<i>Fmr1 KO</i> mice exhibit increased evoked activity in primary somatosensory cortex during whisker stimulation.

<p>(A) Schematic showing experimental set up of intrinsic optical imaging over primary somatosensory cortex (black circle) during periodic whisker stimulation. (B) Pictures of the thin skull preparation and example images collected from a wild-type (WT) mouse (left) and <i>Fmr1</i> KO mouse (right) mouse. Scale bar = 0.4 mm. Rostral (R), Caudal (C), Lateral (L) and Medial (M) coordinates are shown. (C) Representative examples of data collected during a typical imaging session. Above, a time series of pixel values for the cortical location indicated by the asterisk in the <i>Fmr1</i> KO in panel B. Below, a fast-fourier transform (FFT) of the raw trace extracts the magnitude of the change in reflectance (ΔR/R) corresponding to the frequency of whisker stimulation (red square). (D) The number of pixels within the region of response with ΔR/R magnitudes greater than the threshold indicated on the abscissa for WT (n = 10) and <i>Fmr1</i> KO (n = 10) mice. The response to whisker stimulation is elevated in <i>Fmr1</i> KO mice (WT vs. KO, p = .011; 2-way ANOVA).</p

The Gap Cross task is a whisker-dependent sensory learning paradigm.

<p>(A) Schematic of the gap cross learning task. Motion sensors positioned at four points along the 2 platforms (labeled #1–4) track the mouse as it moves from the starting platform across a given gap distance to the target 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).</p

<i>Fmr1</i> KO mice display normal learning on the gap cross assay at shorter gap distances but impaired learning at longer whisker-dependent distances.

<p>(A) The percent successful crosses averaged across the first six sessions and subsequent six sessions across gap distances ranging from 3.0 cm to 6.0 cm for both wild-type mice (black lines, n = 6) and <i>Fmr1</i> KO mice (blue lines, n = 9). For each distance, the line marker on the <i>left</i> is the average success rate of the first six sessions and the connected line marker on the <i>right</i> is the average success rate of the subsequent six sessions. Error bars represent standard error of the mean. (B) At shorter ‘nose’ distances, both wild-type (WT) and Fmr1 KO mice (KO) improve to a greater percentage of successful crosses between the average of the first six sessions (WT, grey, KO light blue) and the last six sessions (WT, black, KO dark blue). This improvement is statistically significant (WT, p = .007; n = 6; KO, p<.001, n = 9; WT; two-way ANOVA) (C) At whisker-dependent distances, wild-type (WT) improve between early sessions (grey line) and subsequent sessions (black line) despite the lower overall success rate at increasing gap distances. However, KO mice do not display significant improvement between early sessions (light blue line) and later sessions (dark blue line) (WT, p = .002, n = 6, KO, p = .14; n = 9, two-way ANOVA). (D) Average improvement for WT and KO mice at shorter ‘nose’ distances and longer ‘whisker’ distances. WT mice display significantly greater improvement at whisker-dependent distances that KO mice (p = .02, two-tailed t-test).</p

Open Field.

<p>A) 5-HTT KO mice show significantly decreased distance traveled throughout the arena in a novel open field. This is seen during the first 3 minutes of the test. B) KO mice show a non significant increase in latency to enter center zone C) There was no significant genotypic differences in number of entries into the center zone D) There was no significant genotypic difference in time spent in the center zone. Error bars represent standard error of the mean.</p

Marble Burying.

<p>5-HTT KO mice compared to WT mice buried significantly less marbles placed in a novel cage; *p<0.001. Error bars represent standard error of the mean.</p

Significant correlation of rCBF with behavioral freezing scores in the left and right hemispheres (L/R).

<p>Arrows (↑, ↓) indicate a positive or negative correlation of rCBF with the behavioral freezing score. Areas significant after correction for multiple comparisons are marked with an *p<0.05.</p

Factorial analysis examining the effect of genotype, conditioning or the interaction.

<p>Depicted are select coronal slices (anterior-posterior coordinates relative to bregma) of the template brain. Colored overlays show statistically significant effects of genotype or conditioning or their interaction, but do not reflect the direction of the effect. Abbreviations are from Franklin and Paxinos mouse atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023869#pone.0023869-Franklin1" target="_blank">[67]</a>: BL (basolateral amygdaloid nucleus), BM (basomedial amygdaloid nucleus), Ce (central amygdala), I (insular cortex), La (lateral amygdaloid nucleus), M1 (primary motor cortex), M2 (secondary motor cortex), MO (medial orbital cortex), PrL (prelimbic cortex), RS (retrosplenial cortex). Mouse brain atlas figures were reproduced from the mouse brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023869#pone.0023869-Franklin1" target="_blank">[67]</a> with modification and with permission from Elsevier.</p

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

Dendritic spine turnover and stability are normal in <i>ngr1</i>−/− mice.

<p>(A) Repeated <i>in vivo</i> two-photon imaging through cranial windows in EGFP-M transgenic mice reveals the turnover and stability of dendritic spines on the apical dendrites of layer V pyramidal neurons in S1 barrel cortex. Scale bar = 10 µm. The boxed region (yellow) is shown at higher magnification in panel B. (B) Dendritic spines were imaged every four days for twelve days. Solid arrowheads (yellow) are examples of spine gains. Outlined arrowheads (yellow) are examples of spines lost. Scale bar = 2 µm. (C) The turnover of dendritic spines every four days in WT (n = 5; 1512 spines) and <i>ngr1</i>−/− mice (n = 4; 1106 spines) is similar across 4-day intervals (p>0.4). The average across all sessions is also comparable (p>0.9). (D) The average percent of spines gained and lost is similar between WT (n = 5) and <i>ngr1−/−</i> mice (n = 4) (gained p>0.2; lost p>0.9). (E) The survival fraction of spines present on day 0 re-examined at days 4, 8, and 12 is nearly identical (p>0.8) (F) The percent of new spines present on day 12 is similar between WT and <i>ngr1−/−</i> mice. (p>0.2) (G) The fraction of new spines appearing on day 4 that are transient (p>0.3), surviving less than 4 days, those lasting less than 8 days (present only on day 4 and 8) (p>0.8), and persistent spines surviving more than 8 days (p>0.1) are similar between WT and <i>ngr1−/−</i> mice. (H) Timeline of acute deletion of <i>ngr1</i> in <i>ngr1flx/flx;Cre-ER</i> mice following tamoxifen injection and imaging schedule as NgR1 protein levels decline. (I) Basal cortical spine dynamics in S1 barrel cortex are unaffected by acute deletion of <i>ngr1</i>. The turnover ratio does not change with the decline or absence of NgR1 protein (n = 3, 6 neurons, 1083 spines) (p>0.9).</p