Fig. 4.
Actl6b−/− mice exhibit autism-related behaviors. (A) Social interaction and control tests: relative interaction time between the adult test mouse and a juvenile mouse (3–5 wk) or novel object. Open-field test measured activity over 20 min as total distance traveled. (B) Schematic of three-chamber sociability assay: Test mice may enter the zone with a novel object or the zone with a novel juvenile mouse. Social preference scores were calculated from time spent in each zone using the formula shown. (C) Box plots showing male and female littermates of Actl6b+/− × Actl6b+/− crosses with gene dosage-dependent impairment in social interaction with a juvenile mouse but not with (D) a control novel object. (E) Actl6b+/− and Actl6b−/− mice of both sexes showed defects in sociability, which were most severe in female knockouts. (F) Single, repeated photobeam breaks or “stereotypic counts” represented by a small red box in an open field. Male Actl6b−/− mice showed repetitive movements indicated from increased stereotypic counts over 120 min. (G) Diagram of Barnes maze test of spatial memory: an elevated, white, circular open field containing 12 holes, with one “target” escape hole leading to a comfortable cage. Visual cues provided the mouse with a frame of reference for the location of the target hole. Male Actl6b−/− mice spent less time around the target hole, indicating impaired memory as to the location of the target hole. (H) Actl6b−/− mice of both sexes showed increased activity, not observed in Actl6b+/−. Tests shown in C, E, and H were conducted at Stanford University on a cohort of mice highly backcrossed to the C57BL/6 background; mice (+/+, +/−, −/−): n = 9, 12, 7 males, n = 11, 12, 10 females for C; n = 10, 13, 6 males, n = 11, 12, 9 females for D; n = 10, 12, 7 males, n = 11, 12, 9 females for E; n = 10, 13, 6 males, n = 11, 11, 5 females for H. Values for each test in this cohort were as follows: F(2,30)Females = 79.87 and F(2,25)Males = 59.49 for C; F(2,29)Females = 0.29 and F(2,26)Males = 0.74 for D; F(2,29)Females = 6.82 and F(2,26)Males = 4.83 for E; and F(2,24)Females = 13.44 and F(2,26)Males = 8.60 for H. Stereotypic counts in F and spatial memory in G were assessed at Fujita Health University on adult male mice that were the F1 offspring of a cross between Actl6b+/− 129S6/SvEv × Actl6b+/− C57BL/6. Mice (+/+, −/−): n = 22, 21 in F, and n = 22, 14 in G. Significance was calculated for B, E, and H using a one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison post hoc. Significance for F and G was calculated using Student’s t test: t41 = 8.68 in F and t34 = 3.99 in G. Whiskers indicate 10th and 90th percentiles. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.