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

Abstract

Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca(2+) 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 Ca(2+) channel, CaV3.3, was deleted (CaV3.3(-/-)). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in CaV3.3(-/-) mice and in mice in which the CaV3.3 gene was silenced predominantly in the TRN. CaV3.3(-/-) mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both CaV3.3 and CaV3.2, 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.

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.

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.

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.

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.

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.