Heterozygous loss of epilepsy geneKCNQ2alters social, repetitive and exploratory behaviors

Figures

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  Figure 1. An overview of behavioral assays performed with KCNQ2 +/+ and KCNQ2 +/− mice. A, The order of behavioral assays performed on KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice. All behavioral assays mice were performed in the dark phase except for the home cage activity during a light:dark cycle (12 hours each) measured over 4 days. B, Sample genotyping results for the male (M) cohorts of WT and Q2+/− mice showing a 240‐bp PCR product from the wild‐type allele (“E” lanes) and a 427‐bp product from the knockout allele (“T” lanes). C, Western blots of Kv7.2, Kv7.3 and GAPDH in the hippocampal membrane P2 fractions of WT and Q2+/− mice at age 3 and 5 months. The Q2+/− mice displayed reduced protein expression of Kv7.2 but not Kv7.3 in the hippocampi compared with the WT mice

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  Figure 2. The KCNQ2 +/− mice show increased home cage activity during the light phase. A‐D, Home cage activity of male and female KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice was examined using continuous video recording for 4 days. A, Sample traces of home cage activity of KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice at the 4th day of recording. The locomotor activities of male mice and female mice were assessed by measuring the distance which they traveled per minute over a 12 hours dark phase (Light OFF) followed by a 12 hours light phase (Light ON). Quantifications of the average total distance traveled during a 24 hours period (B), a 12 hours dark phase (C) and a 12 hours light phase (D). The total distance traveled by Q2+/− mice during the light phase was significantly greater than that of WT mice for both sexes. E, The rotarod tests revealed no difference in latency to fall for WT and Q2+/− mice. Male mice (WT n = 12; Q2+/− n = 11). Female mice (WT n = 5; Q2+/− n = 6). Data shown represent the Ave ± SEM. Student t test results are shown here (*P < .05). Table S2 shows two‐way ANOVA test results with genotype as one factor and sex as the other

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  Figure 3. The KCNQ2 +/− mice display increased exploratory behavior in the center of the open field. A, Sample traces of the open field activity of male KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice for 5 minutes. The traces are shown in red. The mice are shown in white. B, The total distance traveled throughout the open field arena was similar between WT and Q2+/− regardless of sex. B‐F, Quantifications of the entries into the center zone (C) as well as the duration (D), distance (E) and velocity (F) within the center zone. The male Q2+/− mice displayed more entry into the center zone and spent more time and traveled with higher velocity in the central zone than WT mice (WT n = 12; Q2+/− n = 11). The female Q2+/− mice spent more time but traveled slower in the central zone than WT mice (WT n = 5; Q2+/− n = 6). Data shown represent the Ave ± SEM. Student t test results are shown here (*P < .05). Table S3 shows two‐way ANOVA test results with genotype as one factor and sex as the other

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  Figure 4. The KCNQ2 +/− mice display increased exploratory behavior in the open arms of the elevated plus maze. A, A schematic of an elevated plus maze (EPM) and sample traces of locomotor activity of male KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice for 5 minutes in the EPM. The traces are shown in red. The mice are shown in white. B, The total distance traveled throughout EPM was similar between WT and Q2+/− regardless of sex. C‐F, Quantifications of the number entries into the open arms (C), duration at the open arms (D), latency to enter the open arms (E) and total distance traveled in the open arms (F). The male Q2+/− mice had more entry into the open arms with decreased latency and spent more time and traveled longer distance within the open arms compared with the WT mice (WT n = 12; Q2+/− n = 11). The female Q2+/− mice had more entry into the open arms compared with the WT mice (WT n = 5; Q2+/− n = 6). Data shown represent the Ave ± SEM. Student t test results are shown here (*P < .05). Figure S1 shows the quantifications of the number entries into the closed arms, duration at the closed arms, latency to enter the closed arms and total distance traveled in the closed arms. Table S4…

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  Figure 5. The KCNQ2 +/− mice display increased repetitive grooming and marble burying behaviors. A‐B, The grooming event of KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice were recorded for 10 minutes in their home cages without the bedding. A, Both male and female Q2+/− mice groomed themselves for a longer duration of time during their grooming events compared with their WT littermates. B, WT and Q2+/− mice displayed similar number of grooming events regardless of sex. C‐D, The WT and Q2+/− mice were subjected to the marble bearing test for 30 minutes. C, Representative results of the marble burying activity of male WT and Q2+/− mice. D, The male Q2+/− mice buried a larger percentage (%) of the marbles than the WT mice (WT n = 12; Q2+/− n = 11). The female Q2+/− mice buried similar amounts of marbles as the WT mice (WT n = 5; Q2+/− n = 6). Data shown represent the Ave ± SEM. Student t test results are shown here (**P < .01, ***P < .005). Table S5 shows two‐way ANOVA test results with genotype as one factor and sex as the other

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  Figure 6. The KCNQ2 +/− mice display reduced sociability. The KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice were subjected the three‐chamber social interaction test. A, The design of the three‐chamber social interaction test. The test mouse was first habituated in the empty apparatus for 10 minutes (step‐1) and then in the apparatus containing an empty wire cage each in the left and right chambers for 10 minutes (step‐2). During the “sociability” test, a stranger mouse‐1 (age‐matched C57BL/6J) was placed inside the wire cage of the left chamber, and the test mouse was introduced into the center chamber and allowed to explore for 10 minutes (step‐3). During the “social novelty” test, a second stranger mouse‐2 (another age‐matched C57BL6) was placed inside the wire cage of the right chamber, and the test mouse was introduced into the center chamber and allowed to explore for 10 minutes (step‐4). B‐D, Quantifications of the time spent at each chamber (B) as well as the duration (C) and latency (D) of sniffing behavior during the “sociability” test. E‐G, Quantifications of the time spent at each chamber (E) as well as the duration (F) and latency (G) of sniffing behavior during the “social…

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  Figure 7. The KCNQ2 +/− mice display increased social dominance behavior. A‐C, The KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice were subjected to the tube dominance test. A, The schematic of the tube dominance test. The test mouse (WT or Q2+/−) is placed at one end of the tube and the age matched control mouse (CD‐1) is placed at the other end at the same time. A mouse which forces its opponent to retreat backward and exit the tube is deemed the winner and receives +1 point. The retreating mouse is deemed the loser and receives −1 point. If the mice stay in the middle of the tube or pass each other, they receive 0. B‐C, Quantifications of average score (B) and % of win (C). The male and female Q2 +/− mice received higher scores and % of win than the WT mice against CD‐1 mice. D‐G, Male WT and Q2+/− mice were subjected to urine marking test for 30 minutes. D, Schematic of a urine test during which the test mouse and CD‐1 mouse were separated by a wire mesh partition. Quantifications of the number of urine marks (E), the total area of urine marks (F) and the distance of urine marks from the partition (G). Both male and female Q2+/− mice displayed significantly more urine marks with larger areas…

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  Figure 8. The KCNQ2 +/− mice display increased seizure propensity to chemoconvulsant kainate and enhanced hippocampal excitability. KCNQ2 +/+ (WT) and KCNQ2 +/− (Q2+/−) mice at 6‐month of age were intraperitoneally injected with kainate (15 mg/kg) and vehicle control (saline). A, Behavioral seizures were scored based on modified Racine scale and Pinal and Rovner scale for the first 2 hours post injection. B, Quantification of cumulative behavioral seizure scores. C, The average body weights of the WT and Q2+/− mice at 6‐month of age. Both male and female Q2+/− mice displayed significantly increased seizure scores during the first 2 hours post injection compared with the WT mice. D, Left, representative voltage traces to various current injections (1 s,0 to +300pA) in CA1 pyramidal neurons from KCNQ2 +/+ (control) or KCNQ2 +/− (Kcnq2 het) mice. Right, summary graph showing the effect of deleting one copy of KCNQ2 on AP number across a range of current injections (two‐way repeated measures ANOVA; n = 9 neurons from two control mice; n = 9 neurons from four Kcnq2 het mice). Data shown represent the Ave ± SEM. Student t test results for A‐C are shown here (***P < .005). Table S9 shows two‐way…

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  Figure 9. A summary of behavioral assays performed with KCNQ2 +/+ and KCNQ2 +/− mice. Compared with the wild type KCNQ2 +/+ (WT) littermates, KCNQ2 +/− mice (Q2+/−) display abnormalities in multiple behaviors including circadian‐dependent activity, exploratory behavior, social behavior, repetitive and compulsive‐like behavior, in addition to enhanced seizure susceptibility