Article Figures & Data
Figures
- Figure 1.
Generation and validation of a knock-out mice with a complete deletion of Shank3. A, Schematic design for generation of a Shank3Δ4-22 complete knock-out mouse using a Cre-loxP strategy. Bruce4 C57BL/6 embryonic stem cells from a previously generated mouse with two LoxP site located upstream exon 4 and downstream exon 9 (top, red triangles) were retargeted to insert an additional LoxP site 155 pb downstream of exon 22 (green triangle). Floxed mice were crossed with CMV-Cre mice to generate ubiquitous deletion of exons 4–22 (bottom). ANK, ankyrin repeats; SH3, Src homology 3 domain; PDZ, PSD/Dlg1/zo-1 domain; Pro, proline-rich domain; SAM, sterile α-motif domain. The positions of the PCR primers (P1, P2, P3) for genotyping are indicated. B, Expression of Shank3 in PSD fractions. PSD fractions from wild-type, heterozygous, and homozygous mice were subjected to immunoblotting with either the N367/62 anti-Shank3 antibody directed against an epitope in the SH3 domain or the H160 C-terminal antibody. Immunoblots show that all Shank3 protein bands are absent in KO brains. The migration of molecular weight markers is shown on the left (in kilodaltons) and an immunoblot for βIII-tubulin as a loading control is shown below. Original full scans of immunoblots are displayed in Extended Data Figure 1-1. C, RT-PCR analysis for specific Shank3 transcripts in Shank3Δ4-22 mice. Brain-derived mRNAs from wild-type and homozygous mice were subjected to RT-PCR targeting different isoforms. All transcripts were absent in Shank3Δ4-22 homozygous mice. D, Distribution of genotype. A deficit in the number of Shank3Δ4-22 knockout mice was observed at the time of weaning. E, Survival curve of Shank3Δ4-22 wild-type, heterozygous and homozygous mice between 2 and 22 months. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. *: p < 0.05, **: p < 0.1.
- Figure 2.
Delayed developmental milestones of in Shank3Δ4-22-deficient mice. Analysis of markers of developmental milestones revealed genotype differences in Shank3Δ4-22 wild-type, heterozygous, and homozygous pups between postnatal days 1 and 21 on measures of (A) body weight, (B) auditory startle, (C) air righting, (D) wire suspension, (E) grasping reflex, and (F) negative geotaxis. Additional milestones (jar opening, tooth eruption, fur development, eye opening, rooting reflex, cliff aversion, ear twitch, surface righting, open field crossing, and ultrasonic vocalizations) are displayed in Extended Data Figure 2-1. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *: WT versus KO; o: WT versus Het, #: Het versus KO. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 3.
Impaired motor performances in in Shank3Δ4-22-deficient mice. A, Average stance, stride, and sway. Gait analysis showed an increase stride length in Shank3Δ4-22 homozygous mice. B, Distance traveled during a 60-min session in an open field. Spontaneous locomotor activity in the open field was reduced in Shank3Δ4-22 homozygous mice relative to other genotypes. C, Latency to fall over six trials (three trials per day for two consecutive days) in the accelerating rotarod task. Motor learning on the accelerating rotarod was deficient in Shank3Δ4-22 homozygous mice compared to wild-type animals as they failed to improve over time. Heterozygous mice had an intermediate phenotype. D, Percentage of falls and distance crossed during the beam walking test. While not different on the large (L, 1 inch) and medium (M, ½ inch) beams, Shank3Δ4-22 homozygous mice were strongly impaired in the small (S, ¼ inch) beam walking test, as shown by a significant increase of the number of falls and a decrease of the distance crossed. E, Strength and endurance measured in the inverted screen and hanging tests. Endurance strength was significantly impaired in Shank3Δ4-22 homozygous mice as they exhibited significantly shorter latency to fall in both the inverted screen and hanging tests. Additional results of motor tests (hindlimb placing and grip strength) are available in Extended Data Figure 3-1. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *: WT versus KO; o: WT versus Het, #: Het versus KO. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 4.
Altered sensory profile in Shank3Δ4-22-deficient mice. A, Somatosensation evaluated with corneal reflex, toe pinch retraction, pinna reflex, and tail flick. Normal tactile and pain responses were observed in Shank3Δ4-22-deficient mice. B, Auditory functions measured with the Preyer reflex and startle response to increasing sound intensities. No genotype difference was observed for Preyer reflex, however startle response was decreased in both heterozygous and homozygous Shank3Δ4-22 mice compared to their wild-type littermate with genotype differences being more marked for the higher startle intensities. Pre-pulse inhibition results are displayed in Extended Data Figure 4-1A. C, Gross visual function assessed by the visual placing test. Normal visual placing was observed for all genotypes. D, Olfactory abilities evaluated by the time to find hidden food in buried food test and the cumulative time sniffing the applicator without direct interactions during olfactory habituation and dishabituation to nonsocial and social odors. Strong impairments were observed in the buried food test for Shank3Δ4-22 homozygous mice as shown by a significant increase in the latency to retrieve the buried food, compared to their heterozygous and wild-type littermates. Individual performances are available in Extended Data Figure 4-1B. Similarly, a significant lack of interest for nonsocial scents (water, banana, and lemon) was observed in Shank3Δ4-22 homozygous mice but not in heterozygotes and wild-type during olfactory habituation/dishabituation, while they still displayed normal habituation/dishabituation for social scents (unfamiliar male and female bedding). The olfactory habituation and dishabituation to nonsocial and social odors was measured as cumulative time spent sniffing a sequence of identical and novel odors delivered on cotton swabs inserted into a clean cage. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 5.
Social behavior of Shank3Δ4-22-deficient mice. A, Male social interaction in response to the presentation of an unfamiliar conspecific female in estrus and scored by the cumulative sniffing time and latency from the male toward different body regions of the female. No genotype differences were evident in the dyadic male-female social interaction for the overall sniffing time from the male toward the female, however a trend toward a decrease in anogenital sniffing as well as a significant increase of the latency to initiate the first anogenital sniffing event was observed in Shank3Δ4-22 homozygous mice. B, Preference for social stimulus in the three-chambered social interaction test measured by cumulative time interacting with either a mouse or an inanimate object. All three genotypes demonstrated a significant preference for an unfamiliar mouse over a nonsocial object. C, Social transmission of food preference measured by the time spent by the test mouse sniffing the demonstrator mouse and the time spent interacting with both cued and noncued food. All genotypes had a strong preference for the food flavor presented by the demonstrator mouse. USVs and time spent sniffing the demonstrator during the demonstrator interaction phase are displayed in Extended Data Figure 5-1. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 6.
Object avoidance behavior in Shank3Δ4-22-deficient mice. A, Short-term memory measured by the time of interaction with familiar and new object in the novel object recognition test. The test consisted of a training with two identical objects followed 1 h later by a testing session where one of the object was replaced by a novel object. During the testing session, both wild-type and Shank3Δ4-22 heterozygous mice had a strong preference for the novel object over the familiar object, while Shank3Δ4-22 homozygous mice failed to display a preference. However, this failure was due to an avoidance of both objects as shown by the strong decrease in object interaction and the increase in latency to explore any of the object for the first time in Shank3Δ4-22 homozygous animals, rather than to a real lack of object preference. Representative heatmaps for the three genotypes are available in Extended Data Figure 6-1A. B, Repetitive behavior and object avoidance measured in the marble burying test by the number of marble buried during a 30-min session. Shank3Δ4-22 homozygous mice displayed a strongly impaired burying behavior, leaving most of the marbles undisturbed. Representative pictures and individual data are displayed in Extended Data Figure 6-1B. C, Time spend exploring objects in the repetitive novel object contact task. Shank3Δ4-22 homozygous mice spent significantly less time interacting with the objects than their wild-type and heterozygous littermates. D, Nest building scores. Shank3Δ4-22 homozygous mice are building less elaborate nests and use less nesting material than their wild-type and heterozygous littermates. Representative pictures of the nests and individual data are displayed in Extended Data Figure 6-1C. E, Time interacting with the scent applicator (touching, biting, climbing) during the olfactory habituation/dishabituation test. Shank3Δ4-22 homozygous mice are avoiding interaction with the scent applicator for all nonsocial scents and for a social male scent but have interaction level similar to wild-type and heterozygous animals when presented with a female scent. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 7.
Hyper-reactivity and escape behavior in Shank3Δ4-22-deficient mice. A, Hyper-reactivity measured by animal response in touch escape, positional passivity, and catalepsy. Shank3Δ4-22 homozygous mice have hyper-reactive responses as shown by a higher score in the touch escape indicating an escape response to lighter strokes, a lower score in positional passivity indicating that they struggle more when restrained, and a lower latency to get off a rdownod in the catalepsy test. B, Impulsivity in the negative geotaxis and beam walking tests. The latency to start turning in the negative geotaxis test and to start crossing in the beam walking test are significantly lower in Shank3Δ4-22 homozygous mice compared to their wild-type and heterozygous littermates and often associated with higher failure rates (data not shown) thus demonstrating impulsive behavior. C, Escape behavior measured in different tests with increased inanimate object exposure. No escape attempts were observed for any genotype during the habituation phase of the buried food test (empty home cage with clean bedding). Object exposure induced a significant escape behavior in Shank3Δ4-22 homozygous mice with a number of attempts increasing with the number of objects in the cage (same home cage, four objects in the repetitive novel object contact task, 20 objects in the marble burying test). Very little escape attempts were observed in wild-type mice, while an intermediate phenotype was observed in heterozygous mice. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *: WT vs KO; o: WT vs Het. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 8.
Repetitive behavior, stereotypies, and cognitive flexibility in Shank3Δ4-22-deficient mice. A, Repetitive behaviors in the open field test. Shank3Δ4-22 homozygous mice engaged in significantly more self-grooming and rotations relative to the other genotypes. A trend toward an increase amount of head stereotypies was also observed. B, Object preference and pattern of exploration in the repetitive novel object contact task. For each mouse, the time spent interacting with each object was measured and the objects were then ranked from the most (1) to less (4) preferred (left panel). No genotype differences were observed for the proportions of visits to each object. The pattern of object exploration was analyzed by recording specific sequential pattern of visits to three or four specific toys to identify the total number of three-object or four-object sequence investigations, the number of unique sequences, and the percentage of choices of the top, top two, or top three preferred sequences. All groups had identical percentage of their preferred three-object or four-object sequences choices over the total number of sequence choices. C, Cognitive flexibility measured by reversal learning in the Barnes maze. During initial learning (d1 to d4, each point represents the mean of traveled distance for four independent trials), improvement shown by reduction of the travel distance was faster in Shank3Δ4-22 wild-type and heterozygous mice than in homozygous animals; however, by day 4, the three groups were not different anymore and all of them had a strong preference for the escape hole quadrant during the initial probe test. During the reversal training (r1 to r4, each point represents the mean of travel distance for four independent trials), Shank3Δ4-22 homozygous mice initially traveled for longer distances but were still able to learn the new position and performed as well as their littermates on reversal days 2, 3, and 4. However, the reversal probe test at the end of the reversal training showed that while wild-type and heterozygous animals had a significant preference for the new target quadrant, the homozygous mice had a similar preference for the quadrants containing the initial and the reversal escape holes. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *: WT versus KO; #: Het versus KO. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 9.
Learning and memory in Shank3Δ4-22-deficient mice. A, Working memory in Y-maze measured by spontaneous alternation behavior. All genotypes showed comparable number of arm choices, percentage of correct choices (three-way alternation), type 1 error (three consecutive choices where the first and third choices are identical), or type 2 error (three consecutive choices where the second and third choices are identical). B, Contextual and cued fear conditioning in Shank3 mice. A higher percentage of freezing was observed in Shank3Δ4-22 homozygous mice compared to wild-type and heterozygous animals on day 1. While the difference was already present before the sound-shocks associations, it was strongly increased posttraining. No genotype differences were detected in freezing scores in the posttraining session on day 1. Opposite results were observed for contextual conditioning (day 2) and cued conditioning (day 3): Shank3Δ4-22 homozygous mice showed an impairment of contextual learning compared to their wild-type and heterozygous littermates but an enhancement of freezing postcues during the cued testing. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *: WT versus KO; #: Het versus KO. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 10.
Anxiety-like behavior in Shank3Δ4-22-deficient mice. A, Thigmotaxic behavior in open field. No genotype differences were found for the time spent in the center of the open field, the time spent close to the chamber walls (borders), or their ratio. B, Vertical activity in open field. The cumulated time spend in free standing rears and rears against the walls of the open field were both counted. When compared to wild-type and heterozygotes littermates, Shank3Δ4-22 homozygous mice displayed decreased rearing activity due to a decrease of wall rears rather than free standing rears. C, Shank3Δ4-22 homozygous mice spent a lower amount of time in the open area when compared to wild-type and heterozygous mice. Similarly, the number of head dipping from the open arcs to the outside of the maze was reduced in Shank3Δ4-22 homozygous mice. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. *p < 0.05, **p < 0.1, ***p < 0.001.
- Figure 11.
Main features and comorbidities associated with Phelan–McDermid displayed by different mouse models with Shank3 deficits. Green indicates an absence of genotype difference. Blue indicates a decrease of the associated behavior in Shank3-deficient animals. Red indicates an increase of the associated behavior in Shank3-deficient animals. Gray indicates the behavior has not been studied in the corresponding article. Age column: d = days, w = weeks, m = months, * indicates that only the age at the beginning of the testing was provided.
Tables
Strategy Targetedexons Domains Expressed isoforms Original publication Other publications Synonyms Provider Repository Catalog a a(e10-12s) b b(e10-12s) a/b(e11-12s) c d e e-1 f 1 Deletion Ubiquitous CMV-Cre/loxP-mediated excision Exons 4-9 Ankyrin − − − − − + + + + + Bozdagi et al. (2010) Yang et al. (2012);
Bozdagi et al. (2013);
Drapeau et al. (2014)Shank3Δ ex4-9; B6(Cg)-Shank3tm1.2Bux/J Joseph D. Buxbaum JAX #017890 2 Deletion Homologous recombination (replacement of exon 4-9 by NEO cassette) Exons 4-9 Ankyrin − − − − − + + + + + Wang et al. (2011) Bariselli et al. (2017) Shank3e4-9; B6.129S7-Shank3tm1Yhj/J Yong-Hui Jiang JAX #017442 3 Deletion Ubiquitous MV-Cre/loxP-mediated excision Exons 4-9 Ankyrin − − − − − + + + + + Jaramillo et al. (2016) Craig M. Powell NA NA 4 Deletion Homologous recombination (replacement of exon 4-7 by NEO cassette) Exons 4-7 Last three ankyrin repeats − − − − − + + + + + Peça et al. (2011) Shank3A Guoping Feng NA NA 5 Deletion Ubiquitous MV-Cre/loxP-mediated excision Exon 9 Last ankyrin repeat − − − − − + + + + + Lee et al. (2015) Shank3 (Δ9) Eunjoon Kim NA NA 6 Deletion Homologous recombination (introduction of stop codon in exon 11) Exon 11 SH3 − + − + + − + + + + Schmeisser et al. (2012) Vicidomini et al. (2017);
Reim et al. (2017)Shank3αβ, Shank3Δ11 Tobias M. Boeckers NA NA 7 stop codon Insertion of Neo-Stop cassette in intron 12 Exon 13 PDZ − − − − + + − + + + Jaramillo et al. (2017) Shank3E13 Craig M. Powell NA NA 8 Deletion Homologous recombination (replacement of exon 13-16 by NEO cassette) Exons 13-16 PDZ − − − − + − − + + + Peça et al. (2011) Luo et al. (2017);
Copping et al. (2017)Shank3B; B6.129-Shank3tm2Gfng/J Guoping Feng JAX #017688 9 inducible Deletion Homologous recombination (inversion of exons 13-16 and flanking with FLEx cassette) + crossing with CAGGS-CreER mice for tamoxifen rescue Exons 13-16 PDZ −(+) −(+) −(+) −(+) + −(+) −(+) + + + Mei et al. (2016) Shank3fx/fx and Shank3fx/fx:CAGGS-CreER; STOCK Shank3tm5.1Gfng/J; B6.129(Cg)-Shank3tm5.1Gfng/J Guoping Feng JAX #028800 10 Deletion Ubiquitous CMV-Cre/loxP mediated excision Exon 21 PRO − − + + + − − − + − Bangash et al. (2011) (retracted) Cope et al. (2016) Shank3ΔC (Shank3Δ ex21); B6.129S6(Cg)-Shank3tm1.1Pfw/J; B6.Cg-Shank3tm1.1Pfw/J; STOCK Shank3tm1.1Pfw/J Paul Worley JAX #018398 11 Deletion Ubiquitous CMV-Cre/loxP-mediated excision Exon 21 PRO − − + + + − − − + − Kouser et al. (2013) Kloth et al. (2015);
Duffney et al. (2015);
Bidinosti et al. (2016);
Li et al. (2017)Shank3ΔC/ΔC Craig M. Powell NA NA 12 inducible point insertion Insertion of a floxed mutated exon 21 followed by a transcriptional stop (Neo-stop) cassette + crossing with B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J for tamoxifen rescue Exon 21 PRO − − + + + − − − + − Speed et al. (2015) Shank3G/G and Reversible-Shank3GCre+ Craig M. Powell NA NA 13 point insertion Homologous recombination (G insertion at position 3680 causing a frameshift and premature stop codon) Exon 21 PRO −(+) −(+) + + + −(+) −(+) −(+) + −(+) Zhou et al. (2016) Shank3*G3680 knock-in; STOCK Shank3tm3.1Gfng/J Guoping Feng JAX #028778 14 point mutation Homologous recombination (R1117X nonsense mutation) Exon 21 PRO − − + + + − − − + − Zhou et al. (2016) Shank3*R1117X knock-in; STOCK Shank3tm4.1Gfng/J Guoping Feng JAX #028779 15 Deletion Ubiquitous CMV-Cre/loxP-mediated excision Exons 4-22 ANK, SH3, PDZ, PRO, SAM − − − − − − − − − − Wang et al. (2016) Han et al. (2016) Shank3Δe4–22 Yong-Hui Jiang NA NA 16 over-expression EGFP–Shank3 BAC transgenic mice Full gene ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Han et al. (2013) Tg(Shank3-EGFP)1Hzo; B6.FVB-Tg(Shank3-EGFP)1Hzo/J Huda Y Zoghbi JAX #024033 1-5: targeted deletions in the ankyrin repeat domain. Δ4-7: deletion of exons 4 to 7; Δ4-9: deletion of exons 4 to 9; Δ9: deletion of exon 9. 6: targeted deletion in the SH3 (Src Homology 3) domain. Δ11: deletion of exon 11. 7-9: targeted deletions in the PDZ (PSD95/Discs large/zona-occludens-1) domain. Δ13: deletion of exon 13; Δ13-16: deletion of exon 13 to 16). 10-14: targeted deletions of point mutations in the proline-rich domain. Δ21: deletion of exon 21. 15: deletion of all functional domains. Δ4-22: deletions of exons 4 to 22. 16: overexpression of the full Shank3 gene.
Genotype distribution at weaning WT Het KO %WT %Het %KO χ2 (df2) Asymp p value All animals, observed N 365 686 278 27.46 51.62 20.92 12.78 0.0017 All animals, expected N 332.25 664.5 332.25 25.00 50.00 25.00 All animals, residual N 32.75 21.5 -54.25 2.46 1.62 -4.08 Males, observed N 185 357 147 26.85 51.81 21.34 5.10 0.0781 Males, expected N 172.25 344.5 172.25 25.00 50.00 25.00 Males, residual N 12.75 12.5 -25.25 1.85 1.81 -3.66 Females, observed N 180 329 131 28.13 51.41 20.47 8.01 0.0182 Females, expected N 160 320 160 25.00 50.00 25.00 Females, residual N 20 9 -29 3.13 1.41 -4.53 Cohort 1 (10 litters) - developmental milestones WT Het KO Age at testing All animals 14 30 10 P0–P21 Males 7 16 5 P0–P21 Females 7 14 5 P0–P21 Cohort 2 (10 litters) - ultrasonic vocalizations WT Het KO Age at testing All animals 16 32 9 P6 Males 4 15 6 P6 Females 12 17 3 P6 Cohorts 3 and 4 - adult behavior Cohort 3 Cohort 4 WT Het KO Age at testing WT Het KO Age at testing Handling, cage observation, neurological and motor reflexes 11 10 9 P86–P90 8 9 10 P103–P107 15-month weight 8 8 6 P460 5 7 4 P455 20-month weight 7 7 2 P610 4 5 3 P600 Open field 11 10 9 P93–P94 8 9 10 P106–P108 Zero-maze 11 10 9 P95–P96 8 9 10 P109–P110 Y-maze 11 10 9 P99–P101 8 9 10 P114–P122 Beam walking 11 10 9 P102–P103 8 9 10 P124–P125 Grip strength 11 10 9 P104 8 9 10 P125 Gait analysis 11 10 9 P105 8 9 10 P126 Rotarod 11 10 9 P107–P108 8 9 10 P127 Three-chambered social interaction task 11 10 9 P113–P114 8 9 10 P130–P131 Nest building 11 10 9 P120 8 9 10 P137 Novel object 11 10 9 P123–P125 8 9 10 P139–P140 Fear conditioning 11 10 9 P126–P128 8 9 10 P141–P143 Startle response * 11 10 9 P137–P139 3* 4* 4* P155–P157 Pre-pulse inhibition 11 10 9 P137–P139 8 9 10 P155–P157 Tail flick 11 10 9 P144–P145 8 9 10 P158–P159 Olfactory habituation/dishabituation 11 10 9 P149–P157 8 9 10 P162–P165 Buried food 11 10 9 P163–P164 8 9 10 P178 Social transmission of food preference 11 10 9 P206–P215 8 9 10 P185–P192 Marble burying 11 10 8 P227–P228 8 9 10 P197 Four-object repetitivenovelobject contact task 11 10 8 P232 7 9 9 P215 Male-female social interaction 11 10 8 P240–P241 7 9 9 P217–P219 Barnes maze 11 10 7 P247–P274 7 9 8 P222–P250 For adult animals, the age indicated corresponds to the average age of the cohort. For each cohort all mice were born within two weeks of each other. *: missing animals due to technical problems during startle recording.
Weight Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 466.906 0.000 1.000 − − − Day × genotype effect 2.275 0.045 0.754 − − − Day × gender effect 0.363 0.765 0.117 − − − Day × genotype × gender effect 0.569 0.742 0.214 − − − Genotype effect 3.046 0.048 0.560 0.144 0.018 0.147 Gender effect 0.933 0.339 0.157 − − − Genotype × gender 0.686 0.509 0.158 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Weight - P1 Nonnormal 1.47 ± 0.02 1.38 ± 0.02 1.36 ± 0.03 2.244 0.118 0.433 − − − 1.067 0.307 0.173 0.016 0.984 0.052 Weight - P2 Nonnormal 1.51 ± 0.03 1.42 ± 0.03 1.4 ± 0.04 2.010 0.146 0.393 − − − 0.510 0.479 0.108 0.193 0.825 0.078 Weight - P3 Nonnormal 1.62 ± 0.05 1.59 ± 0.04 1.55 ± 0.07 0.451 0.640 0.119 − − − 0.030 0.863 0.053 1.047 0.360 0.221 Weight - P4 Nonnormal 1.95 ± 0.09 1.87 ± 0.06 1.87 ± 0.1 0.610 0.548 0.145 − − − 0.822 0.369 0.144 0.378 0.688 0.107 Weight - P5 Nonnormal 2.34 ± 0.1 2.27 ± 0.07 2.27 ± 0.12 0.305 0.739 0.095 − − − 0.803 0.375 0.142 1.021 0.368 0.217 Weight - P6 Nonnormal 2.77 ± 0.14 2.71 ± 0.08 2.7 ± 0.15 0.320 0.728 0.098 − − − 0.436 0.512 0.099 0.356 0.703 0.104 Weight - P7 Nonnormal 3.29 ± 0.12 3.25 ± 0.09 3.13 ± 0.15 0.682 0.511 0.158 − − − 0.835 0.366 0.145 0.934 0.401 0.201 Weight - P8 Nonnormal 3.8 ± 0.14 3.73 ± 0.1 3.65 ± 0.15 0.493 0.614 0.126 − − − 0.723 0.400 0.132 1.023 0.368 0.217 Weight - P9 Nonnormal 4.26 ± 0.14 4.23 ± 0.1 4 ± 0.17 1.146 0.327 0.239 − − − 3.146 0.083 0.411 0.883 0.421 0.192 Weight - P10 Nonnormal 4.86 ± 0.11 4.72 ± 0.1 4.58 ± 0.16 1.013 0.371 0.215 − − − 0.299 0.587 0.083 0.051 0.951 0.057 Weight - P11 Nonnormal 5.42 ± 0.11 5.21 ± 0.1 5.03 ± 0.18 1.837 0.171 0.363 − − − 0.781 0.382 0.139 0.023 0.978 0.053 Weight - P12 Nonnormal 5.85 ± 0.11 5.7 ± 0.11 5.39 ± 0.13 2.148 0.129 0.417 − − − 0.092 0.764 0.060 0.362 0.698 0.105 Weight - P13 Nonnormal 6.22 ± 0.12 6.01 ± 0.11 5.72 ± 0.2 1.787 0.179 0.354 − − − 0.853 0.361 0.147 0.657 0.524 0.153 Weight - P14 Nonnormal 6.62 ± 0.12 6.42 ± 0.11 5.83 ± 0.17 4.891 0.012 0.777 0.274 0.004 0.016 0.577 0.451 0.115 0.618 0.544 0.147 Weight - P15 Nonnormal 7.01 ± 0.14 6.73 ± 0.12 6.38 ± 0.22 2.504 0.093 0.476 0.175 0.031 0.198 0.595 0.445 0.117 0.238 0.789 0.085 Weight - P16 Nonnormal 7.31 ± 0.14 6.96 ± 0.13 6.69 ± 0.19 2.668 0.081 0.502 0.094 0.030 0.318 0.157 0.694 0.067 0.072 0.931 0.060 Weight - P17 Nonnormal 7.55 ± 0.14 7.2 ± 0.13 6.83 ± 0.22 2.973 0.061 0.549 0.118 0.020 0.192 0.889 0.351 0.152 0.170 0.845 0.075 Weight - P18 Nonnormal 7.76 ± 0.14 7.43 ± 0.14 6.98 ± 0.2 3.160 0.050 0.577 0.152 0.016 0.127 0.790 0.379 0.140 0.187 0.830 0.077 Weight - P19 Nonnormal 7.98 ± 0.13 7.58 ± 0.16 7.1 ± 0.18 3.534 0.038 0.628 0.115 0.011 0.121 1.170 0.285 0.185 0.861 0.430 0.189 Weight - P20 Nonnormal 8.31 ± 0.19 7.69 ± 0.19 7.18 ± 0.2 4.268 0.020 0.716 0.051 0.006 0.146 0.729 0.398 0.133 1.415 0.254 0.287 Weight - P21 Nonnormal 8.67 ± 0.21 8.05 ± 0.27 7.38 ± 0.28 3.366 0.044 0.605 0.127 0.013 0.127 0.263 0.611 0.079 0.839 0.439 0.185 Eye opening Repeated measures, sphericity assumed F p value Power WT vs Het WT vs KO Het vs KO Day effect 192.080 0.000 1.000 − − − Day × genotype effect 1.565 0.190 0.469 − − − Day × gender effect 0.716 0.494 0.169 − − − Day × genotype × gender effect 0.653 0.629 0.544 Genotype effect 1.403 0.257 0.285 − − − Gender effect 1.852 0.181 0.265 − − − Genotype × gender 0.957 0.392 0.205 Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Eye opening score - P9 − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Eye opening score - P10 − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Eye opening score - P11 − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Eye opening score - P12 Nonnormal 0.3 ± 0.2 0.28 ± 0.13 0.1 ± 0.1 0.534 0.590 0.132 − − − 1.917 0.173 0.273 0.496 0.613 0.126 Eye opening score - P13 Nonnormal 1.23 ± 0.34 1.35 ± 0.25 0.6 ± 0.3 1.445 0.247 0.292 − − − 0.707 0.405 0.130 0.032 0.969 0.055 Eye opening score - P14 Nonnormal 2.38 ± 0.18 2.75 ± 0.16 2.1 ± 0.09 4.723 0.014 0.761 0.134 0.167 0.005 2.464 0.124 0.336 2.248 0.118 0.433 Eye opening score - P15 Nonnormal 3 ± 0.25 3.1 ± 0.17 2.7 ± 0.26 0.646 0.529 0.151 − − − 0.043 0.837 0.055 1.262 0.293 0.260 Eye opening score - P16 Nonnormal 4 ± 0 3.85 ± 0.06 3.9 ± 0.1 0.734 0.486 0.166 − − − 3.076 0.087 0.403 1.249 0.297 0.257 Eye opening score - P17 Nonnormal 4 ± 0 3.85 ± 0.06 4 ± 0 1.665 0.201 0.332 − − − 1.155 0.288 0.183 1.971 0.152 0.386 Eye opening score - P18 Nonnormal 4 ± 0 3.89 ± 0.05 4 ± 0 0.957 0.392 0.205 − − − 0.690 0.411 0.128 1.041 0.362 0.220 Eye opening score - P19 Nonnormal 4 ± 0 3.96 ± 0.03 4 ± 0 0.428 0.654 0.115 − − − 0.320 0.575 0.086 0.426 0.656 0.115 Eye opening score - P20 − 4 ± 0 4 ± 0 4 ± 0 − − − − − − − − − − − − Average day of full opening Nonnormal 15.53 ± 0.18 15.57 ± 0.33 15.9 ± 0.17 0.469 0.629 0.122 − − − 1.472 0.232 0.220 0.749 0.479 0.169 Ear opening Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 316.707 0.000 1.000 − − − Day × genotype effect 0.807 0.594 0.361 − − − Day × gender effect 2.150 0.079 0.617 − − − Day × genotype × gender effect 1.056 0.396 0.472 − − − Genotype effect 0.113 0.893 0.066 − − − Gender effect 0.438 0.512 0.099 − − − Genotype × gender 0.676 0.514 0.156 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Ear opening score - P1 Nonnormal 0.23 ± 0.23 0.13 ± 0.09 0 ± 0 0.753 0.477 0.170 − − − 2.371 0.131 0.325 0.669 0.517 0.155 Ear opening score - P2 Nonnormal 2.15 ± 0.15 2.06 ± 0.04 2 ± 0 0.675 0.514 0.156 − − − 2.261 0.140 0.313 0.468 0.629 0.122 Ear opening score - P3 Nonnormal 2.38 ± 0.21 2.31 ± 0.13 2.3 ± 0.21 0.123 0.885 0.068 − − − 1.054 0.310 0.171 0.994 0.378 0.212 Ear opening score - P4 Nonnormal 3.15 ± 0.27 3.27 ± 0.13 3.6 ± 0.22 0.966 0.389 0.207 − − − 2.693 0.108 0.362 1.501 0.234 0.303 Ear opening score - P5 Nonnormal 4.15 ± 0.1 4.2 ± 0.11 4.1 ± 0.1 0.167 0.847 0.074 − − − 0.106 0.746 0.062 0.382 0.685 0.108 Ear opening score - P6 Nonnormal 5.76 ± 0.16 5.93 ± 0.06 6 ± 0 1.052 0.358 0.222 − − − 0.780 0.382 0.139 0.733 0.486 0.166 Ear opening score - P7 − 6 ± 0 6 ± 0 6 ± 0 0.439 0.647 0.117 − − − 0.270 0.606 0.080 0.617 0.544 0.146 Ear opening score - P8 − 6 ± 0 6 ± 0 6 ± 0 − − − − − − − − − − − − Ear opening score - P9 − 6 ± 0 6 ± 0 6 ± 0 − − − − − − − − − − − − Average day of full opening Nonnormal 6.15 ± 0.1 5.93 ± 0.06 6 ± 0 0.622 0.541 0.147 − − − 0.070 0.793 0.058 0.274 0.761 0.091 Tooth eruption Bottom incisor - repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 120.634 0.000 1.000 − − − Day × genotype effect 1.452 0.177 0.648 − − − Day × gender effect 1.873 0.116 0.564 − − − Day × genotype × gender effect 1.671 0.107 0.723 − − − Genotype effect 1.855 0.169 0.366 − − − Gender effect 0.094 0.761 0.060 − − − Genotype × gender 0.637 0.533 0.150 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Bottom incisor score - P7 − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Bottom incisor score - P8 Nonnormal 0.38 ± 0.14 0.06 ± 0.04 0.1 ± 0.1 3.701 0.033 0.650 0.010 0.072 0.746 0.685 0.412 0.128 1.274 0.290 0.262 Bottom incisor score - P9 Nonnormal 0.92 ± 0.07 0.75 ± 0.08 1 ± 0.14 1.247 0.297 0.257 − − − 1.198 0.280 0.188 1.949 0.155 0.382 Bottom incisor score - P10 Nonnormal 1.15 ± 0.1 1.03 ± 0.06 1.1 ± 0.1 0.661 0.521 0.164 − − − 2.184 0.147 0.304 1.371 0.265 0.279 Bottom incisor score - P11 Nonnormal 1.84 ± 0.1 1.65 ± 0.08 1.5 ± 0.16 1.438 0.248 0.291 − − − 0.212 0.647 0.074 0.873 0.425 0.191 Bottom incisor score - P12 Nonnormal 1.92 ± 0.07 1.93 ± 0.04 1.8 ± 0.13 0.795 0.458 0.177 − − − 4.249 0.045 0.523 1.594 0.215 0.319 Bottom incisor score - P13 − 2 ± 0 2 ± 0 2 ± 0 − − − − − − − − − − − − Bottom incisor, day of full eruption Nonnormal 11.07 ± 0.21 11.37 ± 0.15 11.5 ± 0.37 0.720 0.492 0.164 − − − 0.018 0.895 0.052 0.141 0.869 0.070 Top incisor - repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 41.000 0.000 1.000 − − − Day × genotype effect 84.000 0.587 0.355 − − − Day × gender effect 41.000 0.150 0.497 − − − Day × genotype × gender effect 84.000 0.563 0.370 − − − Genotype effect 0.314 0.732 0.097 − − − Gender effect 0.845 0.363 0.147 − − − Genotype × gender 1.028 0.366 0.218 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Top incisor score - P10 − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Top incisor score - P11 Nonnormal 0.23 ± 0.12 0.1 ± 0.05 0 ± 0 1.757 0.184 0.348 − − − 0.021 0.886 0.052 0.903 0.413 0.196 Top incisor score - P12 Nonnormal 0.76 ± 0.12 0.79 ± 0.07 0.8 ± 0.13 0.010 0.990 0.051 − − − 1.285 0.263 0.198 1.558 0.222 0.313 Top incisor score - P13 Nonnormal 1.3 ± 0.17 1.24 ± 0.13 1 ± 0.21 0.590 0.559 0.142 − − − 0.009 0.925 0.051 0.804 0.454 0.179 Top incisor score - P14 Nonnormal 1.76 ± 0.12 1.79 ± 0.07 1.8 ± 0.13 0.010 0.990 0.051 − − − 1.285 0.263 0.198 1.558 0.222 0.313 Top incisor score - P15 Nonnormal 1.92 ± 0.07 1.89 ± 0.05 1.8 ± 0.13 0.487 0.618 0.125 − − − 4.489 0.040 0.545 0.861 0.430 0.189 Top incisor score - P16 − 2 ± 0 2 ± 0 2 ± 0 − − − − − − − − − − − − Top incisor, day of full eruption Nonnormal 13.92 ± 0.26 14.41 ± 0.17 14.2 ± 0.32 1.110 0.339 0.233 − − − 0.816 0.371 0.143 1.006 0.374 0.214 Fur development Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 347.979 0.000 1.000 − − − Day × genotype effect 0.885 0.546 0.458 − − − Day × gender effect 1.948 0.089 0.646 − − − Day × genotype × gender effect 3.234 0.001 0.986 − − − Genotype effect 1.683 0.198 0.335 − − − Gender effect 0.635 0.430 0.122 − − − Genotype × gender 8.265 0.001 0.950 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Fur score - P1 Nonnormal 1 ± 0 0.92 ± 0.07 1 ± 0 0.439 0.647 0.117 − − − 0.270 0.606 0.080 0.617 0.544 0.146 Fur score - P2 Nonnormal 1.76 ± 0.12 1.79 ± 0.07 2 ± 0 1.484 0.238 0.300 − − − 0.000 0.995 0.050 6.724 0.003 0.897 Fur score - P3 Nonnormal 2.53 ± 0.18 2.37 ± 0.1 2.5 ± 0.16 0.898 0.415 0.195 − − − 1.736 0.194 0.252 7.221 0.002 0.918 Fur score - P4 Nonnormal 3.23 ± 0.2 3.13 ± 0.11 3.4 ± 0.16 1.605 0.212 0.321 − − − 5.973 0.019 0.667 9.904 0.000 0.978 Fur score - P5 Nonnormal 3.92 ± 0.13 3.86 ± 0.06 4 ± 0 1.034 0.364 0.219 − − − 0.013 0.909 0.051 4.885 0.012 0.777 Fur score - P6 Nonnormal 3.92 ± 0.13 3.89 ± 0.05 4 ± 0 0.657 0.523 0.153 − − − 0.090 0.766 0.060 4.026 0.025 0.689 Fur score - P7 Nonnormal 4.15 ± 0.19 4.27 ± 0.1 4.5 ± 0.16 1.002 0.375 0.213 − − − 0.353 0.556 0.089 1.378 0.263 0.281 Fur score - P8 Nonnormal 4.69 ± 0.17 4.62 ± 0.09 5 ± 0 2.746 0.075 0.514 − − − 1.116 0.297 0.178 4.075 0.024 0.694 Fur score - P9 Nonnormal 5 ± 0 4.93 ± 0.04 5 ± 0 0.927 0.403 0.200 − − − 0.604 0.441 0.118 1.203 0.310 0.249 Fur score - P10 Nonnormal 5.23 ± 0.12 5.24 ± 0.08 5.3 ± 0.15 0.125 0.882 0.068 − − − 0.007 0.936 0.051 2.343 0.108 0.450 Fur score - P11 Nonnormal 5.53 ± 0.14 5.72 ± 0.08 5.8 ± 0.13 0.906 0.411 0.196 − − − 0.997 0.324 0.164 3.754 0.031 0.656 Fur score - P12 Nonnormal 6 ± 0 5.96 ± 0.03 6 ± 0 0.401 0.672 0.111 − − − 0.274 0.603 0.081 0.479 0.622 0.123 Fur score - P13 − 6 ± 0 6 ± 0 6 ± 0 − − . − − − − − − − − − Fur score - P14 − 6 ± 0 6 ± 0 6 ± 0 − − . − − − − − − − − − Day of full fur Nonnormal 11.3 ± 0.2 10.75 ± 0.37 10.9 ± 0.23 0.460 0.634 0.120 − − − 0.110 0.741 0.062 1.960 0.153 0.384 Auditory startle Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 56.506 0.000 − − − Day × genotype effect 3.280 0.002 − − − Day × gender effect 0.283 0.873 − − − Day × genotype × gender effect 1.321 0.241 − − − Genotype effect 12.867 0.000 0.070 0.000 0.000 Gender effect 0.058 0.811 − − − Genotype × gender 0.358 0.701 − − − Gender effect Gender × genotypeeffect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Percentage of responders - P10 − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Percentage of responders - P11 Nonnormal 15.38 ± 10.41 13.79 ± 6.51 0 ± 0 1.308 0.281 0.268 − − − 0.001 0.971 0.050 3.054 0.057 0.561 Percentage of responders - P12 Nonnormal 53.84 ± 14.39 13.79 ± 6.51 0 ± 0 8.700 0.001 0.959 0.001 0.000 0.178 0.488 0.488 0.105 1.584 0.217 0.318 Percentage of responders - P13 Nonnormal 53.84 ± 14.39 55.17 ± 9.39 10 ± 10 3.045 0.058 0.560 0.969 0.043 0.023 0.238 0.628 0.077 1.082 0.348 0.228 Percentage of responders - P14 Nonnormal 100 ± 0 86.2 ± 6.51 60 ± 16.32 3.161 0.052 0.577 0.265 0.016 0.072 0.000 0.990 0.050 0.009 0.991 0.051 Percentage of responders - P15 Nonnormal 100 ± 0 100 ± 0 70 ± 15.27 8.228 0.001 0.949 0.970 0.001 0.000 1.019 0.318 0.167 0.865 0.428 0.189 Percentage of responders - P16 − 100 ± 0 100 ± 0 100 ± 0 − − − − − − − − − − − − Percentage of responders - average Nonnormal 51.92 ± 1.67 47.41 ± 1.31 36.66 ± 2.83 7.944 0.001 0.995 0.286 0.000 0.002 0.466 0.498 0.056 0.144 0.866 0.104 First day of two consecutive successes Nonnormal 14.07 ± 0.26 14.41 ± 0.16 15.6 ± 0.33 12.867 0.000 0.941 0.070 0.000 0.000 0.058 0.811 0.102 0.358 0.701 0.071 Cliff aversion Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 3.957 0.000 0.995 − − − Day × genotype effect 0.796 0.702 0.580 − − − Day × gender effect 0.613 0.782 0.299 − − − Day × genotype × gender effect 1.266 0.209 0.835 − − − Genotype effect 1.355 0.269 0.276 − − − Gender effect 0.218 0.643 0.074 − − − Genotype × gender 0.116 0.891 0.067 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Time to turn (seconds) - P2 Nonnormal 23.61 ± 2.77 24.44 ± 1.86 21.77 ± 3.56 0.745 0.481 0.168 − − − 0.482 0.491 0.104 0.866 0.428 0.189 Time to turn (seconds) - P3 Nonnormal 15.76 ± 3.37 14.17 ± 2.06 14.7 ± 4.34 0.099 0.906 0.064 − − − 0.056 0.814 0.056 1.513 0.232 0.304 Time to turn (seconds) - P4 Nonnormal 8.61 ± 3 4.93 ± 1.35 7.6 ± 3.73 0.963 0.390 0.206 − − − 0.340 0.563 0.088 1.916 0.160 0.376 Time to turn (seconds) - P5 Nonnormal 6.84 ± 2.86 7.03 ± 1.69 8.4 ± 3.61 0.156 0.856 0.073 − − − 0.898 0.349 0.153 2.242 0.119 0.432 Time to turn (seconds) - P6 Nonnormal 11.15 ± 3.42 8.75 ± 2.07 9.6 ± 3.55 0.126 0.882 0.068 − − − 0.040 0.843 0.054 2.223 0.121 0.429 Time to turn (seconds) - P7 Nonnormal 14.38 ± 3.83 9.75 ± 2.21 10 ± 4.36 1.057 0.356 0.223 − − − 1.259 0.268 0.195 0.368 0.694 0.105 Time to turn (seconds) - P8 Nonnormal 12.61 ± 3.97 4.82 ± 1.34 6.55 ± 3.08 2.580 0.087 0.488 0.144 0.788 0.618 0.436 0.120 0.315 0.731 0.097 Time to turn (seconds) - P9 Nonnormal 10.69 ± 3.19 9.72 ± 2.18 3.8 ± 0.92 1.129 0.333 0.236 − − − 0.011 0.917 0.051 0.285 0.753 0.092 Time to turn (seconds) - P10 Nonnormal 13.46 ± 3.45 7.03 ± 1.62 5.6 ± 2.77 2.447 0.098 0.466 0.048 0.076 0.770 1.109 0.298 0.177 0.316 0.731 0.097 Time to turn (seconds) - P11 Nonnormal 9.3 ± 2.66 11.51 ± 2.38 9 ± 3.55 0.634 0.535 0.149 − − − 0.263 0.611 0.079 1.486 0.238 0.300 Time to turn (seconds) - P12 Nonnormal 8.61 ± 1.52 8.93 ± 1.67 5.3 ± 1.21 0.467 0.630 0.121 − − − 0.001 0.975 0.050 0.651 0.527 0.152 Time to turn (seconds) - P13 Nonnormal 5.46 ± 1.7 6.48 ± 1.42 5.3 ± 2.78 0.125 0.883 0.068 − − − 2.134 0.151 0.298 0.791 0.460 0.176 Time to turn (seconds) - P14 Nonnormal 5.76 ± 2.16 4.1 ± 0.67 4.3 ± 1.67 0.605 0.551 0.144 − − − 1.345 0.253 0.205 0.941 0.398 0.202 Number of falls Nonnormal 1.07 ± 0.53 0.44 ± 0.11 0.6 ± 0.26 1.568 0.220 0.314 − − − 3.688 0.061 0.467 1.125 0.334 0.235 First day of two consecutive successes (10-s cutoff) Nonnormal 4.84 ± 0.29 4.75 ± 0.22 5.2 ± 0.55 0.546 0.583 0.134 − − − 2.726 0.106 0.365 2.174 0.126 0.421 First day of two consecutive successes (30-s cutoff) Nonnormal 4.38 ± 0.33 4.06 ± 0.14 4.5 ± 0.45 1.370 0.265 0.279 − − − 0.037 0.849 0.054 1.044 0.361 0.221 Time to turn (seconds) - mean Nonnormal 11.25 ± 1.25 9.36 ± 0.65 8.5 ± 0.85 1.315 0.279 0.269 − − − 0.259 0.613 0.079 0.119 0.888 0.067 Day effect 5.197 0.000 0.994 − − − Day × genotype effect 0.866 0.581 0.502 − − − Day × gender effect 0.830 0.547 0.325 − − − Day × genotype × gender effect 1.115 0.348 0.637 − − − Genotype effect 2.147 0.129 0.416 − − − Gender effect 0.152 0.698 0.067 − − − Genotype × gender 0.834 0.441 0.184 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Percentage of responders - P7 Nonnormal 46.15 ± 14.39 17.24 ± 7.13 50 ± 16.66 2.610 0.085 0.493 0.076 0.870 0.073 0.347 0.559 0.089 0.888 0.419 0.193 Percentage of responders - P8 Nonnormal 30.76 ± 13.32 6.89 ± 4.78 30 ± 15.27 2.340 0.108 0.449 − − − 0.089 0.766 0.060 0.834 0.441 0.184 Percentage of responders - P9 Nonnormal 46.15 ± 14.39 17.24 ± 7.13 10 ± 10 2.860 0.068 0.532 0.046 0.037 0.551 0.244 0.624 0.077 0.368 0.694 0.105 Percentage of responders - P10 Nonnormal 38.46 ± 14.04 24.13 ± 8.08 30 ± 15.27 0.394 0.677 0.110 − − − 3.054 0.088 0.401 1.829 0.173 0.361 Percentage of responders - P11 Nonnormal 53.84 ± 14.39 62.06 ± 9.16 70 ± 15.27 0.461 0.633 0.121 − − − 0.143 0.707 0.066 2.804 0.071 0.524 Percentage of responders - P12 Nonnormal 46.15 ± 14.39 41.37 ± 9.3 50 ± 16.66 0.214 0.808 0.081 − − − 0.527 0.472 0.109 0.270 0.765 0.090 Percentage of responders - P13 Nonnormal 46.15 ± 14.39 48.27 ± 9.44 60 ± 16.32 0.064 0.938 0.059 − − − 1.507 0.226 0.225 0.890 0.418 0.194 Percentage of responders - P14 Nonnormal 61.53 ± 14.04 72.41 ± 8.44 80 ± 13.33 0.379 0.687 0.107 − − − 0.022 0.882 0.052 0.468 0.629 0.122 Percentage of responders - P15 Nonnormal 100 ± 0 96.55 ± 3.44 90 ± 10 0.497 0.612 0.126 − − − 0.569 0.455 0.114 2.150 0.129 0.417 Percentage of responders - Average Nonnormal 52.13 ± 5.39 42.91 ± 2.62 52.22 ± 3.33 2.147 0.129 0.416 − − − 0.152 0.698 0.067 0.834 0.441 0.184 First day of two consecutive successes Nonnormal 9.23 ± 0.63 10.13 ± 0.36 8.8 ± 0.61 1.851 0.169 0.365 − − − 0.137 0.713 0.065 1.106 0.340 0.232 Rooting reflex Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 8.013 0.000 0.999 − − − Day × genotype effect 1.657 0.107 0.735 − − − Day × gender effect 0.847 0.503 0.276 − − − Day × genotype × gender effect 1.347 0.219 0.625 − − − Genotype effect 1.689 0.196 0.336 − − − Gender effect 4.277 0.045 0.525 − − − Genotype × gender 0.283 0.755 0.092 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Percentage of responders - P2 Nonnormal 23.07 ± 12.16 6.89 ± 4.78 20 ± 13.33 1.259 0.294 0.259 − − − 2.018 0.163 0.285 2.605 0.085 0.492 Percentage of responders - P3 Nonnormal 38.46 ± 14.04 34.48 ± 8.98 20 ± 13.33 0.643 0.531 0.151 − − − 1.878 0.177 0.268 0.771 0.469 0.173 Percentage of responders - P4 Nonnormal 46.15 ± 14.39 58.62 ± 9.3 50 ± 16.66 0.292 0.748 0.093 − − − 0.701 0.407 0.130 2.220 0.121 0.429 Percentage of responders - P5 Nonnormal 61.53 ± 14.04 82.75 ± 7.13 60 ± 16.32 1.489 0.237 0.301 − − − 3.072 0.087 0.403 0.308 0.736 0.096 Percentage of responders - P6 Nonnormal 92.3 ± 7.69 68.96 ± 8.74 70 ± 15.27 1.499 0.234 0.302 − − − 3.120 0.084 0.408 0.395 0.676 0.110 Percentage of responders - P7 Nonnormal 84.61 ± 10.41 68.96 ± 8.74 90 ± 10 1.161 0.323 0.242 − − − 0.959 0.333 0.160 1.595 0.214 0.320 Percentage of responders - P8 Nonnormal 84.61 ± 10.41 58.62 ± 9.3 40 ± 16.32 2.196 0.123 0.425 − − − 0.618 0.436 0.120 0.193 0.826 0.078 Percentage of responders - P9 Nonnormal 76.92 ± 12.16 31.03 ± 8.74 40 ± 16.32 4.400 0.018 0.730 0.005 0.055 0.687 2.183 0.147 0.304 0.616 0.545 0.146 Percentage of responders - P10 Nonnormal 38.46 ± 14.04 13.79 ± 6.51 20 ± 13.33 1.743 0.187 0.346 − − − 0.010 0.919 0.051 1.945 0.155 0.381 Percentage of responders - P11 Nonnormal 0 ± 0 0 ± 0 10 ± 10 2.310 0.111 0.444 − − − 2.619 0.113 0.353 2.258 0.117 0.435 Percentage of responders - P12 − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Day of first observation Nonnormal 4.15 ± 0.45 4.31 ± 0.28 5 ± 0.66 0.843 0.437 0.185 − − − 1.546 0.220 0.229 0.757 0.475 0.170 Day of last observation Nonnormal 9.61 ± 0.56 8.58 ± 0.3 9.1 ± 0.48 1.599 0.214 0.320 − − − 1.428 0.239 0.215 1.200 0.311 0.249 Grasping reflex Repeated measures, sphericity assumed F p value Power WT vs Het WT vs KO Het vs KO Day effect 28.265 0.000 1.000 − − − Day × genotype effect 1.038 0.415 0.591 − − − Day × gender effect 0.534 0.850 0.208 − − − Day × genotype × gender effect 1.356 0.150 0.725 − − − Genotype effect 3.923 0.027 0.677 0.304 0.116 0.008 Gender effect 0.052 0.821 0.056 − − − Genotype × gender 0.320 0.728 0.098 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Grasping score - P5 Nonnormal 1.3 ± 0.23 1.34 ± 0.16 1.6 ± 0.16 0.197 0.822 0.079 − − − 0.877 0.354 0.150 2.268 0.115 0.437 Grasping score - P6 Nonnormal 2.46 ± 0.36 2.93 ± 0.21 2.8 ± 0.38 0.580 0.564 0.140 − − − 0.184 0.670 0.070 0.595 0.556 0.143 Grasping score - P7 Nonnormal 2.92 ± 0.28 3.34 ± 0.17 2.9 ± 0.23 2.260 0.116 0.436 − − − 0.858 0.359 0.148 1.670 0.200 0.333 Grasping score - P8 Nonnormal 3.38 ± 0.28 3.41 ± 0.15 3.2 ± 0.24 0.316 0.731 0.097 − − − 1.283 0.264 0.198 1.183 0.316 0.246 Grasping score - P9 Nonnormal 3.76 ± 0.3 3.79 ± 0.09 3.8 ± 0.41 0.035 0.966 0.055 − − − 0.336 0.565 0.088 1.031 0.365 0.218 Grasping score - P10 Nonnormal 4.38 ± 0.24 4.65 ± 0.19 3.9 ± 0.37 2.102 0.134 0.409 − − − 0.035 0.852 0.054 1.782 0.180 0.353 Grasping score - P11 Nonnormal 5.3 ± 0.23 5.41 ± 0.15 4.9 ± 0.31 1.591 0.215 0.319 − − − 0.016 0.899 0.052 1.314 0.279 0.269 Grasping score - P12 Nonnormal 5.3 ± 0.23 5.37 ± 0.15 5 ± 0.29 0.477 0.624 0.123 − − − 0.028 0.868 0.053 0.393 0.678 0.109 Grasping score - P13 Nonnormal 5.69 ± 0.17 5.86 ± 0.09 5.2 ± 0.24 3.789 0.030 0.660 0.399 0.092 0.009 1.328 0.255 0.204 0.287 0.752 0.093 Grasping score - P14 Nonnormal 5.69 ± 0.13 5.68 ± 0.12 4.6 ± 0.26 10.311 0.000 0.982 0.945 0.000 0.000 0.034 0.855 0.054 0.334 0.718 0.100 Grasping score - Average Nonnormal 4.02 ± 0.13 4.18 ± 0.07 3.79 ± 0.13 3.923 0.027 0.677 0.304 0.116 0.008 0.052 0.821 0.056 0.320 0.728 0.098 First day of two consecutive successes (score 4) Nonnormal 9.38 ± 0.43 8.75 ± 0.29 10.9 ± 0.37 10.068 0.000 0.979 0.233 0.005 0.000 0.131 0.719 0.065 1.318 0.278 0.270 Day effect 21.337 0.000 1.000 − − − Day × genotype effect 0.988 0.460 0.563 − − − Day × gender effect 0.921 0.478 0.356 − − − Day × genotype × gender effect 0.688 0.758 0.390 − − − Genotype effect 1.593 0.215 0.319 − − − Gender effect 0.857 0.360 0.148 − − − Genotype × gender 0.865 0.428 0.189 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Time to turn (seconds) - P2 Nonnormal 18.15 ± 2.97 19.93 ± 2.09 24.2 ± 2.64 0.933 0.401 0.201 − − − 0.382 0.540 0.093 1.161 0.323 0.242 Time to turn (seconds) - P3 Nonnormal 20.76 ± 3.19 18.13 ± 1.98 19.7 ± 3.05 0.398 0.674 0.110 − − − 0.020 0.887 0.052 1.146 0.327 0.239 Time to turn (seconds) - P4 Nonnormal 23.61 ± 2.26 18.03 ± 2.12 22.8 ± 3.71 1.506 0.233 0.304 − − − 0.078 0.781 0.059 1.392 0.259 0.283 Time to turn (seconds) - P5 Nonnormal 18.61 ± 3.27 16.82 ± 2.19 25.5 ± 2.29 2.431 0.100 0.464 − − − 0.284 0.597 0.082 0.129 0.879 0.069 Time to turn (seconds) - P6 Nonnormal 18.69 ± 3.26 14.2 ± 2.28 18.8 ± 3.14 1.242 0.299 0.256 − − − 0.163 0.688 0.068 0.079 0.925 0.061 Time to turn (seconds) - P7 Nonnormal 10.38 ± 3.18 8.37 ± 1.72 9 ± 3.16 0.155 0.857 0.072 − − − 4.992 0.031 0.589 0.741 0.482 0.168 Time to turn (seconds) - P8 Nonnormal 5 ± 1.91 5.34 ± 1.27 4.5 ± 1.43 0.073 0.930 0.060 − − − 3.064 0.087 0.402 0.023 0.978 0.053 Time to turn (seconds) - P9 Nonnormal 3 ± 0.62 4.24 ± 1.09 3.3 ± 0.83 0.397 0.675 0.110 − − − 0.371 0.546 0.092 0.413 0.664 0.113 Time to turn (seconds) - P10 Nonnormal 1.3 ± 0.13 2.06 ± 0.43 1.5 ± 0.16 0.901 0.414 0.196 − − − 0.002 0.966 0.050 0.059 0.943 0.058 Time to turn (seconds) - P11 Nonnormal 1 ± 0 1.27 ± 0.15 1.3 ± 0.15 1.320 0.277 0.270 − − − 0.034 0.854 0.054 0.314 0.732 0.097 Time to turn (seconds) - P12 Nonnormal 1 ± 0 1.03 ± 0.03 1 ± 0 0.398 0.674 0.110 − − − 0.331 0.568 0.087 0.196 0.823 0.079 Time to turn (seconds) - P13 − 1 ± 0 1 ± 0 1 ± 0 − − − − − − − − − − − − Time to turn (seconds) - Mean Normal 10.21 ± 1.03 9.2 ± 0.59 11.05 ± 0.79 1.593 0.215 0.319 − − − 0.857 0.360 0.148 0.865 0.428 0.189 Time to turn (days) - first day of two consecutive successes Nonnormal 9.61 ± 0.26 9.44 ± 0.34 9.7 ± 0.36 0.139 0.871 0.070 − − − 3.774 0.058 0.476 1.174 0.319 0.244 Negative geotaxis Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 12.128 0.000 1.000 − − − Day × genotype effect 1.526 0.086 0.895 − − − Day × gender effect 1.036 0.409 0.488 − − − Day × genotype × gender effect 1.386 0.144 0.855 − − − Genotype effect 2.110 0.133 0.410 − − − Gender effect 0.493 0.486 0.106 − − − Genotype × gender 0.090 0.914 0.063 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Time to turn (seconds) - P2 Nonnormal -9.66 ± 5.21 -18.82 ± 2.48 -11.6 ± 4.73 1.821 0.174 0.360 − − − 3.882 0.055 0.487 1.077 0.350 0.227 Time to turn (seconds) - P3 Nonnormal -8.69 ± 5.99 -15.58 ± 2.88 -11.8 ± 5.61 0.847 0.436 0.186 − − − 0.443 0.509 0.100 0.364 0.697 0.105 Time to turn (seconds) - P4 Nonnormal -6 ± 5.23 -10.82 ± 1.86 -15.2 ± 3.51 1.537 0.226 0.309 − − − 0.656 0.422 0.124 4.540 0.016 0.744 Time to turn (seconds) - P5 Nonnormal 9.69 ± 3.67 -4.2 ± 3.15 -2.2 ± 5.3 4.418 0.018 0.732 0.006 0.033 0.936 0.399 0.531 0.095 0.766 0.471 0.172 Time to turn (seconds) - P6 Nonnormal 6.84 ± 4.38 3.2 ± 2.97 8.5 ± 3.87 0.517 0.600 0.130 − − − 0.010 0.920 0.051 0.905 0.412 0.196 Time to turn (seconds) - P7 Nonnormal 6.3 ± 4.2 6.31 ± 3.13 12.4 ± 5.82 0.281 0.756 0.092 − − − 0.699 0.408 0.129 0.013 0.987 0.052 Time to turn (seconds) - P8 Nonnormal 8.3 ± 4.42 11.44 ± 2.71 6.8 ± 6.1 0.331 0.720 0.100 − − − 0.124 0.726 0.064 0.862 0.429 0.189 Time to turn (seconds) - P9 Nonnormal 12.76 ± 3.75 12.17 ± 2.44 23.1 ± 1.6 3.787 0.030 0.660 0.828 0.035 0.010 0.023 0.880 0.053 1.215 0.307 0.251 Time to turn (seconds) - P10 Nonnormal 9.84 ± 2.94 10.03 ± 2.29 19 ± 3.22 2.707 0.078 0.508 0.959 0.055 0.032 2.235 0.142 0.310 0.497 0.612 0.126 Time to turn (seconds) - P11 Nonnormal 10.92 ± 3.44 13.62 ± 2.17 15 ± 3.88 0.580 0.564 0.140 − − − 2.444 0.125 0.334 4.258 0.020 0.715 Time to turn (seconds) - P12 Nonnormal 11.92 ± 2.92 15.96 ± 1.74 22.2 ± 1.33 3.269 0.047 0.592 0.379 0.014 0.073 0.205 0.653 0.073 2.337 0.109 0.448 Time to turn (seconds) - P13 Nonnormal 15.23 ± 3.04 18.58 ± 1.5 24.1 ± 0.62 3.112 0.054 0.570 0.219 0.017 0.091 1.431 0.238 0.216 1.112 0.338 0.233 Time to turn (seconds) - P14 Nonnormal 22.69 ± 1.43 21.82 ± 1.17 22.3 ± 2.25 0.135 0.874 0.070 − − − 2.267 0.139 0.313 1.471 0.241 0.297 Time to turn (seconds) - Mean Normal 6.99 ± 1.25 4.9 ± 1.06 8.66 ± 1.1 2.110 0.133 0.410 − − − 0.493 0.486 0.106 0.090 0.914 0.063 Falls Nonnormal 9 ± 0.83 10.31 ± 0.48 9.1 ± 0.45 1.527 0.228 0.307 − − − 0.192 0.664 0.071 0.307 0.737 0.096 Air righting Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 21.651 0.000 1.000 − − − Day × genotype effect 3.211 0.001 0.986 − − − Day × gender effect 2.423 0.037 0.760 − − − Day × genotype × gender effect 1.309 0.227 0.664 − − − Genotype effect 3.166 0.052 0.577 0.693 0.070 0.016 Gender effect 0.464 0.499 0.102 − − − Genotype × gender 1.482 0.238 0.299 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Air righting score - P8 Nonnormal 0.61 ± 0.26 0.41 ± 0.13 0.8 ± 0.29 1.160 0.323 0.242 − − − 5.791 0.020 0.653 5.562 0.007 0.831 Air righting score - P9 Nonnormal 0.38 ± 0.21 0.93 ± 0.17 0.4 ± 0.22 2.272 0.115 0.438 − − − 0.004 0.948 0.050 0.439 0.648 0.117 Air righting score - P10 Nonnormal 1.69 ± 0.2 1.44 ± 0.13 0.7 ± 0.3 5.755 0.006 0.844 0.426 0.003 0.005 1.456 0.234 0.219 0.438 0.648 0.117 Air righting score - P11 Nonnormal 1.3 ± 0.26 1.44 ± 0.16 0.4 ± 0.26 5.407 0.008 0.819 0.703 0.015 0.002 2.641 0.111 0.356 0.639 0.533 0.150 Air righting score - P12 Nonnormal 1.84 ± 0.15 1.89 ± 0.05 1.4 ± 0.22 4.066 0.024 0.693 0.742 0.034 0.007 0.332 0.567 0.087 0.647 0.528 0.152 Air righting score - P13 Nonnormal 1.76 ± 0.16 1.68 ± 0.13 1.7 ± 0.21 0.069 0.934 0.060 − − − 2.104 0.154 0.295 0.396 0.675 0.110 Air righting score - P14 Nonnormal 2 ± 0 2 ± 0 2 ± 0 − − . − − − − − . − − . Air righting score - P15 Nonnormal 1.92 ± 0.07 2 ± 0 2 ± 0 1.621 0.209 0.324 − − − 1.716 0.197 0.249 1.464 0.242 0.296 Air righting score - P16 Nonnormal 2 ± 0 2 ± 0 2 ± 0 − − . − − − − − . . Air righting score - P17 Nonnormal 2 ± 0 1.96 ± 0.03 2 ± 0 0.398 0.674 0.110 − − − 0.331 0.568 0.087 0.196 0.823 0.079 Air righting score - P18 Nonnormal 2 ± 0 2 ± 0 2 ± 0 − − . − − − − − . − − . Air righting score - P19 Nonnormal 2 ± 0 2 ± 0 2 ± 0 − − . − − − − − . − − . Air righting score - P20 Nonnormal 2 ± 0 2 ± 0 2 ± 0 − − . − − − − − . − − . Air righting score - Mean Nonnormal 1.65 ± 0.06 1.67 ± 0.03 1.49 ± 0.06 3.166 0.049 0.577 0.693 0.070 0.016 0.464 0.499 0.102 1.482 0.238 0.299 First day of two consecutive successes Nonnormal 11.84 ± 0.5 11.37 ± 0.29 12.9 ± 0.56 2.814 0.071 0.525 0.378 0.184 0.023 0.959 0.333 0.160 1.173 0.319 0.244 Wire suspension Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 16.511 0.000 1.000 − − − Day × genotype effect 3.538 0.000 0.994 − − − Day × gender effect 0.497 0.782 0.186 − − − Day × genotype × gender effect 0.635 0.787 0.333 − − − Genotype effect 13.553 0.000 0.997 0.013 0.000 0.001 Gender effect 0.303 0.585 0.084 − − − Genotype × gender 2.871 0.067 0.534 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Suspension time (seconds) - P11 Nonnormal 5.15 ± 1.44 4.37 ± 0.63 2.7 ± 0.47 1.701 0.194 0.338 − − − 0.045 0.833 0.055 0.287 0.752 0.093 Suspension time (seconds) - P12 Nonnormal 3.23 ± 1.06 3.13 ± 0.45 3.9 ± 1.65 0.220 0.803 0.082 − − − 0.856 0.360 0.148 0.662 0.521 0.154 Suspension time (seconds) - P13 Nonnormal 2.69 ± 0.47 4 ± 0.52 2.8 ± 0.87 1.660 0.202 0.331 − − − 2.518 0.120 0.342 0.036 0.965 0.055 Suspension time (seconds) - P14 Nonnormal 7.61 ± 1.97 5.17 ± 0.58 3.7 ± 1.12 2.196 0.123 0.425 − − − 0.006 0.938 0.051 1.893 0.163 0.372 Suspension time (seconds) - P15 Nonnormal 9.92 ± 2.29 4.82 ± 0.53 1.7 ± 0.42 10.137 0.000 0.980 0.002 0.000 0.054 0.290 0.593 0.082 0.611 0.547 0.146 Suspension time (seconds) - P16 Nonnormal 13.38 ± 2.18 6.41 ± 0.53 3.7 ± 0.91 15.666 0.000 0.999 0.000 0.000 0.100 0.744 0.393 0.135 1.971 0.151 0.386 Suspension time (seconds) - P17 Nonnormal 18.53 ± 2.34 11.82 ± 1.22 9.3 ± 1.6 6.683 0.003 0.896 0.004 0.002 0.288 0.538 0.467 0.111 1.214 0.307 0.251 Suspension time (seconds) - P18 Nonnormal 16.15 ± 2.22 18.34 ± 1.78 11.1 ± 2.37 2.398 0.103 0.459 − − − 0.551 0.462 0.112 0.722 0.491 0.164 Suspension time (seconds) - P19 Nonnormal 18.38 ± 2.11 19.48 ± 1.73 8 ± 1.97 7.474 0.002 0.927 0.921 0.003 0.001 0.000 0.995 0.050 1.609 0.212 0.322 Suspension time (seconds) - P20 Nonnormal 17.07 ± 2.31 12.13 ± 1.54 5.6 ± 0.85 6.858 0.003 0.903 0.053 0.001 0.019 0.646 0.426 0.123 1.283 0.287 0.264 Suspension time (seconds) - Average Nonnormal 11.21 ± 1 8.97 ± 0.55 5.25 ± 0.54 13.553 0.000 0.997 0.013 0.000 0.001 0.303 0.585 0.084 2.871 0.067 0.534 Suspension time (seconds) - Best score Nonnormal 24.3 ± 2.06 23.31 ± 1.37 13.7 ± 1.99 7.828 0.001 0.938 0.525 0.001 0.001 0.168 0.684 0.069 1.205 0.309 0.250 Open field Repeated measures, sphericity violated F p value Power WT vs Het WT vs KO Het vs KO Day effect 31.056 0.000 1.000 − − − Day × genotype effect 0.874 0.572 0.501 − − − Day × gender effect 0.630 0.702 0.247 − − − Day × genotype × gender effect 1.857 0.042 0.887 − − − Genotype effect 0.117 0.890 0.067 − − − Gender effect 0.046 0.831 0.055 − − − Genotype × gender 1.755 0.185 0.348 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Time to escape (seconds) - P8 Nonnormal 26.07 ± 2.09 27.55 ± 1.19 28.9 ± 0.99 0.473 0.626 0.122 − − − 0.928 0.341 0.156 0.010 0.990 0.051 Time to escape (seconds) - P9 Nonnormal 23.3 ± 2.82 21.34 ± 1.83 24.2 ± 2.64 0.305 0.739 0.095 − − − 0.774 0.384 0.138 0.929 0.403 0.201 Time to escape (seconds) - P10 Nonnormal 20.46 ± 2.55 19.96 ± 1.72 22.1 ± 2.34 0.286 0.753 0.093 − − − 0.010 0.922 0.051 1.615 0.210 0.323 Time to escape (seconds) - P11 Nonnormal 22.07 ± 2.18 17.55 ± 1.94 20.2 ± 3.04 0.938 0.399 0.202 − − − 1.030 0.316 0.168 3.307 0.046 0.597 Time to escape (seconds) - P12 Nonnormal 17 ± 2.34 19.27 ± 1.49 16.2 ± 2.09 0.660 0.522 0.154 − − − 0.718 0.401 0.132 1.010 0.372 0.215 Time to escape (seconds) - P13 Nonnormal 15.61 ± 2.38 18.2 ± 1.72 15.6 ± 2.25 0.573 0.568 0.139 − − − 0.015 0.902 0.052 0.685 0.509 0.158 Time to escape (seconds) - P14 Nonnormal 17.23 ± 2.78 12.72 ± 1.6 10.6 ± 1.14 2.349 0.107 0.450 − − − 0.421 0.520 0.097 5.349 0.008 0.815 Time to escape (seconds) - P15 Nonnormal 4.92 ± 0.38 5.65 ± 0.39 7 ± 1.46 1.768 0.183 0.350 − − − 0.215 0.645 0.074 0.019 0.981 0.053 Time to escape (seconds) - P16 Nonnormal 3.61 ± 0.28 3.65 ± 0.25 4.5 ± 0.87 1.119 0.336 0.234 − − − 2.068 0.158 0.290 0.043 0.958 0.056 Time to escape (seconds) - P17 Nonnormal 2.46 ± 0.24 3.48 ± 0.26 3.6 ± 0.37 3.840 0.029 0.667 0.014 0.026 0.745 1.782 0.189 0.257 1.179 0.317 0.245 Time to escape (seconds) - P18 Nonnormal 2.23 ± 0.32 2.1 ± 0.21 2.1 ± 0.4 0.028 0.972 0.054 − − − 0.121 0.730 0.063 0.313 0.733 0.097 Time to escape (seconds) - P19 Nonnormal 2.38 ± 0.33 2.17 ± 0.29 2 ± 0.36 0.011 0.989 0.051 − − − 1.947 0.170 0.276 2.592 0.086 0.490 Time to escape (seconds) - P20 Nonnormal 1.92 ± 0.21 2.13 ± 0.16 1.9 ± 0.17 0.632 0.536 0.149 − − − 0.044 0.834 0.055 0.205 0.816 0.080 Time to escape (seconds) - average Normal 12.25 ± 0.59 11.98 ± 0.49 12.22 ± 0.53 0.117 0.890 0.067 − − − 0.046 0.831 0.055 1.755 0.185 0.348 First day of two consecutive successes (30-s cutoff) Nonnormal 11.92 ± 0.58 11.24 ± 0.32 11.5 ± 0.4 0.589 0.559 0.142 − − − 0.701 0.407 0.130 0.297 0.745 0.094 Ultrasonic vocalizations Number of calls - repeated measures, sphericity assumed F p value Power WT vs Het WT vs KO Het vs KO Day effect 4.600 0.012 0.767 − − − Day × genotype effect 0.991 0.416 0.303 − − − Day × gender effect 1.430 0.244 0.300 − − − Day × genotype × gender effect 0.305 0.874 0.116 − − − Genotype effect 0.533 0.590 0.133 − − − Gender effect 1.697 0.199 0.248 − − − Genotype × gender 0.869 0.426 0.191 − − − Gender effect Gender × genotype effect Multifactiorial ANCOVA WT Het KO F p value Power WT vs Het WT vs KO Het vs KO F p value Power F p value Power Number of calls - minute 1 Nonnormal 13.81 ± 4.24 11.46 ± 2.89 15.88 ± 5.31 0.586 0.562 0.139 − − − 0.163 0.689 0.068 0.090 0.914 0.063 Number of calls - minute 2 Nonnormal 18.68 ± 5.15 11.56 ± 2.73 13.22 ± 4.78 0.567 0.572 0.136 − − − 0.327 0.571 0.086 0.068 0.935 0.059 Number of calls - minute 3 Nonnormal 15.75 ± 4.72 13.96 ± 3.72 8.33 ± 4.2 0.172 0.843 0.074 − − − 3.481 0.071 0.442 0.097 0.908 0.064 Number of calls - total Nonnormal 48.43 ± 13.28 37.06 ± 7.85 37.44 ± 12.08 0.156 0.856 0.072 − − − 1.323 0.258 0.201 0.052 0.949 0.057 Calling time - minute 1 Nonnormal 0.93 ± 0.29 0.75 ± 0.19 1.08 ± 0.38 0.738 0.485 0.165 − − − 0.106 0.746 0.062 0.083 0.920 0.062 Calling time - minute 2 Nonnormal 1.24 ± 0.39 0.76 ± 0.19 0.85 ± 0.3 0.368 0.695 0.104 − − − 0.231 0.634 0.075 0.059 0.943 0.058 Calling time - minute 3 Nonnormal 1.16 ± 0.4 0.96 ± 0.25 0.53 ± 0.27 0.162 0.851 0.073 − − − 4.123 0.050 0.505 0.006 0.994 0.051 Calling time - total Nonnormal 3.41 ± 1.05 2.48 ± 0.52 2.47 ± 0.81 0.189 0.828 0.077 − − − 1.431 0.240 0.213 0.023 0.977 0.053 Average duration - min 1 Nonnormal 0.05 ± 0 0.05 ± 0 0.03 ± 0.01 0.138 0.872 0.069 − − − 0.056 0.815 0.056 0.950 0.400 0.197 Average duration - min 2 Nonnormal 0.06 ± 0 0.06 ± 0 0.03 ± 0.01 0.319 0.730 0.095 − − − 1.879 0.182 0.262 1.561 0.229 0.301 Average duration - min 3 Nonnormal 0.06 ± 0 0.06 ± 0 0.02 ± 0.01 6.759 0.004 0.883 − − − 23.838 0.000 0.997 7.350 0.003 0.909 Average duration - total Nonnormal 0.08 ± 0.01 0.06 ± 0 0.03 ± 0.01 0.515 0.604 0.125 − − − 1.663 0.209 0.237 0.847 0.440 0.179 Latency to first call Nonnormal 77.28 ± 17.98 80.04 ± 12.54 75.57 ± 27.65 0.411 0.665 0.113 − − − 0.155 0.696 0.067 3.311 0.045 0.602 Mean frequency - total Nonnormal 71337.77 ± 1902.04 73128.73 ± 3879.33 75883.81 ± 3957.75 0.024 0.976 0.053 − − − 0.036 0.851 0.054 0.029 0.971 0.054 Mean amplitude - total Nonnormal 78.43 ± 29.11 66.33 ± 20.80 37.15 ± 17.64 0.189 0.829 0.076 − − − 2.440 0.130 0.325 0.442 0.648 0.114 Percentage of noncaller Nonnormal 18.75 ± 10.08 28.13 ± 8.08 33.33 ± 16.67 0.401 0.672 0.111 − − − 0.587 0.447 0.117 1.933 0.155 0.382 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). P: postnatal day.
- Table 5.
Detailed results and statistical analyses related to general health, physical factors, gross appearance, and spontaneous activity
Physical factors and gross appearance Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO 3-months weight (g) 2wANOVA Normal 26.33 ± 0.87 27.18 ± 0.57 26.01 ± 0.54 1.241 0.298 0.258 2.546 0.117 0.347 2.000 0.146 0.394 − − − 15-months weight (g) 2wANOVA Normal 33.41 ± 2 31.06 ± 1.21 29.36 ± 1.73 1.578 0.222 0.310 0.269 0.608 0.079 0.491 0.617 0.123 − − − 20-months weight (g) 2wANOVA Normal 32.9 ± 1.86 31.43 ± 1.2 28.84 ± 1.5 0.982 0.390 0.199 0.034 0.856 0.054 0.018 0.982 0.052 − − − Length 2wANOVA Nonnormal 16.45 ± 0.24 16.84 ± 0.24 16.73 ± 0.25 1.062 0.353 0.226 91.207 0.000 1.000 0.471 0.627 0.123 − − − Coat appearance 2wANOVA Nonnormal 2.63 ± 0.13 2.89 ± 0.07 2.94 ± 0.05 2.558 0.087 0.489 2.615 0.112 0.355 1.424 0.250 0.291 0.119 0.050 0.915 Skin color − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Whisker barbering − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Missing fur on face − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Missing fur on body − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Wounding 2wANOVA Nonnormal 0.15 ± 0.11 0 ± 0 0.05 ± 0.05 1.078 0.348 0.229 0.028 0.869 0.053 0.279 0.758 0.092 − − − Body tone 2wANOVA Nonnormal 1.1 ± 0.07 1.1 ± 0.07 1 ± 0.07 0.563 0.573 0.138 2.225 0.142 0.310 0.563 0.573 0.138 − − − Palpebral closure − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Piloerection − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Jar observation Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Body position 2wANOVA Nonnormal 4.15 ± 0.08 4.1 ± 0.07 4.26 ± 0.1 0.702 0.500 0.162 4.949 0.031 0.588 0.139 0.871 0.070 − − − Spontaneous activity 2wANOVA Nonnormal 1.68 ± 0.1 1.57 ± 0.11 1.63 ± 0.11 0.223 0.801 0.083 0.282 0.598 0.082 0.279 0.758 0.092 − − − Latency to sit/stand (s) − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − − − − Latency to rear (s) 2wANOVA Nonnormal 8.94 ± 1.36 8.05 ± 1.03 5.63 ± 1.02 2.137 0.128 0.418 0.036 0.850 0.054 0.046 0.955 0.057 − − − Repeated jumps (%) 2wANOVA Nonnormal 15.78 ± 8.59 10.52 ± 7.23 26.31 ± 10.37 0.702 0.500 0.162 4.949 0.031 0.588 0.139 0.871 0.070 − − − Circling (%) 2wANOVA Nonnormal 5.26 ± 5.26 10.52 ± 7.23 10.52 ± 7.23 0.289 0.750 0.093 5.177 0.027 0.607 0.289 0.750 0.093 − − − Urination 2wANOVA Nonnormal 0.47 ± 0.19 0.1 ± 0.1 0.15 ± 0.11 1.540 0.224 0.312 0.023 0.881 0.052 1.213 0.306 0.253 − − − Defecation (number) 2wANOVA Nonnormal 1.57 ± 0.35 0.94 ± 0.29 0.94 ± 0.27 1.065 0.352 0.226 0.003 0.958 0.050 2.592 0.085 0.494 − − − Respiration − − 2 ± 0 2 ± 0 2 ± 0 − − − − − − − − − Tremor − − 0 ± 0 0 ± 0 0 ± 0 − − − − − − − − − Cage transfer Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Transfer arousal 2wANOVA Nonnormal 3.21 ± 0.22 3.21 ± 0.22 3.15 ± 0.2 0.037 0.964 0.055 1.470 0.231 0.221 2.055 0.139 0.404 − − − Gait 2wANOVA Nonnormal 0 ± 0 0.15 ± 0.08 0.05 ± 0.05 1.783 0.178 0.356 0.003 0.957 0.050 0.637 0.533 0.151 − − − Pelvic elevation 2wANOVA Nonnormal 2 ± 0 2.15 ± 0.08 2 ± 0.07 1.730 0.188 0.347 0.141 0.709 0.066 0.141 0.869 0.071 − − − Tail elevation 2wANOVA Nonnormal 1.89 ± 0.15 1.73 ± 0.18 1.21 ± 0.18 4.003 0.024 0.691 4.469 0.039 0.545 0.204 0.816 0.080 0.530 0.009 0.043 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 5-1. 2wANOVA: 2-way ANOVA.
Gait analysis Genotype Cohort Genotype ×cohort Pairwisecomparisons Test Datastructure WT Het KO F pvalue Power F pvalue Power F pvalue Power WT vsHet WT vsKO Het vs KO Stance mean (cm) 2wANOVA Normal 3.55 ± 0.09 3.41 ± 0.12 3.27 ± 0.14 1.466 0.240 0.299 40.902 0.000 1.000 1.291 0.284 0.267 − − − Stance variance (cm) 2wANOVA Nonnormal 0.15 ± 0.04 0.15 ± 0.03 0.17 ± 0.05 0.189 0.829 0.078 14.972 0.000 0.967 0.735 0.484 0.168 − − − Stride mean (cm) 2wANOVA Normal 5.39 ± 0.23 5.64 ± 0.21 6.43 ± 0.24 5.443 0.007 0.826 15.476 0.000 0.971 0.499 0.610 0.127 0.674 0.003 0.028 Stride variance (cm) 2wANOVA Nonnormal 0.77 ± 0.13 0.86 ± 0.14 0.83 ± 0.18 0.137 0.873 0.070 12.150 0.001 0.928 3.459 0.039 0.622 − − − Sway mean (cm) 2wANOVA Nonnormal 3.4 ± 0.17 3.55 ± 0.16 3.59 ± 0.22 0.191 0.826 0.078 186.368 0.000 1.000 2.443 0.097 0.470 − − − Sway variance (cm) 2wANOVA Nonnormal 0.08 ± 0.01 0.17 ± 0.04 0.14 ± 0.02 1.909 0.159 0.378 3.781 0.057 0.479 0.400 0.672 0.111 − − − Open field spontaneous activity (traveled distance) Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Total distance (cm) 2wANOVA Normal 13,816.17 ± 828.27 11,273.16 ± 764.09 10,099.24 ± 621.43 6.633 0.003 0.896 12.836 0.001 0.940 0.073 0.930 0.061 0.016 0.001 0.299 Distance Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Time rMeasures Sph.viol 36.350 0.000 1.000 − − − - Time × gen. rMeasures Sph.viol 2.235 0.029 0.917 − − − - Genotype rMeasures Sph.viol 6.633 0.003 0.896 0.029 0.001 0.449 - Cohort rMeasures Sph.viol 12.836 0.001 0.940 − − − - Time × gen. × coh. rMeasures Sph.viol 0.878 0.532 0.461 − − − - gen. × coh. rMeasures Sph.viol 0.073 0.930 0.061 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Individual time bins Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Distance 0-10 min 2wANOVA Normal 2723.06 ± 185.28 2365.11 ± 166.4 2479.64 ± 164.36 0.894 0.415 0.196 7.573 0.008 0.770 0.224 0.800 0.083 − − − Distance 10-20 min 2wANOVA Nonnormal 2516.77 ± 150.09 2064.33 ± 169.75 1684.88 ± 108.32 8.357 0.001 0.954 13.148 0.001 0.945 0.026 0.975 0.054 0.030 0.000 0.069 Distance 20-30 min 2wANOVA Normal 2349.52 ± 168.15 1919.27 ± 159.57 1466.99 ± 150.82 7.936 0.001 0.943 11.962 0.001 0.924 0.362 0.698 0.105 0.051 0.000 0.051 Distance 30-40 min 2wANOVA Normal 2203.27 ± 139.79 1680.09 ± 143.29 1589.41 ± 114.03 6.091 0.004 0.868 11.521 0.001 0.915 0.020 0.980 0.053 0.007 0.002 0.710 Distance 40-50 min 2wANOVA Normal 2090.38 ± 156.71 1657.47 ± 139.76 1380 ± 116.11 6.255 0.004 0.877 5.904 0.019 0.664 0.035 0.965 0.055 0.035 0.001 0.185 Distance 50-60 min 2wANOVA Nonnormal 1933.14 ± 160.59 1586.87 ± 98.6 1498.28 ± 139.38 3.074 0.055 0.568 3.755 0.058 0.477 1.459 0.242 0.298 0.055 0.026 0.733 Rotarod Latency Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Trial rMeasures Sph.viol 9.369 0.000 1.000 − − − - Trial × gen. rMeasures Sph.viol 2.268 0.015 0.921 − − − - Genotype rMeasures Sph.viol 8.888 0.000 0.964 0.123 0.000 0.044 - Cohort rMeasures Sph.viol 2.573 0.115 0.350 − − − - Session × gen. × coh. rMeasures Sph.viol 1.867 0.050 0.848 − − − - Gen. × coh. rMeasures Sph.viol 0.726 0.489 0.166 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Individual trials Test Data structure WT Het KO F pvalue Power F pvalue Power F pvalue Power WT vs Het WT vs KO Het vsKO Latency trial 1 2wANOVA Normal 181.28 ± 15.25 175.51 ± 14.58 149.17 ± 13.24 1.323 0.275 0.273 0.053 0.819 0.056 1.320 0.276 0.273 − − − Latency trial 2 2wANOVA Normal 198.66 ± 19.58 190.76 ± 20.22 135.84 ± 15.82 3.395 0.041 0.614 0.001 0.979 0.050 3.956 0.025 0.685 0.947 0.042 0.085 Latency trial 3 2wANOVA Normal 222.75 ± 20.99 178.3 ± 14.43 128.94 ± 14.01 7.010 0.002 0.913 1.047 0.311 0.171 0.816 0.448 0.182 0.159 0.001 0.106 Latency trial 4 2wANOVA Normal 260.92 ± 18.73 209.71 ± 15.54 168.01 ± 16.99 6.767 0.002 0.902 3.832 0.056 0.484 0.017 0.983 0.052 0.094 0.001 0.203 Latency trial 5 2wANOVA Normal 270.8 ± 21.62 228.95 ± 22.25 172.55 ± 21.59 4.498 0.016 0.744 1.536 0.221 0.229 1.050 0.357 0.224 0.368 0.007 0.168 Latency trial 6 2wANOVA Normal 273.51 ± 19.16 192.29 ± 25.69 133.8 ± 15.59 11.838 0.000 0.992 9.021 0.004 0.838 0.222 0.802 0.083 0.013 0.000 0.094 Latency day 1 2wANOVA Normal 200.9 ± 15.13 181.52 ± 14.32 137.98 ± 11.9 5.026 0.010 0.793 0.108 0.744 0.062 2.253 0.115 0.438 0.578 0.006 0.072 Latency day 2 2wANOVA Normal 268.41 ± 16.68 210.32 ± 19.07 158.12 ± 14.54 10.061 0.000 0.980 5.783 0.020 0.655 0.060 0.942 0.059 0.041 0.000 0.073 Beam walking Genotype Cohort Genotype × cohort Pairwisecomparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO % mice falling (large) 2wANOVA NA 0 ± 0 0 ± 0 0 ± 0 NA NA NA NA NA NA NA NA NA − − − % mice falling (medium) 2wANOVA Nonnormal 0 ± 0 0 ± 0 6.57 ± 2.59 6.339 0.003 0.882 0.395 0.533 0.095 0.396 0.675 0.111 1.000 0.003 0.003 % mice falling (small) 2wANOVA Nonnormal 32.89 ± 7.41 26.31 ± 7.27 78.94 ± 6.12 16.788 0.000 1.000 3.622 0.063 0.463 0.972 0.385 0.210 0.366 0.000 0.000 Distance (large,cm) 2wANOVA Nonnormal 95.05 ± 2.27 90.92 ± 4.66 99.07 ± 0.92 1.819 0.173 0.362 1.416 0.240 0.215 0.242 0.786 0.086 − − − Distance (medium, cm) 2wANOVA Nonnormal 87.17 ± 4.25 90.41 ± 4.35 88.15 ± 5.03 0.129 0.879 0.069 2.525 0.118 0.344 0.482 0.620 0.125 − − − Distance (small, cm) 2wANOVA Normal 47.19 ± 5.45 53.77 ± 8.03 26.24 ± 6.14 4.380 0.018 0.732 0.447 0.507 0.101 0.346 0.709 0.102 0.438 0.044 0.006 % mice fully crossing (large) 2wANOVA Nonnormal 94.73 ± 2.4 89.47 ± 5.83 98.68 ± 1.31 1.567 0.219 0.317 1.486 0.228 0.223 0.107 0.899 0.066 − − − % mice fully crossing (medium) 2wANOVA Nonnormal 76.31 ± 7.01 80.7 ± 7.12 80.26 ± 7.29 0.089 0.915 0.063 4.278 0.044 0.528 0.947 0.394 0.205 − − − % mice fully crossing (small) 2wANOVA Nonnormal 27.63 ± 7.12 38.4 ± 9.57 9.21 ± 5.47 3.568 0.035 0.637 0.692 0.409 0.129 0.316 0.730 0.098 0.278 0.128 0.010 Paw misplacements (large, all mice) 2wANOVA Nonnormal 0.47 ± 0.14 0.56 ± 0.16 1.06 ± 0.14 4.197 0.021 0.712 0.137 0.713 0.065 1.662 0.200 0.334 0.693 0.010 0.026 Paw misplacements (medium, all mice) 2wANOVA Normal 1.71 ± 0.29 1.5 ± 0.26 2.38 ± 0.41 1.762 0.182 0.352 0.525 0.472 0.110 0.055 0.947 0.058 − − - Paw misplacements (small, all mice) 2wANOVA Nonnormal 1.44 ± 0.13 2.48 ± 0.47 1.78 ± 0.18 3.330 0.044 0.605 3.700 0.060 0.471 0.760 0.473 0.172 0.015 0.462 0.082 Paw misplacements (large, crossing mice) 2wANOVA Nonnormal 0.52 ± 0.17 0.6 ± 0.17 1.07 ± 0.14 3.194 0.049 0.585 0.210 0.649 0.073 1.333 0.273 0.275 0.937 0.053 0.119 Paw misplacements (medium, crossing mice) 2wANOVA Nonnormal 1.67 ± 0.33 1.37 ± 0.27 2.33 ± 0.53 1.343 0.271 0.276 0.851 0.361 0.148 0.027 0.973 0.054 − − − Paw misplacements (small, crossing mice) 2wANOVA Nonnormal 1.79 ± 0.23 2.77 ± 0.53 2.81 ± 0.64 1.189 0.327 0.227 2.465 0.134 0.318 0.102 0.904 0.063 − − − Time to cross (large, fully crossing) 2wANOVA Nonnormal 10.05 ± 1.3 9.11 ± 1.91 7.17 ± 1.15 0.911 0.409 0.199 3.043 0.087 0.402 1.213 0.306 0.253 − − − Time to cross (medium, fully crossing) 2wANOVA Nonnormal 28.54 ± 5.32 16.62 ± 3.28 18.14 ± 4.47 1.643 0.204 0.330 2.898 0.095 0.386 2.415 0.100 0.464 − − − Time to cross (small, fully crossing) 2wANOVA Normal 54.56 ± 7.16 44.74 ± 5.25 22.43 ± 6 4.119 0.030 0.667 0.037 0.850 0.054 2.660 0.092 0.473 0.479 0.030 0.169 Time to cross (large, all mice) 2wANOVA Nonnormal 15.75 ± 3.07 20.75 ± 6.73 8.5 ± 2.21 4.443 0.027 0.370 0.327 0.574 0.094 0.427 0.659 0.070 0.015 0.033 0.820 Time to cross (medium, all mice) 2wANOVA Nonnormal 46.34 ± 8.74 36.11 ± 8.58 34.61 ± 8.93 1.157 0.337 0.109 1.444 0.245 0.682 0.472 0.632 0.218 − − − Time to cross (small, all mice) 2wANOVA Normal 99.92 ± 4.67 89.98 ± 7.71 111.65 ± 4.61 3.540 0.051 0.606 0.524 0.478 0.073 3.236 0.063 0.158 0.017 0.428 0.220 Motor reflexes Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Righting reflex 2wANOVA Nonnormal 0.05 ± 0.05 0 ± 0 0 ± 0 0.721 0.491 0.165 0.727 0.398 0.133 0.721 0.491 0.165 − − − Hindlimb placing, score 2wANOVA Nonnormal 5.57 ± 0.24 5.26 ± 0.34 4.21 ± 0.57 2.778 0.072 0.524 0.093 0.762 0.060 0.117 0.890 0.067 0.618 0.029 0.086 Hindlimb placing, latency to climb 2wANOVA Nonnormal 8.29 ± 1.61 7.21 ± 1.66 10.96 ± 2.57 0.836 0.439 0.186 0.333 0.567 0.087 0.117 0.890 0.067 − − − Hindlimb placing, failed attempts 2wANOVA Nonnormal 0.21 ± 0.12 0.36 ± 0.17 0.89 ± 0.28 2.778 0.072 0.524 0.093 0.762 0.060 0.117 0.890 0.067 0.618 0.029 0.086 Inverted screen, latency to fall 2wANOVA Nonnormal 33.78 ± 5.09 37 ± 4.72 9 ± 3.13 11.464 0.000 0.991 0.701 0.406 0.130 0.645 0.529 0.152 0.522 0.000 0.000 Hanging, score 2wANOVA Nonnormal 6.26 ± 0.18 6 ± 0.25 4.84 ± 0.27 10.223 0.000 0.982 2.691 0.107 0.363 0.834 0.440 0.185 0.486 0.000 0.001 Hanging, latency to fall 2wANOVA Nonnormal 25.44 ± 2.27 23.66 ± 2.66 8.48 ± 1.05 18.838 0.000 1.000 0.269 0.606 0.080 0.816 0.448 0.182 0.643 0.000 0.000 Grip strength Latency Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Session rMeasures Sph.viol 3.520 0.033 0.644 − − − - Session × gen. rMeasures Sph.viol 1.971 0.105 0.575 − − − - Genotype rMeasures Sph.viol 0.324 0.725 0.099 − − − - Cohort rMeasures Sph.viol 47.402 0.000 1.000 − − − - Session × gen. × coh. rMeasures Sph.viol 2.687 0.035 0.729 − − − - Gen. × coh. rMeasures Sph.viol 1.044 0.359 0.223 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Individual trials Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Session 1 2wANOVA Normal 0.94 ± 0.05 0.99 ± 0.06 1.03 ± 0.06 0.502 0.608 0.128 25.973 0.000 0.999 1.564 0.219 0.317 − − − Session 2 2wANOVA Normal 0.86 ± 0.07 0.85 ± 0.06 1.01 ± 0.05 2.222 0.119 0.433 36.967 0.000 1.000 1.631 0.206 0.329 − − − Session 3 2wANOVA Normal 0.91 ± 0.06 0.92 ± 0.06 0.89 ± 0.06 0.320 0.728 0.098 23.585 0.000 0.997 1.883 0.163 0.374 − − − Mean strength 2wANOVA Normal 0.9 ± 0.05 0.92 ± 0.05 0.98 ± 0.04 0.324 0.725 0.099 47.402 0.000 1.000 1.044 0.359 0.223 − − − Highest score 2wANOVA Nonnormal 1.08 ± 0.05 1.07 ± 0.05 1.13 ± 0.05 0.243 0.785 0.086 26.793 0.000 0.999 0.821 0.446 0.183 − − − WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 6-1. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Sph.viol: sphericity violated, gen: genotype, coh: cohort.
Reflexes and reactions to simple stimuli Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Pinna reflex 2wANOVA Nonnormal 0.89 ± 0.07 0.68 ± 0.1 0.73 ± 0.1 1.790 0.1773 0.357 13.988 0.000 0.956 1.155 0.323 0.243 − − − Cornel reflex 2wANOVA Nonnormal 1.05 ± 0.05 0.94 ± 0.05 1.05 ± 0.05 1.518 0.2289 0.308 0.389 0.536 0.094 1.518 0.229 0.308 − − − Toe pinch retraction 2wANOVA Nonnormal 2.05 ± 0.37 2.36 ± 0.33 2.26 ± 0.43 0.144 0.8659 0.071 1.073 0.305 0.174 0.072 0.931 0.060 − − − Preyer reflex 2wANOVA Nonnormal 1.47 ± 0.15 1.36 ± 0.13 1.42 ± 0.17 0.135 0.8740 0.070 22.250 0.000 0.996 1.478 0.238 0.301 − − − Visual placing NA NA 9 ± 0 9 ± 0 9 ± 0 − − − − − − − − − Tail flick Latency Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 3.081 0.0500 0.583 − − − - Trial × gen. rMeasures Sph.viol 0.169 0.9535 0.085 − − − - Genotype rMeasures Sph.viol 1.118 0.3347 0.236 − − − - Cohort rMeasures Sph.viol 83.467 0.0000 1.000 − − − - Trial × gen. × coh. rMeasures Sph.viol 0.489 0.7438 0.162 − − − - Gen. × coh. rMeasures Sph.viol 2.162 0.1255 0.422 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Individual trials Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vsKO Het vs KO Latency trial 1 (s) 2wANOVA Normal 11.39 ± 0.97 11.32 ± 0.78 10.28 ± 0.82 0.356 0.7023 0.104 13.680 0.001 0.952 0.145 0.865 0.071 − − − Latency trial 2 (s) 2wANOVA Normal 10.99 ± 1.08 9.67 ± 0.98 8.86 ± 0.82 0.961 0.3892 0.208 36.614 0.000 1.000 2.286 0.112 0.444 − − − Latency trial 3 (s) 2wANOVA Normal 11.05 ± 1.23 10.06 ± 1.01 9.29 ± 0.94 0.526 0.5942 0.132 91.329 0.000 1.000 1.578 0.216 0.319 − − − Shortest latency (s) 2wANOVA Normal 8.03 ± 0.82 7.99 ± 0.87 7.47 ± 0.78 0.064 0.9379 0.059 42.964 0.000 1.000 1.039 0.361 0.222 − − − Longest latency (s) 2wANOVA Nonnormal 14.17 ± 0.94 12.82 ± 0.75 11.91 ± 0.72 2.213 0.1198 0.431 64.815 0.000 1.000 1.659 0.200 0.334 − − − Mean latency (s) 2wANOVA Nonnormal 11.14 ± 0.88 10.35 ± 0.78 9.48 ± 0.72 1.118 0.3347 0.236 83.467 0.000 1.000 2.162 0.126 0.422 − − − Startle response Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vsHet WT vs KO Het vs KO Startle 74 dB 2wANOVA Nonnormal 179.96 ± 20.24 165.57 ± 17.22 158.01 ± 10.36 2.376 0.1077 0.448 9.123 0.005 0.836 2.255 0.120 0.428 − − − Startle 78 dB 2wANOVA Nonnormal 183.48 ± 22 157.4 ± 16.93 160.69 ± 14.89 3.181 0.0538 0.571 5.779 0.022 0.647 3.673 0.036 0.638 0.046 0.025 0.770 Startle 82 dB 2wANOVA Nonnormal 197.45 ± 26.03 160.09 ± 14.07 175.58 ± 13.87 3.254 0.0500 0.582 3.939 0.055 0.488 2.621 0.087 0.487 0.019 0.063 0.591 Startle 86 dB 2wANOVA Nonnormal 246.33 ± 35.36 162.3 ± 15.61 176.15 ± 12.88 3.255 0.0500 0.582 0.082 0.777 0.059 0.271 0.764 0.089 0.024 0.042 0.812 Startle 92 dB 2wANOVA Nonnormal 257.25 ± 40.4 192.63 ± 19.03 201.41 ± 23.01 2.153 0.1313 0.411 0.039 0.845 0.054 1.323 0.279 0.267 − − − Startle response Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Sound intensity rMeasures Sph.ass 2.900 0.0510 0.605 − − − - Sound intensity × gen. rMeasures Sph.ass 0.642 0.6620 0.219 − − − - Genotype rMeasures Sph.ass 3.649 0.0364 0.635 0.022 0.024 0.989 - Cohort rMeasures Sph.ass 1.842 0.1835 0.262 − − − - Sound intensity × gen. × coh. rMeasures Sph.ass 0.822 0.5335 0.276 − − − - Gen. × coh. rMeasures Sph.ass 1.922 0.1614 0.372 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Startle 74 dB norm. to weight 2wANOVA Nonnormal 7.01 ± 0.82 6.18 ± 0.64 6.17 ± 0.32 2.843 0.0718 0.522 5.749 0.022 0.645 2.780 0.076 0.512 0.045 0.042 0.959 Startle 78 dB norm. to weight 2wANOVA Nonnormal 7.19 ± 0.89 5.86 ± 0.64 6.35 ± 0.58 3.051 0.0601 0.553 2.972 0.094 0.389 3.611 0.038 0.630 0.038 0.036 0.964 Startle 82 dB norm. to weight 2wANOVA Nonnormal 7.74 ± 1.06 5.89 ± 0.41 6.98 ± 0.63 3.378 0.0456 0.599 1.700 0.201 0.245 2.365 0.109 0.446 0.015 0.089 0.409 Startle 86 dB norm. to weight 2wANOVA Nonnormal 9.86 ± 1.66 6 ± 0.49 7.03 ± 0.64 2.888 0.0690 0.529 0.030 0.864 0.053 0.268 0.766 0.089 0.028 0.070 0.677 Startle 92 dB norm. to weight 2wANOVA Nonnormal 10.04 ± 1.77 7.09 ± 0.58 8.18 ± 1.15 1.758 0.1873 0.343 0.323 0.573 0.086 1.291 0.288 0.261 − − − Startle response normalized to weight Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Sound intensity rMeasures Sph.ass 2.506 0.0449 0.248 − − − - Sound intensity × gen. rMeasures Sph.ass 0.580 0.7933 0.098 − − − - Genotype rMeasures Sph.ass 3.338 0.0471 0.593 0.127 0.407 0.783 - Cohort rMeasures Sph.ass 0.455 0.5046 0.101 − − − - Sound intensity × gen. × coh. rMeasures Sph.ass 0.716 0.5922 0.079 − − − - Gen. × coh. rMeasures Sph.ass 1.812 0.1783 0.353 − − − Pre-pulse inhibition (PPI) Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO PPI % (74 dB) 2wANOVA Normal 22.47 ± 5.7 15.1 ± 4.72 10.56 ± 6.03 0.625 0.5409 0.212 11.061 0.002 0.519 1.821 0.177 0.190 − − − PPI % (78 dB) 2wANOVA Normal 31.81 ± 4.48 14.84 ± 6.7 21.18 ± 5.03 3.513 0.0407 0.543 21.819 0.000 0.793 1.008 0.375 0.352 0.058 0.312 0.656 PPI % (82 dB) 2wANOVA Normal 30.39 ± 5.51 21.53 ± 6.08 13.34 ± 5.99 0.151 0.8604 0.426 14.892 0.000 0.480 0.042 0.959 0.348 − − − PPI % (86 dB) 2wANOVA Nonnormal 31.73 ± 7.95 21.04 ± 7.37 27.04 ± 4.63 1.411 0.2575 0.176 37.139 0.000 0.909 0.463 0.633 0.217 − − − PPI % (92 dB) 2wANOVA Nonnormal 43.62 ± 7.01 34.35 ± 5.82 29.93 ± 6.97 0.989 0.3821 0.212 54.683 0.000 0.892 2.258 0.120 0.222 − − − PPI % (average) 2wANOVA Normal 32 ± 5.53 21.37 ± 5.48 20.41 ± 5.03 0.155 0.8569 0.305 37.489 0.000 0.867 0.310 0.735 0.278 − − − PPI Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Sound intensity rMeasures Sph.viol 15.396 0.0000 1.000 − − − - Sound intensity × gen. rMeasures Sph.viol 1.409 0.1943 0.603 − − − - Genotype rMeasures Sph.viol 1.502 0.2325 0.305 − − − - Cohort rMeasures Sph.viol 9.806 0.0029 0.867 − − − - Sound intensity × gen. × coh. rMeasures Sph.viol 0.978 0.4540 0.427 − − − - Gen. × coh. rMeasures Sph.viol 1.349 0.2686 0.278 − − − Burried food test Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Latency to retrieve and eat food (s) 2wANOVA Nonnormal 51.11 ± 9.9 94.81 ± 23.69 500.28 ± 94.92 17.848 0.0000 1.000 0.001 0.976 0.050 0.000 1.000 0.050 0.858 0.000 0.000 Olfactory habituation/dishabituation, sniffing only Water Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 8.290 0.0019 0.958 − − − - Trial × gen. rMeasures Sph.viol 1.108 0.3505 0.337 − − − - Genotype rMeasures Sph.viol 2.973 0.0602 0.553 0.660 0.027 0.066 - Cohort rMeasures Sph.viol 0.073 0.7886 0.058 − − − - Trial × gen. × coh. rMeasures Sph.viol 1.739 0.1683 0.515 − − − - Gen. × coh. rMeasures Sph.viol 1.015 0.3696 0.217 − − − Banana Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.ass 10.117 0.0001 0.983 − − − - Trial × gen. rMeasures Sph.ass 3.908 0.0054 0.888 − − − - Genotype rMeasures Sph.ass 5.681 0.0060 0.842 0.433 0.002 0.017 - Cohort rMeasures Sph.ass 11.933 0.0011 0.923 − − − - Trial × gen. × coh. rMeasures Sph.ass 0.486 0.7461 0.161 − − − - Gen. × coh. rMeasures Sph.ass 0.134 0.8752 0.069 − − − Lemon Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 6.699 0.0041 0.908 − − − - Trial × gen. rMeasures Sph.viol 0.047 0.9890 0.059 − − − - Genotype rMeasures Sph.viol 2.715 0.0760 0.513 0.404 0.166 0.025 - Cohort rMeasures Sph.viol 15.327 0.0003 0.970 − − − - Trial × gen. × coh. rMeasures Sph.viol 0.667 0.5828 0.211 − − − - Gen. × coh. rMeasures Sph.viol 0.159 0.8534 0.073 − − − Male scent Test Data structure F pvalue Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 27.903 0.0000 1.000 − − − - Trial × gen. rMeasures Sph.viol 1.089 0.3581 0.332 − − − - Genotype rMeasures Sph.viol 0.739 0.4828 0.168 − − − - Cohort rMeasures Sph.viol 2.500 0.1201 0.341 − − − - Trial × gen. × coh. rMeasures Sph.viol 1.603 0.1976 0.479 − − − - Gen. × coh. rMeasures Sph.viol 0.541 0.5857 0.134 − − − Female scent Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 20.922 0.0000 1.000 − − − - Trial × gen. rMeasures Sph.viol 0.131 0.9321 0.076 − − − - Genotype rMeasures Sph.viol 0.585 0.5609 0.142 − − − - Cohort rMeasures Sph.viol 14.771 0.0003 0.965 − − − - Trial × gen. × coh. rMeasures Sph.viol 0.348 0.7765 0.126 − − − - Gen. × coh. rMeasures Sph.viol 1.651 0.2022 0.332 − − − Genotype Cohort Genotype × cohort Pairwisecomparisons Individual trials Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Water 1 2wANOVA Nonnormal 1.82 ± 0.37 1.81 ± 0.42 0.9 ± 0.16 2.205 0.1208 0.429 0.199 0.658 0.072 1.697 0.194 0.340 − − − Water 2 2wANOVA Nonnormal 1.02 ± 0.15 0.98 ± 0.14 0.7 ± 0.15 1.438 0.2470 0.294 0.078 0.781 0.059 0.711 0.496 0.163 − − − Water 3 2wANOVA Nonnormal 0.97 ± 0.15 0.82 ± 0.14 0.7 ± 0.11 1.724 0.1888 0.345 2.900 0.095 0.386 0.366 0.696 0.106 − − − Banana 1 2wANOVA Nonnormal 1.34 ± 0.12 1.04 ± 0.23 0.44 ± 0.15 8.742 0.0006 0.961 6.201 0.016 0.685 0.466 0.630 0.122 0.322 0.001 0.052 Banana 2 2wANOVA Nonnormal 0.65 ± 0.12 0.65 ± 0.1 0.44 ± 0.13 1.641 0.2040 0.330 6.658 0.013 0.716 0.240 0.787 0.086 − − − Banana 3 2wANOVA Nonnormal 0.52 ± 0.09 0.72 ± 0.19 0.44 ± 0.13 1.167 0.3196 0.245 7.761 0.008 0.780 0.050 0.951 0.057 − − − Lemon 1 2wANOVA Nonnormal 0.58 ± 0.18 0.77 ± 0.15 0.52 ± 0.17 0.720 0.4916 0.165 8.572 0.005 0.819 0.041 0.960 0.056 − − − Lemon 2 2wANOVA Nonnormal 0.39 ± 0.11 0.52 ± 0.1 0.24 ± 0.07 2.679 0.0785 0.507 9.784 0.003 0.866 0.037 0.964 0.055 0.709 0.411 0.099 Lemon 3 2wANOVA Nonnormal 0.35 ± 0.06 0.48 ± 0.09 0.25 ± 0.08 2.547 0.0885 0.486 7.405 0.009 0.761 2.493 0.093 0.478 0.594 0.540 0.106 Male 1 2wANOVA Nonnormal 6.1 ± 1.12 4.66 ± 0.88 4.94 ± 1.07 0.659 0.5219 0.154 1.095 0.300 0.177 1.091 0.344 0.231 − − − Male 2 2wANOVA Nonnormal 2.25 ± 0.34 2.77 ± 0.46 1.26 ± 0.31 4.095 0.0225 0.700 0.564 0.456 0.114 3.078 0.055 0.568 0.752 0.116 0.020 Male 3 2wANOVA Nonnormal 1.54 ± 0.38 1.25 ± 0.24 1.72 ± 0.63 0.322 0.7264 0.098 2.465 0.123 0.337 0.312 0.733 0.097 − − − Female 1 2wANOVA Nonnormal 5.68 ± 0.98 6.43 ± 1.34 6.32 ± 1.93 0.129 0.8790 0.069 11.509 0.001 0.914 0.879 0.422 0.193 − − − Female 2 2wANOVA Nonnormal 2.18 ± 0.3 2.24 ± 0.43 2.62 ± 0.81 0.189 0.8283 0.078 1.936 0.170 0.276 0.289 0.751 0.093 − − − Female 3 2wANOVA Nonnormal 1.42 ± 0.26 1.82 ± 0.52 2.76 ± 0.88 1.600 0.2120 0.323 7.672 0.008 0.775 1.792 0.177 0.357 − − − Olfactory habituation/dishabituation, all interactions Water Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 1.891 0.1664 0.385 − − − - Trial × gen. rMeasures Sph.viol 0.718 0.5478 0.225 − − − - Genotype rMeasures Sph.viol 7.210 0.0017 0.920 0.637 0.001 0.015 - Cohort rMeasures Sph.viol 2.639 0.1104 0.357 − − − - Trial × gen. × coh. rMeasures Sph.viol 0.920 0.4371 0.283 − − − - Gen. × coh. rMeasures Sph.viol 1.133 0.3300 0.239 − − − Banana Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 5.229 0.0133 0.821 − − − - Trial × gen. rMeasures Sph.viol 1.282 0.2866 0.388 − − − - Genotype rMeasures Sph.viol 5.737 0.0057 0.846 0.744 0.060 0.008 - Cohort rMeasures Sph.viol 7.922 0.0070 0.788 − − − - Trial × gen. × coh. rMeasures Sph.viol 0.936 0.4284 0.287 − − − - Gen. × coh. rMeasures Sph.viol 0.990 0.3787 0.213 − − − Lemon Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 1.303 0.2728 0.276 − − − - Trial × gen. rMeasures Sph.viol 0.703 0.5597 0.221 − − − - Genotype rMeasures Sph.viol 4.893 0.0115 0.781 0.295 0.048 0.003 - Cohort rMeasures Sph.viol 5.152 0.0276 0.605 − − − - Trial × gen. × coh. rMeasures Sph.viol 0.527 0.6738 0.172 − − − - Gen. × coh. rMeasures Sph.viol 2.405 0.1006 0.463 − − − Male scent Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.viol 28.652 0.0000 1.000 − − − - Trial × gen. rMeasures Sph.viol 0.790 0.4993 0.246 − − − - Genotype rMeasures Sph.viol 5.722 0.0057 0.845 0.839 0.005 0.022 - Cohort rMeasures Sph.viol 2.953 0.0918 0.392 − − − - Trial × gen. × coh. rMeasures Sph.viol 0.367 0.7697 0.131 − − − - Gen. × coh. rMeasures Sph.viol 0.009 0.9906 0.051 − − − Female scent Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Trial rMeasures Sph.ass 25.044 0.0000 1.000 − − − - Trial × gen. rMeasures Sph.ass 0.884 0.4762 0.264 − − − - Genotype rMeasures Sph.ass 1.119 0.3346 0.236 − − − - Cohort rMeasures Sph.ass 6.400 0.0145 0.699 − − − - Trial × gen. × coh. rMeasures Sph.ass 1.911 0.1143 0.541 − − − - Gen. × coh. rMeasures Sph.ass 0.431 0.6521 0.116 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Individual trials Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Water 1 2wANOVA Nonnormal 3.52 ± 0.9 3.88 ± 1 1.35 ± 0.27 2.357 0.1053 0.455 3.302 0.075 0.429 1.116 0.336 0.235 − − − Water 2 2wANOVA Nonnormal 3.45 ± 0.71 1.78 ± 0.48 0.95 ± 0.23 4.964 0.0109 0.786 0.588 0.447 0.117 1.295 0.283 0.267 0.161 0.006 0.364 Water 3 2wANOVA Nonnormal 2.53 ± 0.54 2.3 ± 0.6 0.95 ± 0.2 2.997 0.0592 0.556 0.033 0.856 0.054 0.373 0.690 0.107 0.949 0.078 0.147 Banana 1 2wANOVA Nonnormal 2.8 ± 0.42 3.38 ± 1.16 0.72 ± 0.43 3.854 0.0279 0.672 6.603 0.013 0.712 1.080 0.348 0.229 − − − Banana 2 2wANOVA Nonnormal 1.46 ± 0.49 1.06 ± 0.35 0.57 ± 0.18 1.902 0.1601 0.376 4.991 0.030 0.591 1.056 0.355 0.224 − − − Banana 3 2wANOVA Nonnormal 1.38 ± 0.55 2 ± 0.63 0.4 ± 0.1 3.927 0.0262 0.680 1.920 0.172 0.274 1.097 0.342 0.232 0.334 0.380 0.020 Lemon 1 2wANOVA Nonnormal 1.47 ± 0.37 2.21 ± 0.74 0.45 ± 0.19 3.707 0.0317 0.653 2.710 0.106 0.365 0.878 0.422 0.193 0.412 0.339 0.024 Lemon 2 2wANOVA Nonnormal 1.21 ± 0.35 0.98 ± 0.36 0.28 ± 0.09 2.536 0.0895 0.484 3.609 0.063 0.461 1.012 0.371 0.216 0.781 0.091 0.315 Lemon 3 2wANOVA Nonnormal 0.8 ± 0.18 0.61 ± 0.15 0.2 ± 0.08 1.362 0.2656 0.280 1.615 0.210 0.238 1.334 0.273 0.275 − − − Male 1 2wANOVA Nonnormal 55.55 ± 6.73 48.98 ± 7.22 27.15 ± 6.11 3.341 0.0436 0.605 2.419 0.126 0.332 0.054 0.948 0.058 0.655 0.030 0.199 Male 2 2wANOVA Nonnormal 27.09 ± 6.01 29.19 ± 5.33 7.48 ± 4.34 4.753 0.0130 0.768 1.196 0.280 0.189 0.012 0.988 0.052 0.986 0.020 0.030 Male 3 2wANOVA Nonnormal 17.57 ± 5.5 14.39 ± 4.43 5.26 ± 1.63 2.323 0.1087 0.449 1.329 0.255 0.204 0.978 0.383 0.210 − − − Female 1 2wANOVA Nonnormal 53.65 ± 5.97 50.59 ± 5.48 31.16 ± 7.21 1.781 0.1792 0.355 6.118 0.017 0.679 1.565 0.219 0.316 − − − Female 2 2wANOVA Nonnormal 23.26 ± 6.37 18.85 ± 6.77 14.61 ± 4.3 0.427 0.6551 0.115 2.116 0.152 0.297 0.350 0.707 0.103 − − − Female 3 2wANOVA Nonnormal 14.57 ± 3.85 11.5 ± 3.69 14.21 ± 4.61 0.269 0.7650 0.090 0.575 0.452 0.115 1.703 0.193 0.341 − − − WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 7-1. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort.
Three chambered social interaction test - social preference Zone comparison, three zones, time in chambers All mice Test Data structure F p value Power C vs M C vs O M vs O - Chamber rMeasures Sph.viol 149.525 0.0000 1.000 0.000 0.000 0.000 - Cohort rMeasures Sph.viol 1.456 0.2328 0.452 − − − - Chamber × cohort rMeasures Sph.viol 2.267 0.1149 0.220 − − − WT Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Chamber rMeasures Sph.ass 78.786 0.0000 1.000 0.000 0.001 0.000 - Cohort rMeasures Sph.ass 5.360 0.0342 0.585 − − − - Chamber × cohort rMeasures Sph.ass 1.546 0.2285 0.297 − − − Het Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Chamber rMeasures Sph.viol 61.909 0.0000 1.000 0.000 0.001 0.000 - Cohort rMeasures Sph.viol 1.252 0.2787 0.184 − − − - Chamber × cohort rMeasures Sph.viol 3.768 0.0508 0.543 − − − KO Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Chamber rMeasures Sph.ass 30.043 0.0000 1.000 0.000 0.002 0.000 - Cohort rMeasures Sph.ass 2.003 0.1751 0.267 − − − - Chamber × cohort rMeasures Sph.ass 0.227 0.7982 0.082 − − − Zone comparison, two zones, Mouse A vs object interaction time Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Chamber rMeasures Sph.ass 40.069 0.0000 1.000 10.622 0.005 0.864 14.120 0.002 0.943 19.123 0.000 0.984 - Cohort rMeasures Sph.ass 1.078 0.3038 0.175 0.561 0.465 0.109 3.631 0.769 0.059 0.434 0.519 0.095 - Chamber × cohort rMeasures Sph.ass 0.921 0.3414 0.156 0.002 0.963 0.050 0.089 0.074 0.436 0.617 0.443 0.115 Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Time in mouse or object chamber 2wANOVA Nonnormal 528.42 ± 10.99 509.46 ± 27.36 501.19 ± 14.27 0.428 0.6540 0.116 2.557 0.116 0.348 0.131 0.878 0.069 − − − Time sniffing mouse or object 2wANOVA Normal 89.02 ± 6.62 103.43 ± 10.43 101.27 ± 11.18 0.670 0.5160 0.156 2.367 0.130 0.326 1.153 0.324 0.242 − − − Time close to mouse or object 2wANOVA Normal 162.11 ± 8.5 168.73 ± 11.02 146.49 ± 10.45 1.165 0.3200 0.244 0.888 0.351 0.152 0.037 0.964 0.055 − − − Male-female social interactions, sniffing Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Anogenital, time (s) 2wANOVA Nonnormal 10.22 ± 1.62 12.36 ± 1.86 9.05 ± 1.43 0.933 0.4000 0.202 0.154 0.696 0.067 0.097 0.908 0.064 − − − Anogenital, number 2wANOVA Nonnormal 12.56 ± 3.44 14.28 ± 4.04 7.77 ± 2.36 1.049 0.3580 0.223 0.050 0.824 0.056 0.653 0.525 0.153 − − − Anogenital, latency to first (s) 2wANOVA Nonnormal 26.91 ± 11.44 19.61 ± 8.63 83.18 ± 25.05 4.238 0.0200 0.715 2.222 0.143 0.309 1.172 0.318 0.245 0.619 0.032 0.008 Nose to body, time (s) 2wANOVA Nonnormal 14.94 ± 2.05 14.94 ± 2.71 16.58 ± 2.86 0.348 0.7080 0.103 0.783 0.381 0.140 1.722 0.190 0.344 − − − Nose to body, number 2wANOVA Nonnormal 10.93 ± 1.54 10.83 ± 1.68 13.91 ± 4.35 0.333 0.7190 0.100 0.483 0.490 0.105 2.192 0.123 0.426 − − − Nose to body, latency to first (s) 2wANOVA Nonnormal 18.89 ± 5.38 20.94 ± 8.22 13.38 ± 3.97 0.332 0.7190 0.100 1.025 0.316 0.168 0.915 0.408 0.199 − − − Nose to nose, time (s) 2wANOVA Nonnormal 8.55 ± 0.85 10.73 ± 1.07 9.58 ± 1.13 0.133 0.8760 0.069 0.717 0.401 0.132 1.107 0.339 0.233 − − − Nose to nose number 2wANOVA Nonnormal 6.31 ± 0.79 6.91 ± 0.63 6.61 ± 1.18 1.118 0.3350 0.235 0.020 0.889 0.052 0.394 0.676 0.110 − − − Nose to nose, latency to first (s) 2wANOVA Nonnormal 34.72 ± 7.85 34.09 ± 9.24 16.58 ± 3.97 1.599 0.2130 0.322 0.062 0.804 0.057 0.417 0.661 0.114 − − − All sniffing, time (s) 2wANOVA Nonnormal 33.77 ± 3.7 38.05 ± 4.77 35.23 ± 4.63 0.155 0.8570 0.073 0.686 0.412 0.128 0.654 0.524 0.153 − − − All sniffing, number 2wANOVA Nonnormal 29.81 ± 4.59 32.03 ± 5.13 28.29 ± 6.45 0.313 0.7330 0.097 0.138 0.712 0.065 1.522 0.229 0.308 − − − All sniffing, latency to first (s) 2wANOVA Nonnormal 5.52 ± 1.85 4.29 ± 1.55 7.41 ± 2.92 0.586 0.5610 0.142 1.267 0.266 0.197 0.042 0.959 0.056 − − − Male-female social interactions, ultrasonic vocalization Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO USV, all calls 2wANOVA Nonnormal 380.11 ± 50.36 378.33 ± 64.78 287.58 ± 31.87 1.345 0.2704 0.276 3.242 0.078 0.422 0.193 0.825 0.078 − − − USV, minute 1 2wANOVA Nonnormal 84.16 ± 12.35 94.11 ± 21.67 64.7 ± 8.21 1.071 0.3507 0.227 1.180 0.283 0.186 0.865 0.428 0.190 − − − USV, minute 2 2wANOVA Nonnormal 68.11 ± 9.01 73.22 ± 14.22 57.41 ± 5.55 0.649 0.5271 0.152 1.150 0.289 0.183 0.070 0.932 0.060 − − − USV, minute 3 2wANOVA Nonnormal 77.61 ± 11.62 68.72 ± 8.59 57.7 ± 8.2 1.363 0.2659 0.279 2.155 0.149 0.301 0.799 0.456 0.178 − − − USV, minute 4 2wANOVA Nonnormal 74.5 ± 14.48 76.44 ± 13 52.7 ± 4.98 1.566 0.2197 0.316 4.139 0.048 0.513 0.276 0.760 0.091 − − − USV, minute 5 2wANOVA Nonnormal 75.72 ± 14.21 65.83 ± 11.73 55.05 ± 8.13 1.092 0.3439 0.230 5.269 0.026 0.613 0.049 0.952 0.057 − − − USV Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Time rMeasures Sph.viol 2.964 0.0210 0.785 − − − - Time × genotype rMeasures Sph.viol 0.558 0.8110 0.254 − − − - Genotype rMeasures Sph.viol 1.345 0.2704 0.276 − − − - Cohort rMeasures Sph.viol 3.242 0.0782 0.422 − − − - Time × gen. × coh. rMeasures Sph.viol 1.245 0.2750 0.564 − − − - Gen. × coh. rMeasures Sph.viol 0.193 0.8248 0.078 − − − Social transmission of food preference Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Demonstrator sniffing time (s) 2wANOVA Nonnormal 29.8 ± 6.01 37.44 ± 6.25 24.69 ± 6.39 0.756 0.4752 0.171 4.407 0.041 0.538 0.202 0.818 0.080 − − − Number of sniffing bouts 2wANOVA Nonnormal 9.68 ± 1.51 13.57 ± 1.48 7.26 ± 1.36 4.064 0.0236 0.695 2.772 0.103 0.371 0.099 0.906 0.064 0.126 0.733 0.021 Time exploring all food (s) 2wANOVA Nonnormal 1533.36 ± 98.47 1456.68 ± 98.14 1715.26 ± 124.97 1.372 0.2635 0.281 0.361 0.551 0.091 0.216 0.806 0.082 − − − Time pre-exposed/all food (%) 2wANOVA Nonnormal 64.89 ± 3.55 59.21 ± 4.8 64.54 ± 4.71 0.589 0.5589 0.142 0.150 0.700 0.067 1.790 0.178 0.356 − − − Time new/all food (%) 2wANOVA Nonnormal 35.1 ± 3.55 40.78 ± 4.8 35.45 ± 4.71 0.589 0.5589 0.142 0.150 0.700 0.067 1.790 0.178 0.356 − − − Ratio time pre-exposed/new 2wANOVA Nonnormal 2.6 ± 0.45 2.31 ± 0.47 3.06 ± 0.64 0.636 0.5338 0.150 1.077 0.305 0.174 0.739 0.483 0.168 − − − Time spent exploring cocoa/all food (%) 2wANOVA Nonnormal 51.21 ± 4.98 50.65 ± 5.26 50.97 ± 5.82 0.003 0.9969 0.050 0.240 0.626 0.077 0.856 0.431 0.188 − − − Time exploring cinnamon/all food (%) 2wANOVA Nonnormal 48.78 ± 4.98 49.34 ± 5.26 49.02 ± 5.82 0.003 0.9969 0.050 0.240 0.626 0.077 0.856 0.431 0.188 − − − Ratio time cocoa/cinnamon 2wANOVA Nonnormal 1.55 ± 0.31 1.67 ± 0.4 2.07 ± 0.64 0.585 0.5611 0.141 0.769 0.385 0.138 0.433 0.651 0.116 − − − Total amount of eaten food (g) 2wANOVA Nonnormal 1.21 ± 0.18 0.82 ± 0.15 0.58 ± 0.06 4.286 0.0195 0.720 1.848 0.180 0.265 0.726 0.489 0.166 0.146 0.011 0.491 Amount of eaten food, pre-exposed (g) 2wANOVA Nonnormal 0.87 ± 0.14 0.67 ± 0.14 0.46 ± 0.07 2.346 0.1068 0.452 2.802 0.101 0.375 1.058 0.355 0.224 − − − Amount of eaten food, new (g) 2wANOVA Nonnormal 0.34 ± 0.08 0.15 ± 0.04 0.13 ± 0.03 4.130 0.0223 0.703 0.063 0.803 0.057 1.108 0.339 0.233 0.065 0.048 0.990 Amount of eaten food, cocoa (g) 2wANOVA Nonnormal 0.59 ± 0.13 0.36 ± 0.11 0.3 ± 0.07 1.887 0.1629 0.373 0.343 0.561 0.089 0.720 0.492 0.165 − − − Amount of eaten food, cinnamon (g) 2wANOVA Nonnormal 0.62 ± 0.13 0.46 ± 0.13 0.3 ± 0.05 1.563 0.2202 0.315 1.125 0.294 0.180 0.165 0.849 0.074 − − − Percentage pre-exposed vs new Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Flavor rMeasures Sph.ass 25.686 0.0000 0.999 18.792 0.000 0.983 4.601 0.045 0.433 9.230 0.007 0.817 - Cohort rMeasures Sph.ass 0.000 1.0000 0.050 0.800 0.384 NA 0.196 0.663 NA 0.531 0.476 0.106 - Flavor × cohort rMeasures Sph.ass 0.009 0.9227 0.051 6.593 0.020 0.678 0.195 0.665 0.070 1.137 0.301 0.172 Time cacao vs cinnamon Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Flavor rMeasures Sph.ass 0.001 0.9745 0.050 0.058 0.812 0.050 0.035 0.854 0.054 0.058 0.812 0.063 - Cohort rMeasures Sph.ass 0.100 0.7525 0.061 0.080 0.780 0.135 0.196 0.663 0.070 0.080 0.780 0.058 - Flavor × cohort rMeasures Sph.ass 0.001 0.9702 0.050 0.004 0.950 0.178 0.957 0.342 0.152 0.004 0.950 0.050 Amount of eaten food, pre-expose vs new Test Data structure All F All p value Power WT F WT p value Power Het F Het pvalue Power KO F KO p value Power - Flavor rMeasures Sph.ass 42.099 0.0000 1.000 13.852 0.002 0.935 13.378 0.002 0.929 13.503 0.002 0.931 - Cohort rMeasures Sph.ass 2.399 0.1276 0.330 0.400 0.537 0.091 2.323 0.147 0.299 0.131 0.722 0.063 - Flavor × cohort rMeasures Sph.ass 3.445 0.0692 0.445 4.346 0.055 0.496 0.872 0.364 0.142 0.080 0.781 0.058 Amount of eaten food, cacao vs cinnamon Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Flavor rMeasures Sph.ass 0.212 0.6473 0.074 0.005 0.945 0.050 0.265 0.614 0.077 0.011 0.918 0.051 - Cohort rMeasures Sph.ass 2.399 0.1276 0.330 0.400 0.537 0.091 2.323 0.147 0.299 0.131 0.722 0.063 - Flavor × cohort rMeasures Sph.ass 0.117 0.7342 0.063 0.178 0.679 0.068 0.052 0.822 0.055 0.271 0.610 0.078 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. C, center chamber; M, mouse chamber; O, object chamber. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 8-1. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort.
Novel object habituation Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Total distance (cm) 2wANOVAA Nonnormal 3616.84 ± 351.4 3111.83 ± 221.31 3118.64 ± 277.29 0.724 0.490 0.166 15.022 0.000 0.967 0.927 0.402 0.202 − − − Time in left side (s) 2wANOVA Normal 314.74 ± 20.15 306.42 ± 15.26 276.81 ± 23.06 1.079 0.348 0.229 0.100 0.753 0.061 2.014 0.144 0.397 − − − Time in right side (s) 2wANOVA Normal 284.74 ± 20.17 293.06 ± 15.23 322.76 ± 23.1 1.086 0.345 0.230 0.112 0.739 0.062 2.009 0.145 0.396 − − − Time spend in left vs right half side Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Side rMeasures Sph.ass 0.001 0.979 0.052 0.445 0.514 0.097 0.145 0.708 0.065 1.249 0.279 0.184 - Cohort rMeasures Sph.ass 12.824 0.001 0.937 3.326 0.086 0.406 10.401 0.005 0.860 3.035 0.100 0.376 - Side × cohort rMeasures Sph.ass 0.044 0.835 0.062 0.096 0.761 0.060 1.352 0.261 0.195 2.506 0.132 0.321 Novel object recognition: training with two identical objects Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vsHet WT vs KO Het vs KO Total distance (cm) 2wANOVA Nonnormal 2421.86 ± 196.94 2175 ± 173.66 1631.78 ± 118.14 5.366 0.008 0.820 2.961 0.091 0.393 0.804 0.453 0.180 0.540 0.004 0.059 Time in left side (s) 2wANOVA Normal 150.86 ± 9.32 148.91 ± 10.25 176.59 ± 16.36 1.489 0.235 0.303 0.040 0.843 0.054 0.905 0.411 0.198 − − − Time in right side (s) 2wANOVA Normal 148.86 ± 9.35 150.82 ± 10.23 122.95 ± 16.33 1.511 0.230 0.307 0.042 0.839 0.055 0.896 0.414 0.196 − − − Number of side switches 2wANOVA Normal 15.94 ± 1.28 14.26 ± 1 13.89 ± 1.21 0.822 0.445 0.183 0.259 0.613 0.079 0.459 0.635 0.121 − − − Number of left object exploration 2wANOVA Nonnormal 21.84 ± 2.83 19.52 ± 2.54 14.1 ± 1.67 2.641 0.081 0.502 0.335 0.565 0.088 1.071 0.350 0.227 0.777 0.070 0.261 Number of right object exploration 2wANOVA Nonnormal 33.42 ± 7.21 28.84 ± 7.87 16.78 ± 2.65 1.558 0.220 0.316 2.295 0.136 0.318 0.443 0.645 0.118 − − − Time exploring left object (s) 2wANOVA Nonnormal 22.2 ± 3.15 19.89 ± 3.1 10.67 ± 2.11 4.527 0.015 0.747 0.536 0.467 0.111 0.993 0.377 0.214 0.834 0.016 0.066 Time exploring right object (s) 2wANOVA Nonnormal 23.97 ± 3.28 23.94 ± 4.06 11.28 ± 2.37 5.322 0.008 0.817 2.582 0.114 0.351 0.277 0.759 0.092 1.000 0.024 0.025 Latency to left object (s) 2wANOVA Nonnormal 15.89 ± 4.3 21.74 ± 7.62 20.67 ± 3.94 0.353 0.704 0.104 0.068 0.796 0.057 0.138 0.872 0.070 − − − Latency to right object (s) 2wANOVA Nonnormal 15.03 ± 3.6 13.99 ± 5.42 17.92 ± 3.01 0.348 0.708 0.103 3.325 0.074 0.432 0.816 0.448 0.182 − − − Total number of object exploration 2wANOVA Nonnormal 55.26 ± 9.69 48.36 ± 9.86 30.89 ± 4.09 1.992 0.147 0.393 0.959 0.332 0.161 0.596 0.555 0.144 − − − Total time exploring objects (s) 2wANOVA Nonnormal 46.18 ± 5.82 43.83 ± 6.59 21.95 ± 4.26 5.733 0.006 0.846 1.680 0.201 0.246 0.041 0.960 0.056 0.954 0.011 0.024 Latency to any object (s) 2wANOVA Nonnormal 8.92 ± 3.11 13.43 ± 5.8 17.5 ± 3.36 0.674 0.514 0.157 1.455 0.233 0.220 0.482 0.621 0.125 − − − Sniffing time,left vs right (s) Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Side rMeasures Sph.ass 2.505 0.119 0.343 1.375 0.257 0.198 1.701 0.209 0.234 0.128 0.725 0.063 - Cohort rMeasures Sph.ass 0.943 0.336 0.159 0.245 0.627 0.075 0.607 0.447 0.114 1.226 0.284 0.182 - Side × cohort rMeasures Sph.ass 1.598 0.211 0.237 7.358 0.015 0.725 0.829 0.375 0.138 1.756 0.203 0.240 Number of interactions, left vs right Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Side rMeasures Sph.ass 8.030 0.006 0.795 4.264 0.055 0.495 2.075 0.168 0.275 3.519 0.078 0.425 - Cohort rMeasures Sph.ass 1.277 0.263 0.199 1.149 0.299 0.173 0.375 0.548 0.089 0.311 0.585 0.082 - Side × cohort rMeasures Sph.ass 6.312 0.015 0.694 1.703 0.209 0.234 2.023 0.173 0.269 6.059 0.025 0.641 Time in left vs right halves (s) Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Side rMeasures Sph.ass 1.555 0.218 0.232 0.000 0.994 0.050 0.013 0.909 0.051 2.506 0.132 0.321 - Cohort rMeasures Sph.ass 0.591 0.445 0.117 0.142 0.711 0.065 3.906 0.065 0.462 0.161 0.693 0.067 - Side × cohort rMeasures Sph.ass 0.110 0.742 0.062 0.371 0.551 0.089 0.224 0.642 0.073 0.856 0.368 0.141 Novel object recognition: test with one new object Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Total distance (cm) 2wANOVA Normal 1973.56 ± 156.94 1482.1 ± 167.46 1057.83 ± 110.93 9.082 0.000 0.968 0.000 0.989 0.050 1.066 0.352 0.227 0.059 0.000 0.117 Time in new object side (s) 2wANOVA Normal 151.59 ± 9.78 143.64 ± 15.18 174.14 ± 19.84 0.985 0.380 0.212 0.004 0.947 0.050 0.545 0.583 0.135 − − − Time pre-exposed object side (s) 2wANOVA Normal 147.79 ± 9.8 155.86 ± 15.26 125.22 ± 19.97 0.983 0.381 0.212 0.001 0.972 0.050 0.533 0.590 0.133 − − − Number of side switches 2wANOVA Normal 14.57 ± 0.77 9.84 ± 1.32 8.52 ± 1.05 8.853 0.001 0.964 0.422 0.519 0.098 1.128 0.332 0.238 0.009 0.001 0.666 Number of new object exploration 2wANOVA Nonnormal 22.73 ± 2.66 17.84 ± 2.68 6.26 ± 0.93 14.115 0.000 0.998 1.316 0.257 0.203 0.241 0.787 0.086 0.289 0.000 0.002 Number of pre-exposed object exploration 2wANOVA Nonnormal 18.52 ± 4.35 13.05 ± 2.92 5.78 ± 0.76 3.898 0.027 0.678 1.389 0.244 0.212 1.540 0.224 0.312 0.413 0.012 0.216 Time exploring new object (s) 2wANOVA Nonnormal 27.84 ± 3.58 21.74 ± 2.89 6.04 ± 1.39 20.724 0.000 1.000 11.516 0.001 0.915 0.182 0.835 0.077 0.225 0.000 0.000 Time exploring pre-exposed object (s) 2wANOVA Nonnormal 12.49 ± 2.05 12.03 ± 2.55 3.8 ± 0.78 6.051 0.004 0.866 0.047 0.829 0.055 0.351 0.706 0.103 0.985 0.009 0.014 Latency new object (s) 2wANOVA Nonnormal 16.68 ± 5.17 50.97 ± 18.18 72.65 ± 14.88 3.295 0.045 0.600 1.589 0.213 0.236 0.299 0.743 0.095 0.193 0.043 0.753 Latency to pre-exposed object (s) 2wANOVA Nonnormal 30.73 ± 8.99 49.62 ± 18.45 68.71 ± 13.88 1.728 0.188 0.346 0.164 0.687 0.068 0.245 0.783 0.087 − − − Total number of object exploration 2wANOVA Nonnormal 41.26 ± 6.59 30.89 ± 5.37 12.05 ± 1.53 8.267 0.001 0.952 0.036 0.850 0.054 0.920 0.405 0.200 0.321 0.000 0.029 Total time exploringobjects (s) 2wANOVA Nonnormal 40.33 ± 5.04 33.77 ± 4.55 9.85 ± 1.88 17.130 0.000 1.000 5.284 0.026 0.616 0.009 0.991 0.051 0.481 0.000 0.000 Novel object, latency to observe any object (s) 2wANOVA Nonnormal 14.17 ± 4.79 16.53 ± 7.27 41.36 ± 11.68 2.538 0.089 0.485 3.004 0.089 0.398 0.853 0.432 0.188 0.581 0.081 0.459 Sniffing time, new vs pre-exposed object Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Side rMeasures Sph.ass 37.818 0.000 1.000 37.629 0.000 1.000 13.312 0.002 0.930 3.302 0.087 0.403 - Cohort rMeasures Sph.ass 2.146 0.149 0.302 1.221 0.285 0.181 1.164 0.296 0.175 14.732 0.001 0.951 - Side × cohort rMeasures Sph.ass 8.100 0.006 0.798 5.648 0.029 0.611 4.628 0.046 0.528 3.660 0.073 0.439 Number of interactions, new vs pre-exposed Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Side rMeasures Sph.ass 9.499 0.003 0.857 3.824 0.067 0.454 11.442 0.004 0.890 0.314 0.582 0.083 - Cohor rMeasures Sph.ass 0.221 0.640 0.075 0.567 0.462 0.110 0.045 0.835 0.055 16.708 0.001 0.970 - Side × cohort rMeasures Sph.ass 9.882 0.003 0.870 5.751 0.028 0.618 5.258 0.035 0.580 1.686 0.211 0.232 Time in new vs pre-exposed halves (s) Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Side rMeasures Sph.ass 0.565 0.456 0.114 0.029 0.867 0.053 0.185 0.673 0.069 1.375 0.257 0.198 - Cohort rMeasures Sph.ass 5.062 0.028 0.599 2.758 0.115 0.347 2.527 0.130 0.323 1.348 0.262 0.195 - Side × cohort rMeasures Sph.ass 0.020 0.889 0.052 0.010 0.920 0.051 0.446 0.513 0.097 0.397 0.537 0.091 Marble burying Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Number of burried marbles 2wANOVA Nonnormal 13.63 ± 1.29 13.78 ± 1 3.77 ± 1.07 18.723 0.000 1.000 0.069 0.793 0.217 0.370 0.693 0.051 0.995 0.000 0.000 Repetitive novel object contact task, exploration Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Time exploring all the objects 2wANOVA Nonnormal 82.08 ± 11.28 84.8 ± 7 53.37 ± 5.01 7.964 0.001 0.943 24.654 0.000 0.998 0.647 0.528 0.152 0.956 0.014 0.006 Total number of object interactions 2wANOVA Normal 83.27 ± 7.92 94.21 ± 6.22 83.47 ± 6.4 2.108 0.133 0.412 73.475 0.000 1.000 1.110 0.338 0.234 − − − Nest building Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Nest shredded 2wANOVA Nonnormal 1.84 ± 0.08 1.94 ± 0.05 1.36 ± 0.15 7.785 0.001 0.939 2.814 0.100 0.377 0.364 0.697 0.105 0.455 0.005 0.000 Nest dispersion 2wANOVA Nonnormal 1.94 ± 0.05 1.94 ± 0.05 1.73 ± 0.14 1.580 0.216 0.320 0.919 0.342 0.156 3.425 0.040 0.618 − − − Nest density 2wANOVA Nonnormal 1.26 ± 0.14 0.73 ± 0.16 0.73 ± 0.21 2.726 0.075 0.515 0.393 0.534 0.094 0.266 0.768 0.090 0.050 0.046 0.966 Nest shape 2wANOVA Nonnormal 2.57 ± 0.19 2.05 ± 0.23 1.36 ± 0.27 5.851 0.005 0.854 3.065 0.086 0.404 0.097 0.908 0.064 0.153 0.001 0.055 Nest walls 2wANOVA Nonnormal 1.21 ± 0.14 1 ± 0.18 0.42 ± 0.17 5.649 0.006 0.840 0.097 0.756 0.061 0.833 0.440 0.185 0.359 0.002 0.023 Nest total score 2wANOVA Nonnormal 8.84 ± 0.45 7.68 ± 0.51 5.63 ± 0.73 7.223 0.002 0.921 1.121 0.295 0.180 0.020 0.980 0.053 0.184 0.000 0.019 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 9-1. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Sph.ass: sphericity assumed, gen: genotype, coh: cohort.
- Table 10.
Detailed results and statistical analyses related to the hyper-reactivity and escape behavior
Reflexes and reactions to simple stimuli Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Touch escape 2wANOVA Nonnormal 1.26 ± 0.1 1.15 ± 0.11 2 ± 0.15 12.962 0.000 0.996 0.046 0.831 0.055 0.862 0.428 0.190 0.648 0.000 0.000 Positional passivity (sum) 2wANOVA Nonnormal 2.15 ± 0.25 1.84 ± 0.23 2.84 ± 0.2 11.737 0.000 0.992 106.722 0.000 1.000 1.993 0.147 0.393 0.034 0.011 0.000 Positional passivity (score) 2wANOVA Nonnormal 1.78 ± 0.24 2.21 ± 0.21 0.94 ± 0.2 14.029 0.000 0.998 44.935 0.000 1.000 3.871 0.027 0.675 0.034 0.004 0.000 Catalepsy (4 trials) 2wANOVA Nonnormal 2.98 ± 0.57 2.75 ± 0.54 0.56 ± 0.25 7.578 0.001 0.933 4.681 0.035 0.565 1.116 0.336 0.236 0.836 0.001 0.002 Trunk curl 2wANOVA Nonnormal 1 ± 0 0.89 ± 0.07 1 ± 0 2.547 0.088 0.487 2.537 0.117 0.346 2.547 0.088 0.487 0.057 1.000 0.056 Negative geotaxis, latency to turn 2wANOVA Nonnormal 6.73 ± 1.06 8.21 ± 1.9 3.22 ± 0.58 4.201 0.020 0.713 2.978 0.090 0.395 0.707 0.498 0.163 0.499 0.042 0.008 Beam walking Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vsHet WT vsKO Het vs KO Latency to cross (large, s) 2wANOVA Nonnormal 8.34 ± 2.79 10.8 ± 4.52 3.96 ± 1.75 1.163 0.321 0.244 0.251 0.618 0.078 1.300 0.281 0.269 − − − Latency to cross (medium, s) 2wANOVA Nonnormal 15.9 ± 4.8 7.17 ± 4.16 7.65 ± 6.13 0.811 0.450 0.181 3.349 0.073 0.435 0.121 0.887 0.068 − − − Latency to cross (small, s) 2wANOVA Nonnormal 54.71 ± 5.89 46.15 ± 7.59 33.72 ± 7.84 2.204 0.121 0.430 0.235 0.630 0.076 0.437 0.648 0.117 − − − Escape behavior Genotype Cohort Genotype × cohort Pairwise comparisons Test Datastructure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Buried food, number of attempts 2wANOVA Nonnormal 0.63 ± 0.35 0.68 ± 0.32 0.42 ± 0.31 0.666 0.519 0.155 1.738 0.194 0.253 0.220 0.804 0.082 Buried food, % of escapers 2wANOVA Nonnormal 21.05 ± 9.6 26.31 ± 10.37 15.78 ± 8.59 0.760 0.473 0.172 3.639 0.062 0.464 0.159 0.853 0.073 Four-object, number of attempts 2wANOVA Nonnormal 0.41 ± 0.21 2.05 ± 0.73 3.88 ± 1.21 5.323 0.008 0.815 5.320 0.025 0.618 3.316 0.045 0.601 0.187 0.002 0.050 Four-object, % of escapers 2wANOVA Nonnormal 23.52 ± 10.6 36.84 ± 11.36 50 ± 12.12 1.502 0.233 0.305 4.351 0.042 0.534 2.575 0.087 0.490 Marble burying, number of attempts 2wANOVA Nonnormal 4.32 ± 1.28 10.63 ± 1.98 16.05 ± 2.38 8.063 0.001 0.946 6.649 0.013 0.715 1.239 0.299 0.257 0.034 0.000 0.055 Marble burying, % of escapers 2wANOVA Nonnormal 47.36 ± 11.76 94.73 ± 5.26 100 ± 0 12.009 0.000 0.993 7.713 0.008 0.777 4.474 0.017 0.740 0.000 0.000 0.598 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Red font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 10-1. 2wANOVA: 2-way ANOVA.
- Table 11.
Detailed results and statistical analyses related to stereotypies, repetitive behavior, perseveration, and cognitive flexibility
Sterotypies in open field Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Grooming, time (s) 2wANOVA Nonnormal 67.01 ± 7.01 62.6 ± 5.58 92.49 ± 10.45 4.929 0.011 0.784 22.806 0.000 0.997 2.530 0.090 0.484 0.883 0.023 0.006 Grooming, number 2wANOVA Normal 25.42 ± 1.51 22.26 ± 1.5 27.1 ± 1.93 2.000 0.146 0.394 1.402 0.242 0.213 1.745 0.185 0.349 − − − Jumping, time (s) 2wANOVA Nonnormal 0.07 ± 0.05 0 ± 0 0.18 ± 0.1 1.666 0.199 0.335 0.033 0.857 0.054 1.149 0.325 0.242 − − − Jumping, number 2wANOVA Nonnormal 0.36 ± 0.23 0 ± 0 0.42 ± 0.23 1.300 0.281 0.269 0.155 0.696 0.067 1.816 0.173 0.362 − − − Rotation, time (s) 2wANOVA Nonnormal 0.39 ± 0.1 1.49 ± 0.81 4.21 ± 2.76 1.560 0.220 0.316 2.069 0.156 0.292 1.038 0.361 0.222 − − − Rotation, number 2wANOVA Nonnormal 1.63 ± 0.39 2.21 ± 0.46 6.15 ± 1.82 5.883 0.005 0.856 3.301 0.075 0.430 3.022 0.057 0.561 0.920 0.010 0.028 Twitching/shaking, time (s) 2wANOVA Nonnormal 0.28 ± 0.07 0.69 ± 0.33 0.63 ± 0.1 1.089 0.344 0.231 0.540 0.466 0.111 0.879 0.422 0.193 − − − Twitching/shaking, number 2wANOVA Nonnormal 1.73 ± 0.42 2.63 ± 0.88 3 ± 0.47 1.194 0.311 0.250 1.484 0.229 0.223 0.589 0.559 0.143 − − − Repetitive novel object contact task, object preference, time Object exploration, time (s) Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Object rMeasures Sph.viol 10.533 0.000 0.999 − − − - Object × gen. rMeasures Sph.viol 2.150 0.069 0.753 − − − - Genotype rMeasures Sph.viol 7.964 0.001 0.943 0.956 0.014 0.006 - Cohort rMeasures Sph.viol 24.654 0.000 0.998 − − − - Object × gen. × coh. rMeasures Sph.viol 0.366 0.859 0.152 − − − - Gen. × coh. rMeasures Sph.viol 0.647 0.528 0.152 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Object exploration Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Dice, time (s) 2wANOVA Nonnormal 16.51 ± 2.49 14.82 ± 1.66 12.14 ± 1.79 1.748 0.185 0.349 4.640 0.036 0.560 0.833 0.441 0.185 − − − Jack, time (s) 2wANOVA Nonnormal 21.78 ± 3.18 27.99 ± 5.1 15.39 ± 2 4.078 0.023 0.697 14.158 0.000 0.958 0.500 0.610 0.127 0.311 0.443 0.025 Lego, time (s) 2wANOVA Nonnormal 24.91 ± 3.59 28.25 ± 2.94 14.97 ± 1.87 8.622 0.001 0.959 20.965 0.000 0.994 0.774 0.467 0.174 0.509 0.031 0.001 Pin, time (s) 2wANOVA Nonnormal 20.8 ± 4.3 13.72 ± 1.82 10.86 ± 1.7 3.199 0.050 0.585 4.532 0.038 0.550 0.057 0.944 0.058 0.244 0.067 0.755 Object exploration, % Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Object rMeasures Sph.ass 8.329 0.000 0.985 − − − - Object × gen. rMeasures Sph.ass 0.721 0.633 0.259 − − − - Genotype rMeasures Sph.ass 0.750 0.478 0.170 − − − - Cohort rMeasures Sph.ass 0.000 1.000 0.050 − − − - Object × gen. × coh. rMeasures Sph.ass 0.652 0.688 0.236 − − − - Gen. × coh. rMeasures Sph.ass 0.000 1.000 0.050 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Object exploration Test Data structure WT Het KO F p value Power F pvalue Power F p value Power WT vs Het WT vs KO Het vsKO Dice, time (%) 2wANOVA Nonnormal 20.09 ± 1.78 19.13 ± 2.11 24.11 ± 3.72 1.259 0.293 0.261 4.635 0.036 0.560 1.179 0.316 0.246 − − − Jack, time (%) 2wANOVA Nonnormal 26.86 ± 3.3 30.29 ± 3.61 28.38 ± 3.26 0.221 0.803 0.083 1.529 0.222 0.228 0.022 0.978 0.053 − − − Lego, time (%) 2wANOVA Normal 29.85 ± 2.19 32.98 ± 2.28 27.98 ± 2.5 1.174 0.318 0.245 0.986 0.326 0.164 0.072 0.931 0.060 − − − Pin, time (%) 2wANOVA Nonnormal 23.18 ± 3.02 17.57 ± 2.65 19.51 ± 2.32 0.701 0.501 0.161 0.186 0.669 0.071 1.684 0.196 0.337 − − − Object exploration ranked by preference Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Object rMeasures Sph.viol 110.887 0.000 1.000 − − − - Object × gen. rMeasures Sph.viol 5.483 0.002 0.996 − − − - Genotype rMeasures Sph.viol 8.054 0.001 0.946 0.948 0.014 0.006 - Cohort rMeasures Sph.viol 24.578 0.000 0.998 − − − - Object × gen. × coh. rMeasures Sph.viol 1.187 0.321 0.457 − − − - Gen. × coh. rMeasures Sph.viol 0.643 0.530 0.152 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Object exploration Test Data structure WT Het KO F p value Power F p value Power F pvalue Power WT vs Het WT vs KO Het vs KO Object #1, time (s) 2wANOVA Nonnormal 31.83 ± 3.59 37.45 ± 4.09 20.53 ± 1.64 9.051 0.000 0.967 15.093 0.000 0.968 0.934 0.400 0.202 0.264 0.048 0.001 Object #2, time (s) 2wANOVA Nonnormal 24.5 ± 3.27 22.95 ± 2.14 15.23 ± 1.78 6.709 0.003 0.899 22.529 0.000 0.996 0.456 0.637 0.120 0.941 0.016 0.033 Object #3, time (s) 2wANOVA Nonnormal 15.68 ± 2.69 15.29 ± 1.56 10.48 ± 1.15 4.224 0.020 0.714 20.037 0.000 0.992 1.306 0.280 0.269 0.998 0.094 0.102 Object #4, time (s) 2wANOVA Nonnormal 12 ± 2.41 9.34 ± 0.96 7.12 ± 0.99 3.763 0.030 0.660 12.155 0.001 0.927 0.630 0.537 0.149 0.451 0.074 0.533 Object exploration,ranked by preference Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Object rMeasures Sph.viol 146.534 0.000 1.000 − − − - Object × gen. rMeasures Sph.viol 0.832 0.490 0.321 − − − - Genotype rMeasures Sph.viol 0.812 0.450 0.181 − − − - Cohort rMeasures Sph.viol 0.783 0.381 0.140 − − − - Object × gen. × coh. rMeasures Sph.viol 1.054 0.377 0.407 − − − - Gen. × coh. rMeasures Sph.viol 0.812 0.450 0.181 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Object exploration Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Object #1, time (%) 2wANOVA Nonnormal 39.5 ± 2.19 43 ± 1.71 40.58 ± 2.74 1.029 0.365 0.219 1.745 0.193 0.254 0.963 0.389 0.207 − − − Object #2, time (%) 2wANOVA Nonnormal 28.98 ± 1.16 27.21 ± 1.2 27.58 ± 1.39 0.805 0.453 0.180 1.231 0.273 0.193 2.400 0.102 0.461 − − − Object #3, time (%) 2wANOVA Normal 17.99 ± 1.18 18.73 ± 1.21 19.28 ± 1.12 0.093 0.911 0.063 0.228 0.635 0.076 0.347 0.709 0.102 − − − Object #4, time (s) 2wANOVA Normal 13.51 ± 0.92 11.62 ± 0.99 12.54 ± 1.16 1.091 0.344 0.230 0.423 0.518 0.098 0.747 0.479 0.169 − − − Repetitive novel object contact task, object preference, number Object interactions, number Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Object rMeasures Sph.viol 2.653 0.051 0.638 − − − - Object × gen. rMeasures Sph.viol 0.858 0.528 0.331 − − − - Genotype rMeasures Sph.viol 2.108 0.133 0.412 − − − - Cohort rMeasures Sph.viol 73.475 0.000 1.000 − − − - Object × gen. × coh. rMeasures Sph.viol 0.459 0.837 0.184 − − − - Gen. × coh. rMeasures Sph.viol 1.110 0.338 0.234 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Exploration numbers Test Data structure WT Het KO F p value Power F pvalue Power F pvalue Power WT vs Het WT vs KO Het vsKO Dice 2wANOVA Nonnormal 20.17 ± 1.7 21.05 ± 1.39 20.76 ± 1.69 0.054 0.948 0.058 15.707 0.000 0.973 0.620 0.542 0.148 − − − Jack 2wANOVA Normal 20.17 ± 2.46 23.42 ± 2.38 20.94 ± 1.95 1.196 0.311 0.249 72.111 0.000 1.000 1.200 0.310 0.250 − − − Lego 2wANOVA Normal 23.94 ± 2.46 26.31 ± 1.71 20.82 ± 2 2.785 0.072 0.523 17.510 0.000 0.984 0.367 0.695 0.106 0.495 0.573 0.091 Pin 2wANOVA Normal 20.17 ± 2.46 23.42 ± 2.38 20.94 ± 1.95 1.196 0.311 0.249 72.111 0.000 1.000 1.200 0.310 0.250 − − − Object interaction % Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Object rMeasures Sph.viol 4.812 0.022 0.897 − − − - Object × gen. rMeasures Sph.viol 0.363 0.762 0.151 − − − - Genotype rMeasures Sph.viol 0.328 0.722 0.099 − − − - Cohort rMeasures Sph.viol 7.375 0.009 0.758 − − − - Object × gen. × coh. rMeasures Sph.viol 0.560 0.627 0.220 − − − - Gen. × coh. rMeasures Sph.viol 0.328 0.722 0.099 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Object interaction number Test Datastructure WT Het KO F p value Power F pvalue Power F pvalue Power WT vs Het WT vs KO Het vsKO Dice 2wANOVA Normal 24.87 ± 1.44 23.13 ± 1.27 25.81 ± 1.7 1.119 0.335 0.235 10.075 0.003 0.875 0.297 0.745 0.094 − − − Jack 2wANOVA Normal 23.14 ± 1.35 24.09 ± 1.09 24.69 ± 0.91 0.164 0.849 0.074 11.956 0.001 0.923 0.546 0.583 0.135 − − − Lego 2wANOVA Nonnormal 28.84 ± 2 28.66 ± 1.57 24.78 ± 1.41 1.507 0.232 0.305 2.901 0.095 0.386 1.286 0.286 0.265 − − − Pin 2wANOVA Nonnormal 33.55 ± 7.69 32.7 ± 6.64 30.85 ± 4.83 0.235 0.792 0.085 10.476 0.002 0.887 0.467 0.630 0.122 − − − Object interaction number, object ranked by preference Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Object rMeasures Sph.viol 74.224 0.000 1.000 − − − - Object × gen. rMeasures Sph.viol 0.867 0.499 0.335 − − − - Genotype rMeasures Sph.viol 2.228 0.119 0.432 − − − - Cohort rMeasures Sph.viol 72.229 0.000 1.000 − − − - Object × gen. × coh. rMeasures Sph.viol 0.653 0.649 0.254 − − − - Gen. × coh. rMeasures Sph.viol 1.142 0.328 0.239 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Object interaction number Test Data structure WT Het KO F p value Power F pvalue Power F p value Power WT vs Het WT vs KO Het vsKO Object #1 2wANOVA Normal 26.88 ± 2.3 29.15 ± 1.88 25.58 ± 1.75 1.682 0.197 0.337 30.522 0.000 1.000 0.173 0.842 0.075 − − − Object #2 2wANOVA Nonnormal 23.23 ± 2.05 25.15 ± 2.07 21.64 ± 1.85 2.474 0.095 0.473 59.810 0.000 1.000 0.752 0.477 0.170 0.421 0.829 0.167 Object #3 2wANOVA Nonnormal 17.94 ± 2.12 21.94 ± 1.92 18.88 ± 1.7 2.307 0.110 0.446 66.844 0.000 1.000 1.460 0.242 0.297 − − − Object #4 2wANOVA Nonnormal 16.41 ± 1.91 17.94 ± 1.34 16.94 ± 1.62 0.441 0.646 0.118 54.987 0.000 1.000 2.377 0.104 0.457 − − − Object interaction %, object ranked by preference Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Object rMeasures Sph.viol 86.885 0.000 1.000 − − − - Object × gen. rMeasures Sph.viol 0.745 0.578 0.288 − − − - Genotype rMeasures Sph.viol 1.010 0.411 0.207 − − − - Cohort rMeasures Sph.viol 0.960 0.390 0.163 − − − - Object × gen. × coh. rMeasures Sph.viol 0.979 0.327 0.390 − − − - Gen. × coh. rMeasures Sph.viol 0.960 0.390 0.207 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Object interaction % Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Object #1 2wANOVA Nonnormal 32.55 ± 1.41 31.48 ± 1.02 31.19 ± 0.99 0.066 0.936 0.059 11.328 0.002 0.910 0.966 0.388 0.208 − − − Object #2 2wANOVA Normal 28.09 ± 0.86 26.68 ± 0.74 25.78 ± 0.8 1.433 0.249 0.292 0.009 0.926 0.051 0.256 0.775 0.088 − − − Object #3 2wANOVA Nonnormal 20.49 ± 0.83 22.71 ± 0.81 22.52 ± 0.79 1.538 0.225 0.311 9.140 0.004 0.842 2.049 0.140 0.402 − − − Object #4 2wANOVA Normal 18.85 ± 0.7 19.11 ± 0.83 20.08 ± 0.64 0.454 0.638 0.120 2.559 0.116 0.348 0.975 0.385 0.210 − − − Repetitive novel object contact task, pattern of object investigation Genotype Cohort Genotype × cohort Pairwise comparisons Three-object sequences Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Total number of 3-object choices 2wANOVA Normal 56.11 ± 3.2 53.57 ± 4.82 53.11 ± 3.29 0.077 0.926 0.061 12.053 0.001 0.925 0.573 0.567 0.140 − − − Number of different 3-object sequences 2wANOVA Normal 26.5 ± 0.57 25.36 ± 1.21 25.47 ± 0.93 0.405 0.669 0.112 7.502 0.009 0.765 0.830 0.442 0.184 − − − Number of repetition of top preferred sequence 2wANOVA Nonnormal 4.88 ± 0.4 4.78 ± 0.37 4.64 ± 0.29 0.012 0.988 0.052 10.796 0.002 0.896 0.052 0.950 0.057 − − − Number of repetition of 2nd preferred sequence 2wANOVA Nonnormal 4.27 ± 0.27 4.15 ± 0.33 4.05 ± 0.26 0.017 0.983 0.052 8.748 0.005 0.826 0.312 0.733 0.097 − − − Number of repetition of 3rd preferred sequence 2wANOVA Nonnormal 3.83 ± 0.23 3.68 ± 0.3 3.7 ± 0.25 0.042 0.959 0.056 6.542 0.014 0.708 0.303 0.740 0.095 − − − Number of repetition of top 3 preferred sequences 2wANOVA Normal 13 ± 0.87 12.63 ± 0.98 12.41 ± 0.78 0.008 0.992 0.051 9.545 0.003 0.857 0.146 0.865 0.071 − − − % top preferred sequence choice 2wANOVA Nonnormal 8.57 ± 0.32 9.41 ± 0.61 8.75 ± 0.19 1.324 0.276 0.272 0.000 0.994 0.050 1.436 0.248 0.293 − − − % top 2 preferred sequence choice 2wANOVA Nonnormal 16.22 ± 0.52 17.48 ± 1.01 16.43 ± 0.32 1.179 0.316 0.246 0.143 0.707 0.066 1.543 0.224 0.312 − − − % top 3 preferred sequence choice 2wANOVA Nonnormal 23.14 ± 0.71 24.69 ± 1.43 23.44 ± 0.51 0.837 0.439 0.185 0.564 0.456 0.114 1.040 0.361 0.221 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Four-object sequences Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Total number of 4-object choices 2wANOVA Normal 55.55 ± 3.26 53.26 ± 4.79 52.58 ± 3.24 0.067 0.935 0.060 10.400 0.002 0.885 0.528 0.593 0.132 − − − Number of different 4-object sequences 2wANOVA Normal 40.77 ± 1.59 39.63 ± 2.78 39 ± 2.03 0.097 0.908 0.064 9.857 0.003 0.868 0.895 0.415 0.195 − − − Number of repetition of top preferred sequence 2wANOVA Nonnormal 3.05 ± 0.2 3.1 ± 0.2 3.41 ± 0.17 1.297 0.283 0.267 4.144 0.047 0.514 0.050 0.951 0.057 − − − Number of repetition of 2nd preferred sequence 2wANOVA Nonnormal 2.83 ± 0.2 2.84 ± 0.2 2.76 ± 0.18 0.034 0.967 0.055 4.324 0.043 0.531 1.214 0.306 0.253 − − − Number of repetition of 3rd preferred sequence 2wANOVA Nonnormal 2.44 ± 0.16 2.47 ± 0.19 2.23 ± 0.13 0.468 0.629 0.122 4.499 0.039 0.547 1.063 0.353 0.225 − − − Number of repetition of top 3 preferred sequences 2wANOVA Nonnormal 8.33 ± 0.53 8.42 ± 0.55 8.41 ± 0.42 0.087 0.916 0.063 5.267 0.026 0.614 0.634 0.535 0.150 − − − % top preferred sequence choice 2wANOVA Nonnormal 5.58 ± 0.3 6.31 ± 0.47 6.66 ± 0.27 2.187 0.123 0.425 2.759 0.103 0.370 1.056 0.356 0.224 − − − % top 2 preferred sequence choice 2wANOVA Nonnormal 10.7 ± 0.44 11.99 ± 0.77 12.09 ± 0.51 1.734 0.187 0.346 2.734 0.105 0.367 1.166 0.320 0.244 − − − % top 3 preferred sequence choice 2wANOVA Nonnormal 15.14 ± 0.54 16.93 ± 1.02 16.43 ± 0.67 1.557 0.221 0.314 3.181 0.081 0.416 1.237 0.299 0.257 − − − Barnes maze initial training - distance Distance Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Day rMeasures Sph.ass 13.695 0.000 1.000 − − − - Day × gen. rMeasures Sph.ass 2.062 0.062 0.684 − − − - Genotype rMeasures Sph.ass 2.663 0.080 0.503 0.659 0.145 0.515 - Cohort rMeasures Sph.ass 11.841 0.001 0.920 − − − - Day × gen. × coh. rMeasures Sph.ass 1.173 0.324 0.416 − − − - Gen. × coh. rMeasures Sph.ass 1.114 0.337 0.234 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Individual days Test Data structure WT Het KO F p value Power F p value Power F pvalue Power WT vsHet WT vsKO Het vs KO Day 1 2wANOVA Normal 501.24 ± 48.28 485.42 ± 47.71 484.49 ± 53.07 0.003 0.997 0.050 4.283 0.044 0.526 0.084 0.919 0.062 − − − Day 2 2wANOVA Normal 427.6 ± 43.5 468.59 ± 40.26 504.18 ± 47.17 1.234 0.301 0.256 9.205 0.004 0.844 1.918 0.158 0.378 − − − Day 3 2wANOVA Normal 292.36 ± 29.11 340.26 ± 31.24 485.74 ± 41.16 11.293 0.000 0.989 6.902 0.012 0.730 3.082 0.055 0.567 0.496 0.000 0.005 Day 4 2wANOVA Normal 311.01 ± 34.75 370.86 ± 29.61 367.31 ± 42.11 1.479 0.239 0.300 5.449 0.024 0.628 0.666 0.519 0.155 − − − Barnes maze reversal - distance Distance Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Day effect rMeasures Sph.ass 26.455 0.000 1.000 − − − - Day × gen. rMeasures Sph.ass 2.612 0.023 0.824 − − − - Genotype rMeasures Sph.ass 1.811 0.175 0.359 − − − - Cohort rMeasures Sph.ass 1.924 0.172 0.274 − − − - Day × gen. × coh. rMeasures Sph.ass 3.192 0.007 0.902 − − − - Genotype × cohort effect rMeasures Sph.ass 0.290 0.750 0.093 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Individual days Test Data structure WT Het KO F p value Power F pvalue Power F p value Power WT vs Het WT vs KO Het vsKO Day 1 2wANOVA Nonnormal 420.93 ± 37.75 437.03 ± 37.86 591.59 ± 38.48 5.592 0.007 0.834 0.793 0.378 0.141 1.475 0.239 0.299 0.948 0.009 0.018 Day 2 2wANOVA Normal 336.81 ± 35.36 413.64 ± 32.4 390.91 ± 45 1.285 0.286 0.265 0.374 0.544 0.092 2.525 0.091 0.481 − − − Day 3 2wANOVA Normal 357.93 ± 35.96 421.04 ± 44.36 395.85 ± 48.06 0.666 0.519 0.155 3.371 0.073 0.436 0.116 0.890 0.067 − − − Day 4 2wANOVA Normal 288.54 ± 39.85 288.65 ± 37.41 337.24 ± 38.59 0.965 0.389 0.207 8.849 0.005 0.829 1.373 0.264 0.281 − − − Barnes maze initial training probe test Genotype Quadrant pairwise comparisons All animals Test Data structure F p value Power T vs L T vs R T vs O L vsR L vs O R vs O - Quadrant rMeasures Sph.viol 296.653 0.000 1.000 0.000 0.000 0.000 0.555 0.201 0.628 - Cohort rMeasures Sph.viol 10.200 0.002 1.000 - Quadrant × coh. rMeasures Sph.viol 11.435 0.000 0.983 WT Test Data structure F p value Power T vs L T vs R T vs O L vs R L vs O R vs O - Quadrant rMeasures Sph.viol 58.318 0.000 1.000 0.000 0.000 0.000 0.057 0.168 0.335 - Cohort rMeasures Sph.viol 9.373 0.007 0.820 - Quadrant × coh. rMeasures Sph.viol 4.241 0.010 0.831 Het Test Data structure F p value Power T vs L T vs R T vs O L vsR L vsO R vs O - Quadrant rMeasures Sph.viol 107.980 0.000 1.000 0.000 0.000 0.000 0.205 0.895 0.278 - Cohort rMeasures Sph.viol 65.390 0.000 1.000 - Quadrant × coh. rMeasures Sph.viol 5.366 0.003 0.915 KO Test Data structure F p value Power T vsL T vsR T vsO L vsR L vs O R vs O - Quadrant rMeasures Sph.viol 378.546 0.000 1.000 0.000 0.000 0.000 0.832 0.278 0.341 - Cohort rMeasures Sph.viol 890.226 0.000 1.000 - Quadrant × coh. rMeasures Sph.viol 1.683 0.186 0.406 Genotype Cohort Genotype × cohort Pairwise comparisons Probe test quadrants Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vsHet WT vsKO Het vsKO Target 2wANOVA Nonnormal 107.29 ± 7.96 125.68 ± 7.96 142.87 ± 4.53 5.342 0.008 0.816 12.144 0.001 0.927 0.935 0.400 0.202 0.112 0.001 0.173 Left 2wANOVA Nonnormal 28.57 ± 4.6 14.87 ± 2.32 11.37 ± 2.49 7.081 0.002 0.914 4.207 0.046 0.519 0.660 0.522 0.154 0.010 0.002 0.740 Right 2wANOVA Nonnormal 16.92 ± 3.86 20.57 ± 4.93 12.28 ± 2.15 1.097 0.342 0.231 7.056 0.011 0.739 1.048 0.359 0.222 0.763 0.678 0.290 Opposite 2wANOVA Nonnormal 22.28 ± 4.49 14.37 ± 3.47 9.2 ± 2.09 2.438 0.099 0.466 10.443 0.002 0.886 1.149 0.326 0.240 0.199 0.024 0.526 Barnes maze reversal probe test Genotype Quadrant pairwise comparisons All animals Test Data structure F p value Power T vs L T vsR T vs O L vsR L vs O R vs O - Quadrant rMeasures Sph.viol 50.865 0.000 1.000 0.000 0.000 0.000 0.242 0.000 0.000 - Cohort rMeasures Sph.viol 24.530 0.000 0.998 - Quadrant × coh. rMeasures Sph.viol 4.443 0.005 0.870 WT Test Data structure F p value Power T vsL T vsR T vsO L vs R L vs O R vs O - Quadrant rMeasures Sph.viol 32.279 0.000 1.000 0.000 0.000 0.000 0.005 0.024 0.003 - Cohort rMeasures Sph.viol 159.377 0.000 1.000 - Quadrant × coh. rMeasures Sph.viol 0.007 0.956 0.051 Het Test Data structure F p value Power T vs L T vs R T vs O L vsR L vs O R vs O - Quadrant rMeasures Sph.viol 28.198 0.000 1.000 0.000 0.000 0.001 0.235 0.001 0.086 - Cohort rMeasures Sph.viol 6.412 0.021 0.666 - Quadrant × coh. rMeasures Sph.viol 10.315 0.000 0.998 KO Test Data structure F p value Power T vsL T vs R T vs O L vs R L vs O R vs O - Quadrant rMeasures Sph.viol 12.026 0.000 0.999 0.000 0.010 0.646 0.070 0.000 0.000 - Cohort rMeasures Sph.viol 397.250 0.000 1.000 - Quadrant × coh. rMeasures Sph.viol 2.273 0.095 0.531 Genotype Cohort Genotype × cohort Pairwise comparisons Probe test quadrants Test Data structure WT Het KO F p value Power F p value Power F pvalue Power WT vs Het WT vs KO Het vsKO Target 2wANOVA Nonnormal 115.33 ± 11.27 105.35 ± 12.55 67.82 ± 11.65 5.430 0.008 0.822 8.183 0.006 0.800 3.469 0.040 0.621 0.773 0.010 0.046 Left 2wANOVA Nonnormal 18.44 ± 3.76 8.03 ± 2.15 9.68 ± 2.79 3.343 0.044 0.604 2.079 0.156 0.292 0.172 0.842 0.075 0.039 0.123 0.923 Right 2wANOVA Nonnormal 9.8 ± 2.46 18.48 ± 7.8 23.17 ± 5.32 1.367 0.265 0.280 1.873 0.178 0.268 2.980 0.061 0.551 0.489 0.229 0.826 Opposite 2wANOVA Nonnormal 32.67 ± 6.87 42.82 ± 10 75.15 ± 10.65 6.632 0.003 0.894 7.210 0.010 0.748 2.097 0.134 0.409 0.662 0.004 0.030 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 11-1. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Norm: normal, No-norm: non-normal, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort.
Y-maze, spontaneous alternation behavior Genotype Cohort Genotype × cohort Pairwise comparisons % of choices Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Arm 1 2wANOVA Normal 32.34 ± 0.87 34.24 ± 0.92 32.77 ± 1.17 0.844 0.436 0.187 0.035 0.852 0.054 0.412 0.664 0.113 − − - Arm 2 2wANOVA Normal 35.17 ± 1.17 32.74 ± 1.1 35.18 ± 1.36 1.548 0.223 0.314 9.976 0.003 0.873 0.119 0.888 0.067 − − - Arm 3 2wANOVA Normal 32.19 ± 1.46 32.98 ± 1.09 32.04 ± 1.02 0.285 0.753 0.093 10.366 0.002 0.885 0.520 0.598 0.131 − − - Chance level comparison Test Data structure All t All p value Power WT t WT p value Power Het t Het p value Power KO t KO p value Power Arm 1 1S-t test Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Arm 2 1S-t test Normal 1.465 0.148 NA 1.578 0.132 NA -0.534 0.600 NA 1.354 0.193 NA Arm 3 1S-t test Normal -1.338 0.186 NA -0.772 0.450 NA -0.312 0.759 NA -1.262 0.223 NA Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Total number of choices 2wANOVA Normal 43.42 ± 3.25 40.26 ± 2.54 38.47 ± 2.75 0.612 0.546 0.147 0.164 0.687 0.068 2.244 0.116 0.437 − − - Number of correct choice 2wANOVA Normal 57.46 ± 1.59 60.68 ± 1.93 57.52 ± 1.61 1.227 0.302 0.256 2.987 0.090 0.396 0.927 0.402 0.202 − − - Number of type 1 errors 2wANOVA Normal 37.8 ± 1.43 34.09 ± 1.59 38.29 ± 1.79 2.296 0.111 0.445 0.300 0.586 0.084 5.165 0.009 0.804 − − - Number of type 2 errors 2wANOVA Nonnormal 4.04 ± 1.11 5.47 ± 1.02 4.51 ± 1.07 0.397 0.674 0.111 4.402 0.041 0.539 3.449 0.039 0.621 − − - Fear conditioning Training Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Time rMeasures Sph.viol 43.998 0.000 1.000 − − − - Time × genotype rMeasures Sph.viol 3.194 0.002 0.970 − − − - Genotype rMeasures Sph.viol 14.505 0.000 0.998 0.809 0.000 0.000 - Cohort rMeasures Sph.viol 12.351 0.001 0.932 − − − - Time × gen. × coh. rMeasures Sph.viol 0.602 0.782 0.281 − − − - Gen. × coh. rMeasures Sph.viol 0.494 0.613 0.127 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Training, individualtime bins Test Data structure WT Het KO F pvalue Power F pvalue Power F pvalue Power WT vs Het WT vs KO Het vsKO Habituation 2wANOVA Nonnormal 16.85 ± 2.93 10.45 ± 2.37 21.6 ± 5.3 2.081 0.135 0.408 0.015 0.903 0.052 0.061 0.941 0.059 − − - Pre-tone 0-120 2wANOVA Nonnormal 10.45 ± 1.72 8.15 ± 1.43 21.01 ± 3.97 6.546 0.003 0.892 0.330 0.568 0.087 0.041 0.960 0.056 0.820 0.021 0.004 Tone/shock 120-140 2wANOVA Nonnormal 10.58 ± 3.26 6.85 ± 2.82 19.94 ± 6.06 2.361 0.105 0.456 0.020 0.887 0.052 0.199 0.820 0.079 − − - Post-tone 140-260 2wANOVA Nonnormal 19.74 ± 3.73 18.83 ± 3.86 47.68 ± 6.71 13.149 0.000 0.996 5.506 0.023 0.634 2.222 0.119 0.433 0.990 0.000 0.000 Tone/shock 260-280 2wANOVA Nonnormal 15.07 ± 4.7 24.58 ± 5.22 47.23 ± 7.47 7.613 0.001 0.934 0.026 0.871 0.053 0.762 0.472 0.173 0.507 0.001 0.027 Tone/shock 260-280 2wANOVA Nonnormal 31.06 ± 5.14 37.88 ± 6.6 65.23 ± 6.72 12.505 0.000 0.995 18.006 0.000 0.986 0.640 0.532 0.151 0.650 0.000 0.002 Tone/shock 400-420 2wANOVA Nonnormal 31.03 ± 5.86 40.59 ± 7.61 65.21 ± 5.94 9.728 0.000 0.977 12.565 0.001 0.935 0.061 0.941 0.059 0.503 0.001 0.015 Post-tone 420-540 2wANOVA Nonnormal 36.71 ± 6.53 47.61 ± 7.36 61.74 ± 6.78 7.880 0.001 0.942 45.207 0.000 1.000 0.135 0.874 0.070 0.303 0.003 0.139 Context Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Time rMeasures Sph.ass 4.558 0.004 0.880 − − − - Time × genotype rMeasures Sph.ass 0.675 0.670 0.262 − − − - Genotype rMeasures Sph.ass 1.788 0.178 0.357 − − − - Cohort rMeasures Sph.ass 0.542 0.465 0.112 − − − - Time × gen.× coh. rMeasures Sph.ass 0.918 0.481 0.355 − − − - Gen. × coh. rMeasures Sph.ass 1.026 0.366 0.219 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Context, individual time bins Test Datastructure WT Het KO F p value Power F pvalue Power F p value Power WT vs Het WT vs KO Het vs KO 0-60 2wANOVA Nonnormal 63.09 ± 5.13 57.19 ± 5.63 44.3 ± 6.21 2.643 0.081 0.502 0.582 0.449 0.116 0.558 0.576 0.137 0.750 0.063 0.261 60-120 2wANOVA Nonnormal 66.34 ± 6.83 66 ± 6.94 59.6 ± 7.41 0.230 0.795 0.084 0.676 0.415 0.127 2.233 0.118 0.435 − − - 120-180 2wANOVA Nonnormal 62.65 ± 7.14 62.11 ± 6.47 42.81 ± 7.83 2.263 0.114 0.440 0.392 0.534 0.094 0.958 0.390 0.207 − − - 180-240 2wANOVA Nonnormal 56.12 ± 6.56 54.45 ± 7.58 43.33 ± 6.29 0.944 0.396 0.205 0.073 0.788 0.058 0.109 0.897 0.066 − − - mean 2wANOVA Nonnormal 62.05 ± 5.57 59.94 ± 5.31 47.51 ± 5.84 1.788 0.178 0.357 0.542 0.465 0.112 1.026 0.366 0.219 − − - Cued Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Time rMeasures Sph.viol 25.753 0.000 1.000 − − − - Time × genotype rMeasures Sph.viol 3.101 0.002 0.968 − − − - Genotype rMeasures Sph.viol 5.657 0.006 0.841 0.645 0.007 0.065 - Cohort rMeasures Sph.viol 4.255 0.044 0.525 − − − - Time × gen. × coh. rMeasures Sph.viol 4.116 0.000 0.995 − − − - Gen. × coh. rMeasures Sph.viol 1.616 0.209 0.326 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Cued, individual time bins Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Pre-tone 0-60 2wANOVA Nonnormal 1.64 ± 1.14 0.4 ± 0.28 4.9 ± 2.59 1.897 0.160 0.376 5.996 0.018 0.671 1.527 0.227 0.310 0.841 0.311 0.114 Pre-tone 60-120 2wANOVA Nonnormal 0.84 ± 0.38 1.96 ± 0.89 2.37 ± 0.88 1.056 0.355 0.225 18.576 0.000 0.988 0.747 0.479 0.170 0.461 0.238 0.896 Tone 120-140 2wANOVA Nonnormal 10.94 ± 4.37 12.14 ± 4.53 25.23 ± 6.38 3.005 0.058 0.558 7.144 0.010 0.746 1.298 0.282 0.268 0.984 0.106 0.150 Post-tone 140-200 2wANOVA Nonnormal 8.52 ± 2.11 7.41 ± 1.76 13.09 ± 3.74 1.367 0.264 0.281 1.610 0.210 0.238 0.118 0.889 0.067 − − - Post-tone 200-260 2wANOVA Nonnormal 2.29 ± 1.03 6.59 ± 1.53 10.03 ± 4.8 1.551 0.222 0.314 1.595 0.212 0.236 1.203 0.309 0.251 − − - Tone 260-280 2wANOVA Nonnormal 13.87 ± 5.15 19.36 ± 5.92 39.08 ± 7.75 7.219 0.002 0.921 15.352 0.000 0.970 7.888 0.001 0.942 0.733 0.003 0.025 Post-tone 280-340 2wANOVA Nonnormal 8.92 ± 2.71 19.61 ± 5.1 26.7 ± 6.01 3.891 0.027 0.677 5.448 0.024 0.629 1.180 0.316 0.247 0.240 0.024 0.527 Post-tone 340-400 2wANOVA Nonnormal 5.09 ± 1.34 9.96 ± 2.78 20.99 ± 4.81 6.189 0.004 0.874 1.570 0.216 0.233 1.068 0.351 0.227 0.549 0.003 0.054 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 12-1. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, one sample t test: 1S-t test, Norm: normal, No-norm: non-normal, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, one sample t test: 1S-t test, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort.
Open field thigmotaxis Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Border distance (cm) 2wANOVA Normal 10507.07 ± 558.17 8848.23 ± 548.19 7942.17 ± 394.99 6.537 0.003 0.892 10.443 0.002 0.887 0.080 0.923 0.062 0.022 0.001 0.235 Border distance (cm) Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Time rMeasures Sph.viol 52.599 0.000 1.000 − − − - Time × genotype rMeasures Sph.viol 2.496 0.007 0.877 − − − - Genotype rMeasures Sph.viol 6.537 0.003 0.892 0.043 0.001 0.373 - Cohort rMeasures Sph.viol 10.443 0.002 0.887 − − − - Time × gen. × coh. rMeasures Sph.viol 0.923 0.492 0.399 − − − - Genotype × cohort rMeasures Sph.viol 0.080 0.923 0.062 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Center distance (cm) 2wANOVA Nonnormal 3276.58 ± 335.66 2390.56 ± 291.66 2139.7 ± 246.94 3.932 0.026 0.682 9.890 0.003 0.870 0.049 0.952 0.057 0.036 0.011 0.622 Center distance (cm) Test Data structure F p value Power WT vs Het WT vs KO Het vs KO - Time rMeasures Sph.viol 1.158 0.330 0.343 − − − - Time × genotype rMeasures Sph.viol 1.327 0.237 0.571 − − − - Genotype rMeasures Sph.viol 3.932 0.026 0.682 0.070 0.015 0.798 - Cohort rMeasures Sph.viol 9.890 0.003 0.870 − − − - Time × gen. × coh. rMeasures Sph.viol 0.695 0.683 0.302 − − − - Genotype × cohort rMeasures Sph.viol 0.049 0.952 0.057 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vsHet WT vs KO Het vs KO Border/total distance 2wANOVA Normal 76.87 ± 1.51 79.57 ± 1.69 79.69 ± 1.29 0.950 0.393 0.206 5.570 0.022 0.639 0.049 0.952 0.057 − − − Border/total distance Test Data structure F p value Power WT vs Het WT vsKO Het vs KO - Time rMeasures Sph.viol 5.035 0.001 0.957 − − − - Time × genotype rMeasures Sph.viol 1.182 0.312 0.531 − − − - Genotype rMeasures Sph.viol 1.017 0.369 0.218 − − − - Cohort rMeasures Sph.viol 5.820 0.019 0.658 − − − - Time × gen. × coh. rMeasures Sph.viol 0.679 0.705 0.305 − − − - Genotype × cohort rMeasures Sph.viol 0.094 0.911 0.064 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Center/total distance 2wANOVA Normal 22.88 ± 1.51 20.15 ± 1.68 20.16 ± 1.27 0.939 0.398 0.204 4.471 0.039 0.546 0.048 0.953 0.057 − − − Center/total distance Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Time rMeasures Sph.viol 5.177 0.001 0.962 − − − - Time × genotype rMeasures Sph.viol 1.177 0.315 0.527 − − − - Genotype rMeasures Sph.viol 1.001 0.375 0.215 − − − - Cohort rMeasures Sph.viol 4.757 0.034 0.571 − − − - Time × gen. × coh. rMeasures Sph.viol 0.652 0.728 0.292 − − − - Genotype × cohort rMeasures Sph.viol 0.088 0.916 0.063 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Border/Center distance 2wANOVA Nonnormal 3.78 ± 0.38 4.87 ± 0.74 4.34 ± 0.36 0.990 0.379 0.213 3.522 0.066 0.453 0.216 0.807 0.082 − − − Border/Center distance Test Data structure F pvalue Power WT vs Het WT vs KO Het vsKO - Time rMeasures Sph.viol 5.177 0.001 0.240 − − − - Time × genotype rMeasures Sph.viol 1.177 0.315 0.456 − − − - Genotype rMeasures Sph.viol 1.001 0.375 0.309 − − − - Cohort rMeasures Sph.viol 4.757 0.034 0.469 − − − - Time × gen. × coh. rMeasures Sph.viol 0.652 0.728 0.196 − − − - Genotype × cohort rMeasures Sph.viol 0.088 0.916 0.230 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F pvalue Power F pvalue Power WT vs Het WT vsKO Het vs KO Border time (s) 2wANOVA Normal 2969.88 ± 70.88 2993.1 ± 77.46 3067.4 ± 62.2 0.481 0.621 0.124 1.088 0.302 0.176 0.582 0.563 0.141 − − − Border time (s) Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Time rMeasures Sph.viol 2.960 0.023 0.773 − − − - Time × genotype rMeasures Sph.viol 0.836 0.568 0.374 − − − - Genotype rMeasures Sph.viol 0.481 0.621 0.124 − − − - Cohort rMeasures Sph.viol 1.088 0.302 0.176 − − − - Time × gen. × coh. rMeasures Sph.viol 0.792 0.606 0.354 − − − - Genotype × cohort rMeasures Sph.viol 0.582 0.563 0.141 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F pvalue Power F p value Power WT vs Het WT vs KO Het vsKO Center time (s) 2wANOVA Normal 612.08 ± 71.17 587.82 ± 77.5 517.59 ± 62.2 0.451 0.640 0.119 0.871 0.355 0.150 0.589 0.559 0.143 − − − Center time (s) Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Time rMeasures Sph.viol 3.200 0.016 0.807 − − − - Time × genotype rMeasures Sph.viol 0.836 0.568 0.363 − − − - Genotype rMeasures Sph.viol 0.481 0.621 0.119 − − − - Cohort rMeasures Sph.viol 1.088 0.302 0.150 − − − - Time × gen. × coh. rMeasures Sph.viol 0.792 0.606 0.090 − − − - Genotype × cohort rMeasures Sph.viol 0.582 0.563 0.143 − − − Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Border/center time 2wANOVA Nonnormal 6.95 ± 1.32 8.3 ± 1.88 9.76 ± 2.27 0.476 0.624 0.124 0.533 0.469 0.111 0.107 0.898 0.066 − − − Border/center time Test Data structure F p value Power WT vs Het WT vs KO Het vsKO - Time rMeasures Sph.viol 0.290 0.822 0.103 − − − - Time × genotype rMeasures Sph.viol 1.575 0.163 0.575 − − − - Genotype rMeasures Sph.viol 0.429 0.653 0.116 − − − - Cohort rMeasures Sph.viol 1.546 0.220 0.230 − − − - Time × gen. × coh. rMeasures Sph.viol 0.575 0.740 0.219 − − − - Genotype × cohort rMeasures Sph.viol 1.594 0.213 0.322 − − − Vertical activity in open field Genotype Cohort Genotype × cohort Pairwise comparisons Test Data structure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vsKO Free rears duration (s) 2wANOVA Nonnormal 4.66 ± 1.25 7.93 ± 1.62 6.18 ± 1.48 1.159 0.322 0.243 0.036 0.850 0.054 1.480 0.237 0.301 − − − Free rears number 2wANOVA Nonnormal 8.42 ± 2.13 10.63 ± 1.66 7.57 ± 1.21 0.837 0.439 0.186 1.988 0.165 0.283 1.369 0.264 0.281 − − − Wall rears duration (s) 2wANOVA Nonnormal 14.68 ± 1.77 16.13 ± 2.02 9.01 ± 0.84 5.023 0.010 0.793 1.924 0.171 0.275 0.397 0.675 0.111 0.805 0.045 0.009 Wall rears number 2wANOVA Nonnormal 27.26 ± 2.68 27.52 ± 2.86 19.36 ± 1.88 3.576 0.035 0.638 19.306 0.000 0.991 0.414 0.663 0.113 0.996 0.036 0.030 All rears duration (s) 2wANOVA Nonnormal 19.34 ± 2.3 24.07 ± 3.01 15.2 ± 1.99 3.140 0.052 0.578 0.646 0.425 0.124 1.240 0.298 0.258 0.374 0.468 0.038 All rears number 2wANOVA Nonnormal 35.68 ± 3.83 38.15 ± 3.94 26.94 ± 2.08 3.240 0.047 0.592 16.104 0.000 0.976 1.267 0.290 0.263 0.829 0.107 0.028 Zero-maze Genotype Cohort Genotype ×cohort Pairwise comparisons Test Datastructure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Closed arc time, d1 2wANOVA Normal 428.66 ± 18.61 441.68 ± 13.82 456.86 ± 18.31 0.400 0.672 0.111 13.684 0.001 0.952 0.311 0.734 0.097 − − − Closed arc time, d2 2wANOVA Nonnormal 448.33 ± 20.43 492.29 ± 16.58 487.59 ± 29.63 3.652 0.033 0.646 36.459 0.000 1.000 0.114 0.893 0.066 0.138 0.009 0.489 Closed arc time, m 2wANOVA Normal 438.5 ± 18.27 466.99 ± 13.34 472.23 ± 20.15 1.917 0.158 0.379 28.873 0.000 1.000 0.253 0.778 0.088 − − − Open arc time, d1 2wANOVA Normal 166.03 ± 18.25 153.09 ± 13.6 134.72 ± 17.99 0.562 0.574 0.138 10.417 0.002 0.886 0.420 0.659 0.114 − − − Open arc time, d2 2wANOVA Nonnormal 143.08 ± 21.29 102.14 ± 16.74 78.39 ± 17.83 3.063 0.056 0.566 37.315 0.000 1.000 0.151 0.860 0.072 0.194 0.015 0.502 Open arc time, m 2wANOVA Normal 154.55 ± 18.53 127.61 ± 13.25 106.56 ± 16.4 1.863 0.166 0.369 26.343 0.000 0.999 0.340 0.713 0.101 − − − Close/open time, d1 2wANOVA Nonnormal 4.08 ± 0.85 3.74 ± 0.98 5.76 ± 1.6 0.820 0.447 0.182 9.172 0.004 0.843 0.438 0.648 0.117 − − − Close/open time, d2 2wANOVA Nonnormal 6.95 ± 1.95 25.36 ± 18.5 28.98 ± 11.02 0.969 0.387 0.209 5.901 0.019 0.663 0.746 0.479 0.169 − − − Close/open time, m 2wANOVA Nonnormal 4.61 ± 1 5.55 ± 1.95 8.57 ± 2.42 1.207 0.308 0.251 11.378 0.001 0.911 0.538 0.587 0.134 − − − Open arc entries, d1 2wANOVA Nonnormal 48.47 ± 4.88 50.52 ± 4.46 59.36 ± 7.73 1.870 0.165 0.371 14.588 0.000 0.963 0.603 0.551 0.145 − − − Open arc entries, d2 2wANOVA Normal 42.42 ± 5.18 36.52 ± 4.83 29.31 ± 5.07 1.238 0.299 0.257 19.130 0.000 0.990 0.137 0.872 0.070 − − − Open arc entries, m 2wANOVA Normal 45.44 ± 4.16 43.52 ± 4.04 44.34 ± 5.81 0.150 0.861 0.072 24.720 0.000 0.998 0.447 0.642 0.119 − − − Open entering arc latency 2wANOVA Nonnormal 39.29 ± 13.16 47.08 ± 31.35 22.99 ± 10.38 0.310 0.735 0.097 0.684 0.412 0.128 1.282 0.287 0.265 − − − Open arc crossing latency 2wANOVA Nonnormal 149.34 ± 34.87 139.69 ± 35.2 95.2 ± 32.99 0.796 0.457 0.178 1.515 0.224 0.226 0.395 0.676 0.110 − − − Close arc dipping number, d1 2wANOVA Nonnormal 51.73 ± 6.81 55.42 ± 6.48 51.47 ± 7.31 0.303 0.740 0.096 54.807 0.000 1.000 0.035 0.965 0.055 − − − Close arc dipping number, d2 2wANOVA Nonnormal 31.94 ± 3.01 28.94 ± 3.69 22.21 ± 3.87 2.182 0.124 0.425 54.920 0.000 1.000 0.593 0.556 0.143 − − − Close arc dipping number, m 2wANOVA Nonnormal 41.84 ± 4.31 42.5 ± 4.48 36.84 ± 5.05 0.239 0.788 0.085 94.671 0.000 1.000 0.179 0.836 0.076 − − − Close arc dipping time, d1 2wANOVA Nonnormal 131.15 ± 14.57 145.82 ± 14.24 117.88 ± 13.74 0.984 0.381 0.211 57.892 0.000 1.000 0.648 0.527 0.153 − − − Close arc dipping time, d2 2wANOVA Nonnormal 94.51 ± 10 100.08 ± 13.98 64.33 ± 12.46 2.111 0.132 0.413 26.004 0.000 0.999 0.342 0.712 0.102 − − − Close arc dipping time, m 2wANOVA Normal 112.83 ± 11.57 124.49 ± 12.37 91.1 ± 10.18 2.386 0.103 0.459 72.828 0.000 1.000 0.811 0.450 0.181 − − − Open arc dipping number, d1 2wANOVA Nonnormal 27.15 ± 3.63 20.26 ± 2.46 19.15 ± 3.92 1.441 0.247 0.294 8.140 0.006 0.799 0.567 0.571 0.139 − − − Open arc dipping number, d2 2wANOVA Nonnormal 16.94 ± 3.04 12.22 ± 2.34 8.84 ± 2.54 1.938 0.155 0.383 17.131 0.000 0.982 0.332 0.719 0.100 − − − Open arc dipping number, m 2wANOVA Nonnormal 22.05 ± 3.15 16.97 ± 2.15 14 ± 2.9 2.060 0.138 0.404 15.216 0.000 0.969 0.502 0.609 0.128 − − − Zero-maze Genotype Cohort Genotype ×cohort Pairwise comparisons Test Datastructure WT Het KO F p value Power F p value Power F p value Power WT vs Het WT vs KO Het vs KO Open arc dipping time, d1 2wANOVA Nonnormal 62.85 ± 10.83 42.28 ± 5.15 25.57 ± 5.27 5.700 0.006 0.843 13.928 0.000 0.955 1.730 0.188 0.346 0.091 0.001 0.245 Open arc dipping time, d2 2wANOVA Nonnormal 51.3 ± 10.52 35.3 ± 8.44 17.87 ± 5.34 3.798 0.029 0.665 20.104 0.000 0.993 0.427 0.655 0.115 0.278 0.008 0.269 Open arc dipping time, m 2wANOVA Nonnormal 57.07 ± 9.63 39.17 ± 5.86 21.72 ± 4.71 6.448 0.003 0.886 23.422 0.000 0.997 1.299 0.282 0.268 0.088 0.001 0.157 Time open vs close, d1 Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Zone rMeasures Sph.ass 277.319 0.000 1.000 73.861 0.000 1.000 125.301 0.000 1.000 83.255 0.000 1.000 - Cohort rMeasures Sph.ass 8.156 0.006 0.935 5.563 0.031 0.604 8.257 0.011 0.773 3.298 0.087 0.403 - Zone × cohort rMeasures Sph.ass 12.518 0.001 0.801 7.248 0.015 0.718 3.063 0.098 0.379 2.143 0.161 0.282 Time open vs close, d2 Test Data structure All F All p value Power WT F WT p value Power Het F Het p value Power KO F KO p value Power - Zone rMeasures Sph.ass 440.281 0.000 1.000 94.767 0.000 1.000 278.317 0.000 1.000 119.843 0.000 1.000 - Cohort rMeasures Sph.ass 0.578 0.450 0.218 1.269 0.276 0.186 2.497 0.132 0.320 0.921 0.351 0.148 - Zone × cohort rMeasures Sph.ass 25.848 0.000 1.000 12.054 0.003 0.905 18.624 0.000 0.982 3.749 0.070 0.447 Time open vs close, m Test Data structure All F All p value Power WT F WT pvalue Power Het F Het pvalue Power KO F KO pvalue Power - Zone rMeasures Sph.ass 440.281 0.000 1.000 103.409 0.000 1.000 274.392 0.000 1.000 131.582 0.000 1.000 - Cohort rMeasures Sph.ass 0.578 0.450 0.116 0.006 0.941 0.051 0.422 0.524 0.094 0.484 0.496 0.101 - Zone × cohort rMeasures Sph.ass 25.848 0.000 0.999 11.720 0.003 0.897 12.720 0.002 0.919 3.786 0.068 0.451 WT, wild-type mice; Het, heterozygous mice; KO, homozygous knock-out mice. Group values are reported as mean ± SEM. Bold font indicates significant results (p < 0.05). Individual results and statistical analyses for cohorts 1 and 2 are available in Extended Data Table 13-1. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Norm: normal, No-norm: non-normal, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort, d1: day 1, d2: day 2, m: day 1 - day 2 mean. 2wANOVA: 2-way ANOVA, rMeasures: repeated measures, Sph.ass: sphericity assumed, Sph.viol: sphericity violated, gen: genotype, coh: cohort, d1: day 1, d2: day 2, m: day 1 - day 2 mean.
Extended Data Figure 1-1
Validation of a knockout mice with a complete deletion of Shank3 (A) Genotyping of Shank3∆4-22 mice by PCR. The P1-P3 primer pair produced a 490 bp band identifying the ∆4-22 allele, while the P2-P3 primer pair amplified the 390 bp product from the wild-type allele. (B) Original full scans of immunoblots related to Figure 1. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Download Figure 1-1, TIF file.
Extended Data Figure 2-1
Normal developmental milestones of Shank3Δ4-22 deficient mice. Analysis of the markers of developmental milestones that revealed no major genotype differences in Shank3 wild-type, heterozygous and homozygous pups between postnatal days 1 and 21 on measures of (A) ear opening, (B) tooth eruption, (C) fur development, (D) eye opening, (E) rooting reflex, (F) cliff aversion, (G) ear twitch, (H) surface righting and (I) open field crossing. (K) Only non-significant differences were detected in the number and quality of ultrasonic vocalizations emitted by 6-day old pups. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. *: WT vs KO; o: WT vs Het, #: Het vs KO. *: p < 0.05, **: p < 0.1, ***: p < 0.001. Download Figure 2-1, TIF file.
Extended Data Table 5-1
Individual results and statistical analyses for cohorts 1 and 2 related to general health, physical factors, gross appearance and spontaneous activity. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 5-1, DOCX file.
Extended Data Figure 3-1
Motor functions in in Shank3Δ4-22 -deficient mice. (A) Number of failed attempts (falls or hanging without being able to pull itself up in less than 60 seconds) in the hind placing test. The number of failed attempts was more important in Shank3Δ4-22 homozygous mice. (B) Strength measured in the grip strength. No genotype difference was found for acute grip strength. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. *: WT vs KO; o: WT vs Het, #: Het vs KO. *: p < 0.05, **: p < 0.1, ***: p < 0.001. Download Figure 3-1, TIF file.
Extended Data Table 6-1
Individual results and statistical analyses for cohorts 1 and 2 related to motor functions. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 6-1, DOCX file.
Extended Data Table 7-1
Individual results and statistical analyses for cohorts 1 and 2 related to the sensory profile. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 7-1, DOCX file.
Extended Data Figure 4-1
Altered sensory profile in Shank3 complete knockout mice. (A) Pre-pulse inhibition. A non-significant decrease of pre-pulse inhibition was observed in both heterozygous and homozygous Shank3Δ4-22 mice compared to wild-type animals. (B)Individual scores in buried food test showing that 50% of Shank3Δ4-22 homozygous mice fail to retrieve buried food (cut-off of 900 seconds). WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Download Figure 4-1, TIF file.
Extended Data Figure 5-1
Social interactions in ShankΔ4-22-deficient mice. (A) Male female social interaction. A non-significant decrease of the number of ultrasonic vocalization was observed in males Shank3Δ4-22 homozygous mice upon exposure to an estrus female. (B)Social transmission of food preference. A trend toward a reduction of sniffing during the demonstrator interaction phase was observed in in Shank3Δ4-22 homozygous mice. Download Figure 5-1, TIF file.
Extended Data Table 8-1
Individual results and statistical analyses for cohorts 1 and 2 related to social behavior. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. C, center chamber; M, mouse chamber; O, object chamber. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 8-1, DOCX file.
Extended Data Table 9-1
Individual results and statistical analyses for cohorts 1 and 2 related to the avoidance behavior. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 9-1, DOCX file.
Extended Data Figure 6-1
Pictures and individual scores representative of object avoidance behavior. (A) Heatmaps of the novel object recognition task showing object interactions in wild-type and heterozygous Shank3Δ4-22 mice and object avoidance in homozygous Shank3Δ4-22 mice. (B) Representative picture of marble positions and individual scores in the marble burying test showing that in many instance Shank3Δ4-22 homozygous mice left the marbles completely undisturbed.(C) Representative picture of marble positions and individual scores in the nest building test showing a lower quality of nest building in Shank3Δ4-22 homozygous mice with some animals only coarsely shredding the nestlets without building a real nest and others even leaving the nestlets completely untouched. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Download Figure 6-1, TIF file.
Extended Data Table 10-1
Individual results and statistical analyses for cohorts 1 and 2 related to the hyper-reactivity and escape behavior. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 10-1, DOCX file.
Extended Data Table 11-1
Individual results and statistical analyses for cohorts 1 and 2 related to stereotypies, repetitive behavior, perseveration and cognitive flexibility. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 11-1, DOCX file.
Extended Data Table 12-1
Individual results and statistical analyses for cohorts 1 and 2 related to learning and memory. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 12-1, DOCX file.
Extended Data Table 13-1
Individual results and statistical analyses for cohorts 1 and 2 related to anxiety-like behaviors. WT, wild-type mice; Het, heterozygous mice; KO, homozygous knockout mice. Group values are reported as means ± s.e.m. Red font indicates significant results (p < 0.05), orange font indicates trends (0.1 < p < 0.05). Download Table 13-1, DOCX file.
In this issue
Behavioral Phenotyping of an Improved Mouse Model of Phelan–McDermid Syndrome with a Complete Deletion of the Shank3 Gene
