Axonal bouton turnover and stability are normal in <i>ngr1</i>−/− mice.

<p>(A) Examples of axons imaged repeatedly by repeated <i>in vivo</i> two-photon microscopy through cranial windows. Solid arrowheads (yellow) are examples of new boutons. Outlined arrowheads (yellow) are examples of boutons lost. Scale bar = 10 µm (B) Higher magnification of the boxed region (yellow) in panel A. Scale bar = 5 µm. (C) The turnover of axonal boutons every four days in WT (n = 4, 424 boutons) and <i>ngr1</i>−/− mice (n = 5, 749 boutons) is similar across 4-day intervals in S1 barrel cortex (p>0.2). (D) The average percent of axonal boutons gained and lost is similar between WT and <i>ngr1−/−</i> mice (gained p>0.5; lost p>0.6). (E) The survival fraction of boutons present on day 0 is similar at days 4, 8, and 12. (F) The percent of persistent boutons (p>0.1) and new boutons (p>0.3) present on day 12 is comparable between WT and <i>ngr1−/−</i> mice.</p

Cranial windows are properly positioned over S1 barrel cortex and are a stable preparation for imaging cortical spine dynamics.

<p>(A) An example of optical imaging of intrinsic signals reveals the cortical region responsive to stimulation of the C2 whisker. Scale bar = 0.5 mm (B) Apical dendrites of layer V neurons in the boxed region (yellow) are shown at higher magnification in panels C and D. Scale bar = 50 µm (C) Higher magnification images of the boxed region (yellow) in panel B at day 0 (D) Higher magnification images of the boxed region (yellow) in panel B at day 12 (E) Higher magnification image of the boxed region in panel C on day 0. (F) Higher magnification image of the boxed region in panel D on day 12.</p

Myelination is extensive in S1 barrel cortex and does not reflect the distribution of Nogo-A.

<p>(A) Immunostaining for myelin basic protein (αMBP) of coronal sections of S1 barrel cortex reveals extensive myelination in cortical layers IV–VI at P26 when cortical spine dynamics are elevated relative to P40. Myelination increases in S1 barrel cortex by P40 to extend into layer II/III. (B) Immunostaining of V1 reveals myelination in cortical layers V–VI at P26 that extends into layer IV by P40. The approximate positions of cortical layers II/III to VI are indicated at right. The scale bar corresponds to 200 µm (C) Quantification of the relative distribution of staining intensity for myelin basic protein in S1 and V1 at P24 and P40 at increasing depths from the pial surface to the underlying white matter. Pixel intensity is normalized by the intensity of white matter (grey box). Error bars represent the standard deviation between at least 5 sections and 3 mice per group. (D) Higher magnification image of the distribution of myelinated fibers in layer I of S1 and V1 at P40. Few myelinated fibers are present in layer I of S1 relative to V1. The scale bar corresponds to 50 µm. (E) Nogo-A intensely labels the soma of putative oligodendrocytes but is also evident in cortical neurons. The pattern of expression is comparable at P26 and P40. The approximate positions of cortical layers II/III to VI are indicated at right. The scale bar corresponds to 200 µm.</p

Mice lacking <i>ngr1</i> perform better on the gap cross assay but display normal tactile learning across sessions.

<p>(A) A schematic of the gap cross assay. The movement of a mouse from the starting, or ‘home’, platform to the target platform across a given gap distance is detected with motion sensors positioned at the back and edge of each platform. (B) Activation of each sensor (grey box) indicates the position of the mouse. (C) Successful crosses are defined as the movement of the mouse from the starting platform to the target platform (green circles). Failures are defined as trials in which the mouse approaches the edge of the home or target platform and returns to the back of the home platform (red crosses). (D) <i>ngr1−/−</i> mice cross ‘whisker’ distances at a significantly higher success rate (WT, n = 19; <i>ngr1−/−</i>, n = 14; p>.01 for distances 5.5 and 6 cm; p>.32 for distances 3.5 and 4 cm, two-way ANOVA). This greater success rate is most significant at longer distances, 5.5 cm and 6 cm (**, p>.01 with Bonferroni correction for multiple comparisons). (E) Despite better overall performance, the percent improvement for a given gap distance from the first 4 sessions (left value for each distance) to the second 4 sessions (right value for each distance) is similar for WT and <i>ngr1−/−</i> mice for a given gap distance. (F) WT mice improve with experience at ‘whisker’ gap distances (WT, n = 19, *, p<.05, two-way repeated measures ANOVA) from the first 4 sessions (Early, grey line) to the second 4 sessions (Late, black line). (G) <i>Ngr1</i> mutant mice improve with experience at ‘whisker only’ gap distances (<i>ngr1−/−</i>, n = 14, *, p<.05, two-way repeated measures ANOVA) from the first 4 sessions (Early, pink line) to the second 4 sessions (Late, red line).</p

Motor learning is normal in NgR1 mutant mice but consolidation of fear extinction is impaired.

<p>(A) <i>Ngr1</i> mutant mice display a deficit in overall performance on the rotarod, but the rate of improvement is similar in WT and <i>ngr1−/−</i> mice (WT n = 8, <i>ngr1−/−</i> n = 9) (**, p<.01; *, p<.05). (B) <i>Ngr1</i> mutant mice also exhibit a mild deficit at a slower acceleration rate at the conclusion of training (p<.05, unpaired two-tailed t-test with Welch's correction). (C) The average percent improvement plotted as the percent difference in average latency to fall of the second two trials and last two trials. Improvement is similar between WT and <i>ngr1−/−</i> mice (p>.22, Kolmogorov-Smirnov test) (D) Schematic for fear conditioning and extinction protocol. On day 1, adult female mice were conditioned to an acoustic tone that co-terminated with a 1 second foot shock (0.6 mA). On days 2 and 3, mice were presented with 12 unpaired tones during a 30′ period. (E) Extinction of the fear response is plotted at percent time spent freezing averaged across two consecutive trials on day 2 and day 3 (n = 16 WT, n = 12 <i>ngr1−/−</i>). Extinction between the two genotypes differs across trials during day 2 but not day 3 by RM-ANOVA (bracket, day 2, p<.005; day 3, p>.42). (F) Acquisition of the freezing response is similar between WT and <i>ngr1−/−</i> mice on consecutive trials of the conditioned (tone) and unconditioned (0.6 mA shock) stimulus (n = 16 WT, n = 12 <i>ngr1−/−</i>). (G) Acquisition of the freezing response is similar between WT and <i>ngr1−/−</i> mice on consecutive trials of the conditioned (tone) and a milder unconditioned (0.3 mA shock) stimulus (WT, n = 8; <i>ngr1−/−</i>, n = 6).</p