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. 2021 Oct;46(11):2011-2020.
doi: 10.1038/s41386-021-00985-9. Epub 2021 Mar 3.

Autistic-like behavior, spontaneous seizures, and increased neuronal excitability in a Scn8a mouse model

Jennifer C Wong  1 , Steven F Grieco  2 , Karoni Dutt  3 , Lujia Chen  2 , Jacquelyn T Thelin  4 , George Andrew S Inglis  4 , Shangrila Parvin  5 , Sandra M Garraway  5 , Xiangmin Xu  2 , Alan L Goldin  3 , Andrew Escayg  4
Affiliations

Autistic-like behavior, spontaneous seizures, and increased neuronal excitability in a Scn8a mouse model

Jennifer C Wong et al. Neuropsychopharmacology. 2021 Oct.

Abstract

Patients with SCN8A epileptic encephalopathy exhibit a range of clinical features, including multiple seizure types, movement disorders, and behavioral abnormalities, such as developmental delay, mild-to-severe intellectual disability, and autism. Recently, the de novo heterozygous SCN8A R1620L mutation was identified in an individual with autism, intellectual disability, and behavioral seizures without accompanying electrographic seizure activity. To date, the effects of SCN8A mutations that are primarily associated with behavioral abnormalities have not been studied in a mouse model. To better understand the phenotypic and functional consequences of the R1620L mutation, we used CRISPR/Cas9 technology to generate mice expressing the corresponding SCN8A amino acid substitution. Homozygous mutants exhibit tremors and a maximum lifespan of 22 days, while heterozygous mutants (RL/+) exhibit autistic-like behaviors, such as hyperactivity and learning and social deficits, increased seizure susceptibility, and spontaneous seizures. Current clamp analyses revealed a reduced threshold for firing action potentials in heterozygous CA3 pyramidal neurons and reduced firing frequency, suggesting that the R1620L mutation has both gain- and loss-of-function effects. In vivo calcium imaging using miniscopes in freely moving RL/+ mutants showed hyperexcitability of cortical excitatory neurons that is likely to increase seizure susceptibility. Finally, we found that oxcarbazepine and Huperzine A, a sodium channel blocker and reversible acetylcholinesterase inhibitor, respectively, were capable of conferring robust protection against induced seizures in RL/+ mutants. This mouse line will provide the opportunity to better understand the range of clinical phenotypes associated with SCN8A mutations and to develop new therapeutic approaches.

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Figures

Fig. 1
Fig. 1. Generation and characterization of the R1620L line.
A The R1620L mutation is located in the S4 segment of the DIV transmembrane domain. B Gel electrophoresis image of the digested PCR product (left), confirmed by Sanger sequencing (right). Taq 1 digestion of the PCR product yielded two fragments (610 and 176 bp) from the mutant allele, while the WT allele was identified by the uncut 786 bp PCR product (Fig. 1B). C Homozygous mutants die by postnatal day 22 (P22). Approximately 20% of heterozygous mutants die by P60. D Nav1.6 protein levels were not significantly different between WT littermates, RL/+ mutants, and RL/RL mutants; Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data are presented as normalized to WT levels with the average WT density = 1. N = 5–7/genotype. Data are presented as mean ± SEM.
Fig. 2
Fig. 2. RL/+ mutants exhibit abnormal behaviors.
A, B RL/+ mutants traveled significantly farther and faster than WT littermates in the open field paradigm; unpaired student’s t test. C RL/+ mutants and WT littermates spent similar lengths of time in the center of the open field; unpaired student’s t test. D RL/+ mutants and WT littermates spent similar amounts of time in the lit side of the light/dark box; unpaired student’s t test. E Motor learning is comparable between RL/+ mutants and WT littermates; two-way rANOVA. F RL/+ mutants exhibit impaired learning and memory in the novel object recognition paradigm; unpaired student’s t test. G RL/+ mutants exhibit significantly less freezing behavior during the training (Day 1), context (Day 2), and cue (Day 3) sessions of the fear conditioning paradigm; two-way rANOVA followed by Sidak’s multiple comparisons test and Mann–Whitney test. A–G N = 9–10/genotype. H RL/+ mutants and WT littermates spent more time exploring the stranger mouse than the empty cage. I WT littermates spent more time exploring the stranger vs. familiar mouse while RL/+ mutants spent similar lengths of time exploring the stranger and familiar mouse. H, I, N = 9–10/genotype; two-way ANOVA followed by Sidak’s multiple comparisons test. J RL/+ mutants exhibit a significantly longer latency to interact and K interact significantly less compared to WT littermates in the reciprocal interaction paradigm. J, K, N = 4–5 pairs/genotype; Mann–Whitney test. *P ≤ 0.05, **P ≤ 0.01, **P ≤ 0.001, ****P ≤ 0.0001. Data are presented as mean ± SEM.
Fig. 3
Fig. 3. RL/+ mutants exhibit greater susceptibility to induced seizures.
Male (A) and female (B) RL/+ mutants are significantly more susceptible to 6 Hz-induced seizures compared to WT littermates (16 mA). A, B N = 8–11/genotype/sex; Mann–Whitney test. C Greater separation between female RL/+ mutants and WT littermates was observed at 14 mA. D Female RL/+ mutants take a significantly longer time to recover following a 6 Hz-induced seizure compared to WT littermates, C, D N = 18/genotype; Mann–Whitney test. E Male RL/+ mutants exhibit a significantly shorter latency to the first myoclonic jerk (MJ) and F generalized-tonic–clonic seizure (GTCS) compared to WT littermates. E, F N = 8–10/genotype; Mann–Whitney test. G Female RL/+ mutants exhibit a significantly shorter latency to the first MJ and H GTCS compared to WT littermates. G, H N = 10/genotype; unpaired student’s t test. I All P13-P14 homozygous RL/RL mutants (8/8) exhibited a GTCS, compared to 14/17 (82%) of RL/+ mutants and 0/6 WT littermates. GTCS generation also occurred at lower average temperatures in RL/RL compared to RL/+ mutants. N = 6–17/genotype. *compared to WT, #compared to RL/+ ; log-rank (Mantel–Cox) test. J In an older cohort of mice (P21–P23), all heterozygous RL/+ mutants exhibited a MJ, and 5/7 (71%) of these mutants had a GTCS. WT littermates were taken to the maximum temperature (42.5 °C) but did not exhibit either a MJ or GTCS. N = 7–8/genotype. *compared to WT; log-rank (Mantel–Cox) test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are presented as mean ± SEM.
Fig. 4
Fig. 4. RL/+ mutants exhibit significantly increased neural excitability and seizure susceptibility.
A Representative action potential firing pattern in WT (left) and RL/+ (right) neurons observed during injection of 10 and 70 pA. B The number of action potentials fired during a 2-s current injection is plotted against the corresponding level of current. RL/+ mutants had significantly less firing compared to WT; Friedman’s test. C Cartoon depicting viral injection (AAV-CAMK2a-GCaMP6f) into visual cortex and placement of GRIN lens at the injection site to allow for miniscope recordings. D Experimental timeline. Recordings were first conducted at baseline (data shown in E–H). One week later, mice were subjected to 5 min of baseline recordings followed by PTZ administration. Recordings continued for 15 min after PTZ (data shown in I–L). E Representative raster plot of neural calcium response of three WT littermates (432 total number of neurons), and F 3 RL/+ mutants (420 total number of neurons). G Distribution of single cell firing rates (WT: mean ± SD = 0.28 ± 0.11 Hz, RL/+ : mean ± SD = 0.25 ± 0.08 Hz). Firing rate is calculated by (total number of calcium peaks)/(total time); Mann–Whitney test. H Distribution of single cell sum peak amplitudes (WT: mean ± SD = 6.02 ± 3.26 dF/F, RL/+ : mean ± SD = 11.82 ± 5.35 dF/F); Mann–Whitney test. I, J Raster plots of neural calcium response from two WT (289 total number of neurons) and two RL/+ mice (289 total number of neurons) before and after PTZ administration (vertical red dotted line). J RL/+ mutants exhibited large synchronous neural responses during seizures (white arrow heads). K Distribution of single cell firing rates during baseline or PTZ sessions for RL/+ mutants. RL/+ baseline: mean ± SD = 0.16 ± 0.12 Hz, RL/+ PTZ: mean ± SD = 0.21 ± 0.0811 Hz. WT littermates did not show significantly increased firing rates after PTZ treatment; Mann-Whitney test. L Distribution of single cell sum peak amplitudes in baseline or PTZ sessions for RL/+ mice; RL/+ baseline: mean ± SD = 7.89 ± 5.10 Hz, RL/+ PTZ: mean ± SD = 11.15 ± 5.98 Hz. WT littermates did not exhibit significantly increased amplitudes after PTZ administration; Mann–Whitney test. ***P ≤ 0.001, ****P ≤ 0.0001. Data are presented as mean ± SD.
Fig. 5
Fig. 5. Oxcarbazepine and Huperzine A increase resistance to 6 Hz- and PTZ-induced seizures in RL/+ mutants.
A OXC significantly increases resistance to 6 Hz-induced seizures in the RL/+ mutants. N = 9–13/group; Kruskal–Wallis test followed by Dunn’s multiple comparisons test. B OXC (50 mg/kg) significantly increases the latency to PTZ-induced GTCS in RL/+ mutants and WT littermates; however, the number of RL/+ mutants exhibiting a GTCS was comparable to vehicle-treated mutants. N = 8–9/group; log-rank (Mantel–Cox) test. C Hup A (1 mg/kg) confers robust protection against 6 Hz-induced seizures in RL/+ mutants. N = 9–10/group; Friedman’s test. D Hup A (1 mg/kg) significantly increased the latency to PTZ-induced GTCS in RL/+ mutants and WT littermates and reduced the number of mutants and WT littermates that exhibited a GTCS. N = 7–8/group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are presented as mean ± SEM.
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