In Vivo Volume Dynamics of Dendritic Spines in the Neocortex of Wild-Type and Fmr1 KO Mice

Apical dendritic spines of M-line mice in the mouse visual cortex imaged at an interval of 2 d. A, Sample images of a mouse with considerable pulsation artifacts obtained with conventional and resonant Galvano scanners. The resonant scanner was relatively robust in response to motion artifacts. Scale bar, 2 μm. B, C, Examples of a time-lapse series of dendrite images from WT (B) and KO (C) mice. Spine numbers correspond to the respective traces in the volume–time courses in Figure 2, A and B. Scale bar, 3 μm. D, Spines 3 and 7 in C are displayed at a different brightness level from that used in C. Scale bar, 1 μm.

Changes in spine volumes in the mouse visual cortex per 2 d. A, B, Time course of spine volume changes for the spines shown in Figure 1, B and C. Trace numbers correspond to the spines numbered in Figure 1, B and C. C, D, Spine-head volume changes (ΔV) per 2 d in WT (C) and Fmr1 KO (D) mice. Data were obtained from 754 spines from 15 dendrites in five WT mice and from 878 spines from 20 dendrites in five KO mice.

Slow dynamics of spine-head volume measured per 2 d. A, B, The dynamics of spine-head volumes in WT (A) and KO (B) mice plotted against V. Each plotted point represents the SD of spine-head volume changes in 32 pooled spines of similar volume. Error bars represent 95% confidence intervals of the estimated SD. The red lines represent the least squares fits. The red line for WT mice (A) is represented by 0.21V ^2/3 + 0.041, and for KO mice (B) it is represented by 0.32V ^2/3 + 0.041. Note that the values of σ were below the red lines for small spines. C, D, Double logarithmic plots of the dynamics of spine-head volumes shown in A and B. Each plotted point represents the SD of spine-head volume changes in 32 pooled spines of similar volume. Error bars represent 95% confidence intervals of the estimated SD. The black lines with a slope of two-thirds fit well with the data. The red lines are reference lines with a slope of 1. E, F, Slow spine-head volume changes in WT (E) and Fmr1 KO (F) mice plotted against V ^2/3 (note that the numbers and scales on the x-axis represent V). Each plotted point represents the SD of spine-head volume changes in 32 pooled spines of similar volume. The solid line is a fit to the data, and can be decomposed into a slow component (dashed lines; Eq. 6, confidence interval of the slope 0.18–0.22) and a fast component, obtained using measurements at 10 min intervals (dotted lines; Eq. 4) for E, and Equation 7 (95% confidence interval of the slope 0.24–0.32) for F. The slope of the fitted line for KO mice is significantly larger than that for WT mice (generalized linear model, t statistic = −3.67, p = 0.0006^a). G, H, The drift of the spine-head volume changes in WT (G) and Fmr1 KO (H) mice plotted against the initial V value. The abscissa is linear in V ^2/3, while the numbers and scales represent V as in E and F. Each plotted point represents the mean of spine-head volume changes in 32 pooled spines of similar volume. Error bars represent the SEM. The lines are the same for the pooled data in WT and KO mice (Eq. 8).

Fast volume changes in apical dendritic spines in the mouse visual cortex. A, B, Spine-head volume changes in WT (A) and KO (B) mice measured at an interval of 10 min. C, Spine-head volume changes in a fixed sample imaged at an interval of 2 d. D, E, Head volume changes (ΔV) per 10 min in WT (D) and Fmr1 KO (E) mice. Data were obtained from 475 spines from two dendrites in two WT mice, and from 1433 spines from four dendrites in three KO mice.

Fast dynamics of spine-head volume. A, B, The fast component of the dynamics of spine-head volume in WT (A) and KO (B) mice plotted against V ^2/3. Each plotted point represents the SD of spine-head volume changes in 30 pooled spines of similar volume. Error bars represent 95% confidence intervals of the estimated SD. The line represents the least squares fit of the pooled data for WT and KO (Eq. 4). C, D, Fast component of the drift of spine-head volume in WT (C) and KO (D) mice plotted against V ^2/3. Each point represents the mean of spine volume changes in 30 spines of similar volume. Error bars represent SEM values. Because the drifts were very small, they were fitted by zero (line).

Elimination of dendritic spines per 2 d. A, B, Example of a time-lapse series of images of a dendrite in WT (A) and KO (B) mice at an interval of 2 d. Arrowheads indicate eliminated spines. Scale bar, 2 μm. C, The elimination rates of dendritic spines per 2 d. Solid bars represent measurement data. Error bars represent SEM values from 6 and 12 dendrites for WT and Fmr1 KO mice, respectively. KO mice seemed to have a higher elimination rate, although the difference was not significant (Mann–Whitney U test, U = 17, p = 0.08^b). Hatched bars represent elimination rates calculated from the model shown with the dashed lines (Fig. 3E,F; Eqs. 6–8). Error bars represent 95% confidence intervals.