Fig 2: NSG1 KO affects motor coordination but not motor learning. (A) The latency to fall from the rotating rod (4–40 RPM) is presented. Means from both groups increased across trials and there were no difference between wild‐type and KO animals. Results are expressed as mean ± SEM. (B) Representative compliant runs of wild‐type (upper) and NSG1 KO (lower) animals depicting automated detection of left hind (LH), right hind (RH), left front (LF), and right front (RF) paws. (C) Pooled data reveal significant differences between genotypes specifically for crucient (Ca, Cb) step patterns but not for alternate (Aa, Ab) or rotary (Ra, Rb) step sequences. (D) Genotypes also differed significantly in total print area, with statistical significance reached for RF, but where all paws showed ~16% reduced print size. Genotypes did not differ in stride length (E), swing time (F), or swing speed (G). n = 12/genotype

Fig 3: NSG1 KO selectively increases diurnal activity. (A) Homecage behavioral monitoring using photocell beam breaks to measure horizontal activity across 48 h. The first 9 h were considered acclimation and excluded from subsequent analyses. There was a highly significant effect of phase (light vs. dark) where animals showed significantly more activity during dark periods (p < 0.0001). (B) NSG1 KO animals were significantly more active than wild‐type animals during the dark phase (p < 0.05), while no difference was found for the light phase (p > 0.05). n = 11 controls and 12 NSG1 KOs

Fig 4: Loss of NSG1 does not cause alterations in open field exploration. (A) Representative heat maps for wild‐type (upper panel) and NSG1 KO (lower panel) animals. (B‐C) Pooled data reveal no significant differences in open field task for measures of total distance traveled (B), time spent along the border (C), nor time spent in the center (D). Results are expressed as mean ± SEM. n = 12/genotype

Fig 5: Validation of global NSG1 knockout (KO) in null animals. (A) Agarose gel showing PCR‐based genotype analysis of a NSG1 heterozygote (lane 2), four NSG1 wild‐type (lanes 3–6), and four NSG1 KO animals (lanes 7–10). DNA ladder (lane 1) is show for molecular weight reference. (B) Western blot illustrating labeling of NSG1 (green) and NSG2 (red) from four wild‐type (left‐most lanes) and four KO animals (right‐most lanes) used in behavioral testing. (C‐D) Immunohistochemical staining of NSG1 (green), NSG2 (red), and DAPI (blue) on parasagittal brain sections from a wild‐type (C) and KO animal (D). Note the robust cerebellar staining of NSG1 antibody in wild‐type brain section while NSG2 signal is relatively low, as well as lack of NSG1 signal in a section from an NSG1 KO animal (bottom right panel in D)