3.3^−/− mice, measured in the presence of the glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2, 3-dione and 2-Amino-5-phosphonopentanoic acid, revealed monosynaptic IPSCs in response to electrical stimulation of TRN. There was no significant difference in the amplitude of IPSCs evoked in TC neurons by single stimuli (wild type, 274 ± 117 pA, n = 8; Ca_V3.3^−/−, 321 ± 131 pA, n = 9, P = 0.8). This indicates that basal synaptic strength is unchanged in the mutant. However, differences were observed in response to multiple TRN stimuli. We used five stimuli at 100 Hz or 500 Hz to mimic tonic or burst firing, respectively (Fig. 5E). Previous in vivo observations show that TRN neurons fire bursts composed of 5–15 spikes at frequencies ranging from 250 to 550 Hz (24). Kim et al. (25) demonstrated that a 100 Hz tonic discharge or a 500 Hz burst frequency of perigeniculate nucleus generates IPSPs in postsynaptic TC cells in vitro. For tonic-frequency stimulation, synaptic strength, measured as the integrated charge of IPSCs, was significantly larger in Ca_V3.3^−/− compared with wild-type mice (Fig. 5F). However, there was no difference between the two genotypes in their responses to burst-frequency stimulation. As a result, in the mutant IPSC, responses to tonic-frequency stimulation were significantly larger than responses to burst-frequency stimulation. To reveal how tonic-frequency stimulation increased inhibitory synaptic transmission in Ca_V3.3^−/− mice, we analyzed the paired-pulse ratio (PPR; ratio of second/first response) of IPSCs. The PPR evoked by tonic-frequency stimulation was greater (P = 0.038) in Ca_V3.3^−/− (0.74 ± 0.6) than in wild type (0.55 ± 0.75) (Fig. 5G). This indicates that the increase in inhibitory synaptic transmission in Ca_V3.3^−/− mice is activity-dependent and presynaptic in origin, perhaps caused by the decreased mAHPs of TRN neurons. The change in PPR may reflect changes in presynaptic release probability.

Discussion

TRN neurons are thought to be pacemakers for the initiation of thalamocortical oscillations (3, 11). Both pharmacological and lesion studies have suggested that rhythmic bursts in the TRN are a key element in generating these oscillations (21, 26). However, our results show that tonic firing of TRN neurons in the absence of TRN burst firing is sufficient to generate SWDs, which we believe is a novel observation.

The Role of TRN in the Enhancement of SWDs.

Our results raise the possibility that tonic firing in the TRN has a function in regulating the thalamocortical rhythms in certain pathological states, such as absence epilepsy. Both genetic elimination and viral-mediated, TRN-specific knockdown of the T-type Ca²⁺ current caused similar and significant increases in both tonic firing and GBL-induced SWDs. This shows that the level of tonic firing is highly correlated with the degree of SWDs and with susceptibility to absence seizures. Moreover, the results from the Ca_V3.2^−/−/3.3^−/− mice, which completely lack burst firing in TRN neurons, indicate that SWDs can be initiated in the complete absence of burst firing. Therefore, we propose that the excitability of TRN neurons can play an important modulatory role in maintaining SWDs. This proposal is based on the facts that both Ca_V3.3^−/− and Ca_V3.2^−/−/3.3^−/− mice lacked burst firing, yet had enhanced tonic firing and enhanced SWDs. Tonic firing in the TRN neurons might be a more potent regulator of the thalamocortical circuit than burst firing. Indeed, we observed that stimulation of TRN neurons at a tonic frequency evoked stronger summed inhibition of TC neurons than stimulation at burst frequency. This suggests that tonic firing might even be more efficient in mediating the TRN-to-TC signals and initiating thalamocortical rhythms. On the other hand, the gain of Ca_V3.2 channel function has been reported in the mutated channel identified in absence epilepsy patients (27). However, there is still a controversy on the role of Ca_V3.2 in absence epilepsy because no consistent sequence variant has been found in Ca_V3.2 in absence patients (28, 29).

It is important to note that there were no spontaneous SWDs in the mutant mice with the increased tonic firing of TRN neurons. In contrast, genetic enhancement or elimination of the T current in TC neurons is known to induce or abolish spontaneous SWDs, respectively (30, 31), which provides convincing evidence that Ca_V3.1 in TC neurons plays a key role in the genesis of absence seizure. Until the current studies, however, effects of manipulations of TRN neurons have not been tested before.

Pharmacological Model of Absence Seizure.

GBL is a precursor of the GABA analog, gamma-hydroxybutyric acid (GHB), and is widely used as a pharmacological model of absence seizures in mice. The established target of GBL (or GHB) action is the presynaptic GABA_B receptor that regulates corticothalamic excitatory synapses onto TC neurons. We used a GBL concentration (70 mg/kg) that is known to preferentially influence the presynaptic GABA_B receptors on cortical and TRN neurons, rather than the postsynaptic GABA_B receptors of TC (32). Therefore, we did not bypass the TC GABA_B activation. It is also known that the dose we used does not induce sleep or coma in mice (33). To test the role of burst firing in TRN in the generation of absence seizures, we applied GBL to both Ca_V3.3^−/− KO and Ca_V3.2^−/−/3.3^−/− DKO mice. Because the enhancement of TRN excitability in both mice was insufficient to induce spontaneous SWDs, additional increases in TC excitability might be required for GBL to cause the thalamocortical oscillations that underlie SWDs. Consistent with our current observations, it has been reported that TRN neurons in epileptic genetic absence epileptic rats from Strasbourg (GAERS) rats displayed higher excitability, higher firing frequency, and regularity (34). In parallel with these results from GAERS rat, stargazer mice (another genetic model for absence seizures) showed hyperexcitable TRN neurons due to an enhanced NMDA receptor (35). Thus, several gene mutation models of absence seizures show altered TRN excitability.

Increased Tonic Firing in TRN in the Absence of Ca_V3.3 T-Type Channels.

The maximum frequency of tonic firing in neurons is determined by AHPs that arise from the interplay of K⁺ and Ca²⁺ channels. Our data showed increased mAHPs but not fAHPs in TRN neurons of Ca_V3.3 KO and Ca_V3.2/3.3 DKO mice. Previous papers have reported that Ca_V3 channels are coupled to Kv4 channels (LVA A-type potassium channels), which is essential to influence firing properties in cerebellar stellate cells. Also Kv4 and Ca_V3 channels are linked by an accessory calcium-sensing protein, K⁺ channel-interacting protein 3 (KChIP3), but not KChIP1 or KChIP2, in stellate cells (36). However, KChIP3 proteins are not expressed in TRN neurons (37). Therefore, it is unlikely that Kv4 is involved in mAHPs in TRN neurons.

Compared with fAHPs, which are mediated by voltage-gated K⁺ channels, mAHPs occur when Ca²⁺-activated K⁺ channels are activated by Ca²⁺ influx through T-type Ca²⁺ channels (38). This is consistent with our results of no change in fAHP in the TRN of Ca_V3.3 KO and Ca_V3.2/3.3 DKO mice. The mAHPs prevent an excessive increase in axon fiber excitability and contribute to the gradual decrease in tonic firing rate during a continuous activity. In TRN neurons, the mAHP is largely dependent on Ca_V3.3 T-type channels that are the major T-type Ca²⁺ channel in this region (22). In Ca_V3.3^−/− and Ca_V3.2^−/−/3.3^−/− mice, the mAHP is greatly reduced due to the lack of T-type channels. This has a significant effect on signal transfer between TRN and TC neurons. Two possible effects of reduced mAHP include (i) increased axon fiber excitability in the TRN neurons of the Ca_V3.3^−/− mice, enabling recruitment of abgreater number of axon fibers from the surrounding TRN neurons, and (ii) increased probability of release during the tonic-frequency activity in Ca_V3.3^−/− mice. Consequent increases in GABA release onto TC fibers will lead to an augmentation of signals from the TRN, which might account for the increased SWDs in Ca_V3.3^−/− mice. The current model for SWD generation postulates that inhibitory inputs from the TRN onto the TC hyperpolarize TC neurons, which leads to the activation of LT T-type Ca²⁺ channels and subsequent burst firing in the TC. Our current results agree with the hypothesis that TRN inputs to TC are critical for SWD generation. However, our study indicates that TRN tonic firing, in absence of burst firing, is also quite capable of delivering effective inhibitory synaptic input onto TC neurons. Another potential explanation could be that enhanced inhibitory inputs onto the TC may influence tonic inhibition in the TC, which has been observed in typical absence epilepsy (39, 40).

Enhanced understanding of the detailed pathophysiological mechanism of SWD generation in the thalamocortical circuit will help improve the treatment of absence epilepsy. Our findings about the novel function of tonic firing in the TRN neurons indicates a need to revise the current model of thalamocortical circuit function and opens up a previously unidentified venue for the development of remedies for absence epilepsy.

Materials and Methods

Animals.

Ca_V3.3 KO was generated on a hybrid background of 129/SvJ and Black Swiss strains and backcrossed to the desired inbred C57BL/6 or 129S4/SvJae for 10 generations. Then, each background line was used to generate desired homozygotes for different experiments. Double heterozygotes of Ca_V3.2^+/−/3.3^+/− were first generated by intercrossing Ca_V3.2 heterozygotes and Ca_V3.3 heterozygotes, and then double heterozygous outcomes were interbred to generate Ca_V3.2^−/−/3.3^−/− DKO mice. For details, see SI Materials and Methods.

Electrophysiology.

For whole-cell patch clamp recordings in TRN cells, horizontal slices 280 μm thick were prepared from 3- to 4-wk-old B6129Ca_V3.3^−/−, B6129Ca_V3.3^−/−/GAD65GFP^tg, and wild-type littermates. Details of electrophysiological recording conditions, data acquisition, and analysis are given in SI Materials and Methods.

AAV-shRNA Vector Design.

To design the AAV-shCa_V3.3 vector, we digested out the U6 promoter, Ca_V3.3 shRNA, and mCherry fragment from the pSicoR lentiviral vector and cloned it into the transfer plasmid AAV containing multiple cloning site (Agilent Technologies cat. no. 240071). The scrambled shRNA-containing AAV (AAV-ctrl) construct was used as the control. For details, see SI Materials and Methods.