Dysfunction of parvalbumin-expressing cells in the thalamic reticular nucleus induces cortical spike-and-wave discharges and an unconscious state

Abstract

Spike-and-wave discharges and an accompanying loss of consciousness are hallmarks of absence seizure, which is a childhood generalized epilepsy disorder. In absence seizure, dysfunction of the cortico-thalamo-cortico circuitry is thought to engage in abnormal cortical rhythms. Previous studies demonstrated that the thalamic reticular nucleus has a critical role in the formation of normal cortical rhythms; however, whether thalamic reticular nucleus dysfunction leads directly to abnormal rhythms, such as epilepsy, is largely unknown. We found that expressing the inhibitory opsin, archaerhodopsin, including in the thalamic reticular nucleus, caused abnormal cortical rhythms in Pvalb-tetracycline transactivator::tetO-ArchT (PV-ArchT) double transgenic mice. We validated the PV-ArchT line as a new mouse model of absence seizure through physiological and pharmacological analyses, as well as through examining their behavioural features. We then discovered that archaerhodopsin expression exclusively in thalamic reticular nucleus parvalbumin-positive neurons was sufficient to induce cortical spike-and-wave discharges using adeno-associated virus-mediated thalamic reticular nucleus targeting. Furthermore, we found that archaerhodopsin expression impaired rebound burst firing and T-current in thalamic reticular nucleus parvalbumin-positive cells by slice physiology. Although T-current in the thalamic reticular nucleus was impaired, the T-current blocker ethosuximide still had a therapeutic effect in PV-ArchT mice, suggesting a gain of function of T-type calcium channels in this absence seizure model. However, we did not find any over- or misexpression of T-type calcium channel genes in the thalamus or the cortex. Thus, we demonstrated that thalamic reticular nucleus dysfunction led to an absence seizure-like phenotype in mice. In a final set of experiments, we showed that the archaerhodopsin-mediated absence seizure-like phenotype disappeared after the removal of archaerhodopsin by using a time-controllable transgenic system. These data may provide a hint as to why many absence seizures naturally regress.

Keywords: T-type calcium channel; animal model of absence seizure; inhibitory opsin; rebound burst firing; tetracycline-controllable gene induction.

Graphical abstract

Figure 1

ArchT-GFP expression in PV-positive cells using a Tet-Off system. (A) ArchT-GFP was expressed by a Tet-Off system. tTA protein-induced ArchT-GFP expression in PV-positive neurons occurred in the absence of DOX. (B) Direct fluorescent image of a sagittal section from a PV-ArchT mouse fed normal chow (n = 3). TRN, thalamic reticular nucleus; CL, cerebellum; RSC, retrosplenial cortex. Scale bar, 500 μm. (C) Confocal images showing PV and GFP immunoreactivities in the TRN of a mouse fed normal chow (n = 3 mice). PV-positive cells (magenta) expressed ArchT-GFP (green). Yellow arrows confirm that PV-positive cells express GFP. Top scale bar, 200 μm; bottom scale bar, 20 µm.

Figure 2

PV-ArchT mice show abnormal EEG. (A) Cortical EEG traces. Top: control; bottom three traces: PV-ArchT mice aged 2 months. EEG of PV-ArchT mice often included epileptiform discharges (blue traces). (B) Spectrograms of the power of EEG for controls and PV-ArchT. The dashed lines indicate the individual data, and the solid lines with shaded area indicate the mean ± SEM. PV-ArchT mice aged 2 months showed high theta power (4–11 Hz, purple highlighted, unpaired t-test, t₁₀ = 2.799, P = 0.005). (C) Typical shapes of epileptiform discharges in PV-ArchT mice.

Figure 3

Characterization of SWD in PV-ArchT mice. (A) PV-ArchT mice aged >2 months exhibited SWDs (red traces) and other types of epileptiform discharges (blue traces). (B) The EEG power of SWD events was significantly higher than that of baseline (4–11 Hz, paired t-test, t₇ = 2.32, P = 0.03). (C) Age-dependent shows the worsen of SWDs events (SWDs; red traces). (D) Line plots show a significant increase in SWD duration (left panel) and frequencies (right panel) with age from P60 to P150 [one-way ANOVA, duration: n = 5 mice; F(3,12) = 23, P = 2 × 10⁻⁵; frequency: n = 5 mice; F(3,12) = 30, P = 7 × 10⁻⁶] from P60 to P150. (E) Line plots show no changes of high theta power with age from P60 and P150 [one-way ANOVA, n = 5 mice: F(3,812) = 0.01, P = 0.999].

Figure 4

PV-ArchT mice exhibited an absence seizure phenotype. Cortical EEG and EMG in PV-ArchT mice at P90 (A) and P120 (B). Both ages show SWDs (red traces) and other types of epileptiform discharges (blue traces). SWD events (98.8% from 85 SWDs at P90 and 91.5% from 150 SWDs at P120) were associated with low EMG amplitude. (C) Before (top) and after (bottom) administering ethosuximide to PV-ArchT mice (3–6 months). Ethosuximide abolished SWDs (before 23 ± 7.6/h versus after treatment 1.8 ± 0.7/h, n = 4 mice). (D) Before (top) and after (bottom) baclofen administration. Baclofen increased the frequency (before 32 ± 9.7/h versus after treatment 63 ± 15/h, n = 4 mice). (E) Line plots (left panel) show a significant decrease and an increase in SWD frequency after ethosuximide and baclofen administration, respectively (ethosuximide: paired t-test, t₃ = 2.9, P = 0.03; baclofen: paired t-test, t₃ = 2.4, P = 0.02). A line plot (right panel) shows significantly increased duration after baclofen (2.3 ± 0.8 versus 6.2 ± 2.4 s, paired t-test, t₃ = 2.4, P = 0.04). (F) Line plots (left panel) show no changes of the theta power after ethosuximide (paired t-test, t₃ = −1.54, P = 0.164). The line plots (right panel) show significantly decreased high theta power after baclofen (paired t-test, t₃ = 2.21, P = 0.038).

Figure 5

ArchT expression in the TRN-induced SWDs. (A) A strategy of ArchT-GFP expression only in the TRN. PV-Cre mice were injected with AAV carrying CAG-DIO-ArchT-GFP, resulting in ArchT-GFP expression in TRN PV-positive cells (n = 4 mice). (B) AAV injection targeted the middle portion of the dotted line of TRN. Scale bar, 200 μm. (C) Confocal images showing the localization of ArchT-GFP signals (green) and PV signals (magenta). Scale bar, 20 μm. (D–F) Cortical EEG waveforms after the virus injection. (D) Cortical EEG traces at 7 days (top grey) and 30 days (bottom black) after virus injection. The line plots show increased theta power 30 days after virus injection (paired t-test, t₃ = −2.34, P = 0.019). (E) Cortical EEG at 60 days after virus injection show epileptiform discharges (blue traces). (F) Baclofen administration significantly induced typical SWDs (red traces) with epileptiform discharges (blue traces). (G) Line plots show increased frequency (left panel) and duration (middle panel) of epileptiform discharges after baclofen administration (frequency: before 2.5 ± 1.2/h versus after 13 ± 4.1/h, paired t-test, t₃ = 2.35, P = 0.04; duration: before 1.7 ± 0.6 versus after 2.7 ± 0.9 s, paired t-test, t₃ = 2.35, P = 0.04); and right panel shows no changes of high theta power (paired t-test, t₃ = 1.9, P = 0.265).

Figure 6

ArchT expression in the TRN impaired rebound bursting and T-current. (A) Representative traces of rebound burst firing in PV neurons following hyperpolarization from PV-ArchT mouse brain slices (top trace, 13 cells with single bursts; middle trace, 4 cells did not evoke burst firing) and from controls (bottom trace, 10 cells evoked burst firing). The data were from three PV-ArchT mice and three controls. Neurons were injected with negative current (−0.5 nA) to induce hyperpolarization to −112 mV. Scale indicates 0.5 s and 40 mV. (B) Number of rebound bursts after hyperpolarization (10 cells from 3 control and 20 cells from 3 PV-ArchT mice). (C) Number of action potentials (APs) in the first burst (10 cells from control and 20 cells from PV-ArchT). (D) Duration required for first bursts to evoke action potentials (10 cells from 3 mice control and 16 cells from 3 PV-ArchT). (E) Frequency of APs from first bursts. (F) T-current was evoked by hyperpolarizing steps (500 ms, ranging from −110 to −50 mV, with 10 mV increments) in PV-ArchT (top trace) and control (bottom trace). Inset shows an example trace of the light-evoked response from a PV-ArchT neuron at a holding potential of −80 mV (G) T-current amplitude. Data are shown as mean ± SEM. *P < 0.05 and ***<0.01, unpaired t-test comparing PV-ArchT and control groups.

Figure 7

Cells expressing T-type Ca²⁺ channel mRNA transcripts in PV-ArchT mouse brains compared with controls. ISH of T-type Ca²⁺ channel transcripts is shown. Left, PV-ArchT; right, control. (A) Cav3.1 (Cacna1g) was expressed in all cortical layers and in the thalamus, but not in the TRN. (B) Cav3.2 (Cacna1h) was strongly expressed in Layer V and in the TRN. (C) Cav3.3 (Cacna1i) was found in all layers of the cortex and in the TRN. The inset of the black square shows the magnification of the cortex, and the inset of the white square shows the magnification of the thalamus and TRN. Note that the expression of T-type Ca²⁺ channel transcripts did not show difference in the cortex, thalamus and TRN comparing PV-ArchT with the control (n = 4 mice). (D) Graphs show transcript levels, as measured with quantitative RT-PCR, of the three types of T-type Ca²⁺ channels in the cortex and thalamus. No significant changes between PV-ArchT and control, respectively (Cav3.1; 0.72 ± 0.11 versus 1 ± 0.14; Cav3.2; 0.96 ± 0.07 versus 1 ± 0.05; Cav 3.3; 0.94 ± 0.09 versus 1 ± 0.05; Cav3.1 of the thalamus 0.79 ± 0.16 versus 1 ± 0.07, n = 3 mice for each group). Data are shown as mean ± SEM.

Figure 8

SWDs were dose- and time-dependently induced by ArchT. Summary graphs (left) showing the feeding regimen (DOX or normal chow) and the corresponding level of ArchT-GFP expression; charts (middle) presenting the average SWDs/h; and IHC images (right) of the TRN showing GFP (green, representing ArchT) in PV neurons (magenta) and the merged image of the two, scale bar, 200 μm. (A) Mice under a normal chow their entire lives (n = 4 mice). (B) Mice with neonatal induction of ArchT (27 ± 3.8 times/h, P60, n = 4 mice). (C) Mice under DOX chow their entire lives (n = 3 mice). (D) Mice with adult induction of ArchT (9.1 ± 0.9 times/h, n = 3 mice). Data are shown as mean ± SEM. *P < 0.05 based on paired t-tests comparing two time points.