CaV 3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons

Abstract

Objective: Genetic alterations have been identified in the CACNA1H gene, encoding the Ca_V 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of Ca_V 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN.

Methods: Pathophysiological contributions of Ca_V 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings.

Results: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a Ca_V 3.2 mutation-sensitive splice variant. In vivo knock-down of Ca_V 3.2 using direct thalamic injection of lipid nanoparticles containing Ca_V 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures.

Significance: This supports a role for TRN Ca_V 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs.

Keywords: T-type calcium channel; absence epilepsy; low threshold spike; thalamocortical.

Figure 1

Adult GAERS TRN neurons display sustained oscillatory burst‐firing at 6‐9 Hz. A, Representative traces showing response of adult NEC (A—upper panel) and GAERS (A—middle panel) TRN neurons to a 2 second sinusoidal current injection stimulus (A—lower panel). Stimulus magnitude was incrementally increased until bursting (>3 action potentials) occurred on 3 initial bursts. Scale bars = 30 mV, 100 msec. B, The sinusoidal stimulus was applied at variable frequencies (5‐10 Hz), with the number of action potentials per burst decreasing with time. Insets show representative voltage traces of the last burst in a series. 6 Hz: NEC = 31.2 ± 6.2%, GAERS = 52.8 ± 7.8, P = .034 t test; 7 Hz: NEC = 23.6 ± 4.4%, GAERS = 43.3 ± 7.2%, P = .044 t test; 8 Hz: NEC = 22.7 ± 5.1%, GAERS = 40.3 ± 6.4%, P = .047 t test; 9 Hz: NEC = 30.1 ± 5.9%, GAERS = 56.2 ± 3.7%, P = .005 t test. NEC (n = 20 cells, n = 11 animals), GAERS (n = 15 cells, n = 10 animals). Scale bars represent 20 msec and 10 mV. *P < .05

Figure 2

P15‐P20 GAERS TRN neurons display sustained oscillatory burst‐firing at 8‐9 Hz. A, Representative traces showing response of P15‐P20 NEC (A—upper panel) and GAERS (A—middle panel) TRN neurons to a 2‐second sinusoidal current injection stimulus (A—lower panel). Stimulus magnitude was incrementally increased until bursting (>3 action potentials) occurred on 3 initial bursts. Scale bars = 30 mV, 100 msec. B, The sinusoidal stimulus was applied at variable frequencies (5‐10 Hz), with the number of action potentials per burst decreasing with time. Insets show representative voltage traces of the last burst in a series. 8 Hz: NEC = 32.5 ± 2.9%, GAERS = 51.2 ± 2.8%, P = .00001 t test; 9 Hz: NEC = 30.5 ± 2.7%, GAERS = 47.2 ± 2.8%, P = .00006 t test; NEC (n = 32 cells, n = 20 animals), GAERS (n = 32 cells, n = 16 animals). Scale bars = 20 msec and 10 mV. *P < .05

Figure 3

GAERS show a developmental increase in the expression of the Cav3.2(+25) splice variant. A, The relative mRNA expression of the T‐type Ca_v3.1‐Ca_v3.3 and the high‐voltage activated Ca_v1.1‐Ca_v1.4 and Ca_v2.1‐Ca_v2.3 Ca²⁺ channels was determined using qRT‐PCR, comparing isoform expression between P10 and P120 in thalamic tissue from GAERS and NEC animals. Amounts are relative to Actin‐B as a control. B, Using splice variant–specific probes for Ca_v3.2(+25) and Ca_v3.2(−25), the relative number of transcript copies of Ca_v3.2(+25) and Ca_v3.2(−25) splice variants was determined across development in GAERS and NEC animals (n = 3 animals per strain): Ca_v3.2(+25): P10 NEC = 8601.2 ± 475.7, P10 GAERS = 7944.6 ± 443.8; P20 NEC = 8985.0 ± 726.4, P20 GAERS = 7278.1 ± 654.4; P120 NEC = 13177.8 ± 487.5, P120 GAERS = 13692.3 ± 424.0; [P10 NEC vs P120 NEC] P = .003, [P20 NEC vs P120 NEC] P = .005 ANOVA; [P10 GAERS vs P120 GAERS] P = .0006, [P20 GAERS vs P120 GAERS] P = .0003 ANOVA. Ca_v3.2(−25): P10 NEC = 14279.6 ± 1421.0, P10 GAERS = 8910.9 ± 1125.8, P = .04 t test; P20 NEC = 9246.1 ± 1006.7, P20 GAERS = 5524.7 ± 214.0, P = .02 t test; P120 NEC = 10170.6 ± 599.2, P120 GAERS = 7814.7 ± 513.8, P = .04 t test; [P10 NEC vs P20 NEC] P = .035, [P10 NEC vs P120 NEC] P = .045 ANOVA; [P10 GAERS vs P20 GAERS] P = .036, [P20 GAERS vs P120 GAERS] P = .04 ANOVA. C, The ratio of Ca_v3.2(+25) to Ca_v3.2(−25) was determined from copy number analysis for each of the developmental time points from both GAERS and NEC animals. All experiments were performed using thalamus samples from 3 rats for each age group (P10, P20, or P120) and from each of GAERS or NEC animals: P10 NEC = 0.61 ± 0.04, P10 GAERS = 0.91 ± 0.07, P = .02 t test; P20 NEC = 0.98 ±0.04, P20 GAERS = 1.31 ± 0.09, P = .03 t test; P120 NEC = 1.30 ± 0.08, P120 GAERS = 1.76 ± 0.06, P = .01 t test; [P10 NEC vs P20 NEC] P = .009, [P10 NEC vs P120 NEC] P = .0004, [P20 NEC vs P120 NEC] P = .017; [P10 GAERS vs P20 GAERS] P = .02, [P10 GAERS vs P120 GAERS] P = .0005, [P20 GAERS vs P120 GAERS] P = .014 ANOVA. *P < .05

Figure 4

Sustained oscillatory burst‐firing in P15‐P20 GAERS TRN neurons is normalized by pharmacological Ca_V3.2 blockade. Representative traces show an oscillatory burst series in response to a 2‐second sinusoidal current injection at 8 Hz in P15‐P20 NEC (A, B—upper panels) and GAERS (A, B—lower panels) TRN neurons. Traces show burst‐firing before (control; A) and after 10‐minute application of Ni²⁺ (100 μM, B) via the perfusate to block Ca_V3.2. Histograms display mean data for burst threshold (C, E) and number of action potentials on the last burst of a 2‐second stimulation, relative to the first burst (D, F) before (control) and after application of Ni²⁺ (100 μM, C, D; Control: NEC = 29.7 ± 7.9%, GAERS = 50.7 ± 4.5% [n = 8], P = .03 t test, Ni²⁺: NEC = 56.2 ± 5.7%, GAERS = 63.7 ± 7.3%, P = .15 t test, NEC [n = 8 cell, n = 3 animals], GAERS [n = 8 cells, n = 3 animals]) and ascorbate (300 μM, E, F; Control: NEC = 17.6 ± 15.3%, GAERS = 52.3 ± 1.7%, P = .001 t test), Asc: NEC = 47.5 ± 5.7%, GAERS = 58.1 ± 5.5%, P = .22 t test, NEC (n = 8 cells, n = 3 animals), GAERS (n = 6 cells, n = 3 animals) to block Ca_V3.2 in NEC and GAERS TRN neurons. *P < .05

Figure 5

Selective knock‐down of thalamic Ca_v3.2 in vivo. A, Dicer siRNA (DsiRNA) was designed to selectively anneal to the Ca_v3.2 channel and target for degradation. DsiRNA was packaged with DiI into LNPs for ApoE‐mediated delivery to brain cells. B, Selective Ca_v3.2 knock‐down was first confirmed in vitro by applying to cultured cortical neurons for 48 hours and expression levels assessed with qPCR. C, For in vivo analyses, P120‐P150 GAERS rats were stereotaxically injected with luciferase control (Luc) or Ca_v3.2 DsiRNA LNPs bilaterally into the ventroposterior lateral thalamus to deliver DsiRNA to TRN neurons without damaging or lesioning the TRN. D, Following a 7‐ to 8‐day recovery period, animals were euthanized, the brain removed, and LNP invasion of TRN was confirmed by DiI fluorescence ex vivo (scale bars: top panels = 400 μm, bottom panels = 20 μm). E, Ca_v3.1‐Ca_v3.3 T‐type isoform expression in TRN tissue was assayed using qPCR relative to the control gene CASC3. Histograms display mean data for expression of T‐type calcium channel isoforms in TRN tissue dissected from 3 sequential 300‐μm‐thick horizontal brain slices for GAERS injected with Luc (n = 6 animals) and Ca_v3.2 DsiRNA (n = 8 animals); Ca_V3.1 isoform: Luc DsiRNA = 1.00 ± 0.22, Ca_V3.2 DsiRNA = 0.79 ± 0.25, P = .55 t test; Ca_V3.2(+25) splice variant: Luc DsiRNA = 1.00 ± 0.16, Ca_V3.2 DsiRNA = 0.35 ± 0.07, P = .0017 t test; Ca_V3.2(−25) splice variant: Luc DsiRNA = 1.00 ± 0.19, Ca_V3.2 DsiRNA = 0.39 ± 0.06, P = .0067 t test; Ca_V3.3 isoform: Luc DsiRNA = 1.00 ± 0.12, Ca_V3.2 DsiRNA = 0.81 ± 0.08, P = .21 t test. F, Oscillatory burst‐firing was assessed in TRN neurons from acute thalamic brain slices in P19‐P20 NEC and GAERS, 6‐8 d following injection with either Luc (n = 3 animals, n = 6 cells) or Ca_v3.2 (n = 3 animals, n = 7 cells) siRNA. Bar charts show percentage number of action potentials on the last burst relative to the first burst from a 2‐s oscillating wave stimulation at 8 Hz. *P < .05

Figure 6

Thalamic Ca_v3.2 knock‐down reduces seizure duration in GAERS. Adult GAERS rats were stereotaxically injected with Ca_v3.2 or Luc DsiRNA LNPs bilaterally into the VB thalamus, transfecting the TRN and implanted with skull screw electrodes on the somatosensory cortex connected to a custom EEG interface. A, Representative ECoG recordings from GAERS injected with Luc (left panel) or Ca_v3.2 (right panel) DsiRNA LNPs. Lower panels show ECoG data with expanded time resolution. Histograms show mean data with (B) % of total time spent in seizure state (Luc = 20.2 ± 4.0%, Ca_V3.2 = 6.9 ± 1.8%, P = .005), (C) seizure duration (Luc = 7.9 ± 1.1 s, Ca_V3.2 = 3.0 ± 0.6 s, P = .002), (D) number of seizures per minute (Luc = 1.65 ± 0.19, Ca_V3.2 = 1.28 ± 0.24%, P = .27), and (E) SWD spike cycle frequency (Luc = 7.7 ± 0.3 Hz, Ca_V3.2 = 7.7 ± 0.4 Hz, P = .99) for GAERS rats injected with Luc (n = 6 animals) and Ca_v3.2 DsiRNA (n = 8 animals). *P < .05