Extended Data Fig. 4: In vitro characterization of Ania3 shRNA construct and in vivo characterization of developmental knockdown (KD_dev) manipulation.

Related to Fig. 4. a, Representative histology by in situ RNA hybridization of unilateral developmental knockdown injection at p15. Image is representative of 3 independent samples. Left: 4x merged image showing Homer1a and shRNA expression, scale bar: 1000 µm. White box indicates the region used for higher magnification images. Center: 20x image of Homer1a expression only (left), mCherry expression only (center), and both Homer1a and mCherry expression (right), scale bars: 100 µm. For all images, Homer1a is shown in green and mCherry is shown in red. b, ex vivo validation of developmental knockdown manipulation assessed by quantification of Homer1a (left), Ania3 (center) and Homer1b/c (right) levels measured by qPCR in PFC samples dissected from Scramble (n = 12) and KD_dev (n = 15), (two-way ANOVA showed significant main effects for group, p < 0.0001, and Homer1 isoform expression, p < 0.0001; post hoc Holm–Sidak’s test for multiple comparisons shows a significant difference in Homer1a, p = 0.0038, and Ania3, p = 0.0451, expression). c, Western blot for Homer1b/c in Scramble and KD_dev mice (n = 4 per group) 4 months after injection. Raw western blot images can be found in Supplementary Fig. 1. d, Startle response in Scramble (n = 12 M + 8 F) and KD_dev, (n = 10 M + 8 F). e-g, Correlations between (e) startle response and PPI, measured as percent inhibition, at 3 dB (PP3), 6 dB (PP6), and 12 dB (PP12) above background, (f) weight (g) and startle response, and (g) weight and PPI. h-i, Performance during nosepoke shaping, where the motor activities required are the same as the SDT training but with no attentional component (that is cue) for scramble (n = 12 M + 7 F) and KD_dev (n = 12 M + 8 F) mice, showing (h) the number of nosepokes per mouse on the day of nosepoke shaping when the mice met criteria to proceed to SDT training and (i) the average latency to nosepoke after retrieving a reward for each mouse that retrieved rewards on the first nosepoke shaping day. No significant interaction between sex and group by two-way ANOVA.j, Auditory brainstem response measured as minimum thresholds in Scramble (n = 4 M + 1 F) and KD_dev (n = 4 M + 1 female), as sound pressure level (dB) in response to increasing frequencies (4, 8, 16, 32 kHz). k, Motor coordination in the Rotarod test for Scramble (n = 13 M + 8 F) and KD_dev (n = 12 M + 8 F), measured as latency (s) to fall from the rod averaged across 4 consecutive trials. Significant difference between sexes but no significant interaction between sex and group by two-way ANOVA. l, Gross motor activity measured as distance moved (inch) by Scramble (n = 13 M + 8 F) and KD_dev (n = 12 M + 8 F) in a square open field arena during a 5-min test. m, Schematic of head-fixed SDT setup (left) and task structure (right). n, Quantification of the latency to first lick (sec) within the decision windows across cue lengths. Each point is the average latency to first lick for the first 3 Go trials per animal (2 s cue: Scramble n = 7 M, KD_dev n = 8 M; 1 s and 0.5 s cues: Scramble n = 8 M, KD_dev n = 7 M). o, Quantification of the latency to first lick jitter across cue lengths. Jitter is quantified as the standard deviation of first lick latencies across the first 3 Go trials (two-way ANOVA showed a significant main effect for group, p = 0.007, and post hoc Holm–Sidak’s test for multiple comparisons showed significant differences between groups at 1 and 0.5 s cues, p = 0.04 for both cue lengths, 2 s cue: Scramble n = 7 M, KD_dev n = 8 M; 1 s and 0.5 s cues: Scramble n = 8 M, KD_dev n = 7 M). p, Schematic of the Attentional Set Shift setup and experiment protocol. q, Latency (s) to retrieve the chocolate pellet measured in Scramble (n = 14 M) and KD_dev, (n = 13 M) mice during the 4 trials of the Attentional Set Shift test. Significant interaction between trial and group, p = 0.04, by repeated-measures two-way ANOVA. r, Working memory performance assessed in a Y-maze apparatus for Scramble (n = 12 M + 7 F) and KD_dev, (n = 13 M + 8 F) mice, measured as correct alternations performed, expressed as a percentage total alternations. Significant difference between sexes but no significant interaction between sex and group by two-way ANOVA. s, Short-term memory tested by a novel object recognition test in Scramble (n = 7 M) and KD_dev, (n = 7 M) mice, measured as time spent exploring the novel object vs the familiar one and expressed as a percentage of total exploration time during a 5 min test. significant main effect for novelty (p < 0.001), but not for group by two-way ANOVA. t-u, Anxiety-like behavior measured as (t) time (in seconds) spent in the center of an open field arena during a 5 min test in Scramble (n = 13 M + 8 F) and KD_dev, (n = 12 M + 8 F) mice, and (u) percentage of time spent in the open arm of an elevated plus maze during a 5 min test in Scramble (n = 12 M + 8 F) and KD_dev, (n = 13 M + 8 F) mice. Significant difference between sexes but no significant interaction between sex and group by two-way ANOVA for both t and u. Data in h-l, n-o, and q-u are expressed as mean ± SEM, and for b and d, upper and lower box limits indicate 75^th and 25^th percentiles, centerline indicates the median, upper and lower whiskers are the maximum and minimum data points.