Research ArticleNEUROSCIENCE

Stable but not rigid: Chronic in vivo STED nanoscopy reveals extensive remodeling of spines, indicating multiple drivers of plasticity

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Science Advances  09 Jun 2021:
Vol. 7, no. 24, eabf2806
DOI: 10.1126/sciadv.abf2806
  • Fig. 1 Repetitive superresolution of the mouse motor cortex using STED microscopy.

    (A) Microscope design: A custom-made STED microscope is attached to a microscope stand. The pulsed 483-nm excitation is temporally synchronized electronically with the 595-nm STED light pulses and merged spatially by dichroic mirrors (DM). After passing two galvanic mirrors in the scan-head, the light is imaged by a scan (SL) and tube lens (TL) before being focused by a glycerol immersion objective (numerical aperture, 1.3) with a correction collar. The mouse is mounted via a head bar on an adjustable heating plate. Vital functions are controlled by a pulse oximeter (MouseOx). (B) After window implantation and a 3-week recovery period, the mouse was imaged twice a week. (C) Representative raw data example of an apical dendrite of a pyramidal neuron in the motor cortex of a Thy1-GFP-M mouse imaged at day 1 (left) and day 4 (right). An axon captured in the same field of view is marked by (*). (D) Magnification of marked region in (C). Images are maximum-intensity projections of six frames. APD, avalanche photodiode detector; BP, band-pass filter; MMF, multimode fiber; QW, quarter wave plate; SMF, single-mode fiber. Colorbar, 0 to 212 photon counts. Photo credit: Waja Wegner, University Medical Center Göttingen.

  • Fig. 2 Chronic STED imaging of dendritic stretches in layer 1 of the motor cortex.

    (A) Superresolution reveals changes of spine nanoplasticity of large, mushroom-type, stable spines (inset). Images are maximum-intensity projections of raw data. Colorbar, 0 to 120 photon counts. (B and C) Trajectories of neck length (B) and head size (C) of spines marked in (A).

  • Fig. 3 Spine morphometric parameters are largely uncorrelated, while head size and neck width, but not neck length, exhibit multiplicative dynamics.

    (A) Features assessed of stable spines are head size, spine neck length, and neck width. (B to D) Histogram of the logarithmic spine head sizes (B), spine neck lengths (C), and neck width (D). (B and D) The log10 data are normally distributed indicated by a Gaussian fit (black line). (E to G) Correlation between spine parameters. Spine head size (E) and neck width (F) as a function of neck length, and head size as function of neck width (G); linear regression (black) and Pearson’s correlation coefficient r. (H to J) The angle between the dendrite and the spine neck (α, neck-dendrite angle) (I) and between the neck and the spine head (β, neck-head angle) (J) is measured. (K) Principal components (PC) analysis of five morphological parameters. (B to G and I to K) All data of all time points pooled. Number of analyzed spines are listed in table S1.

  • Fig. 4 In stable spines, head size and neck length fluctuate independently and are more persistent than fluctuations in neck width, while changes in head size and neck width do correlate.

    (A) Representative examples of changes in spine head size. (B) The morphological parameters are stable over the observation period of 24 days. Data are means ± SEM. (C) Cumulative distribution of relative changes of neck length (gray), neck width (red), and spine head size (blue) over 3 to 4 days. Fraction of spines that changed spine head size within ±10% (gray area) is indicated by blue horizontal lines, while the same for neck length is indicated by gray and for neck width by red horizontal lines (light red area denotes relative change in size to lower than −10%, while changes exceeding 10% are indicated by the green area). (D and E) Head size (D) and neck length (E) after four different time intervals Δt plotted against their size at time t. Straight line is a linear regression, r is the Pearson’s correlation coefficient, and dashed is the line of unity. (F to H) Pearson’s correlation coefficient plotted over lag time Δt for head size (F), neck length (G), and neck width (H). (I) Normalized changes of head size and neck length are not correlated. (J to L) Pearson’s cross-correlation between different spine parameter for different lag times. Neck length to head size (J) and neck length to neck width (L) are uncorrelated, while neck width to head size (K) shows a significant correlation for up to 14 days. Inset scatter plot and linear regression at lag time Δt = 0 for illustration. (F to H and J to L). Error bars are SD of bootstrapped data. (B to L) All data of the same time interval were pooled. Numbers of analyzed spines are listed in table S1.

  • Fig. 5 Spine parameters differ between transient and stable spines.

    (A) Spine categories based on lifetime (open circle, spine not present; filled circle, spine present). (B to D) Gained and lost spines show significantly smaller head sizes (B), smaller neck length (C), and thinner neck width (D). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are median ± 95% CI. Numbers of analyzed spines are listed in table S1.

  • Fig. 6 Nanoscopy in SOD transgenic mice.

    (A) Representative example of a GFP-expressing dendrite in a CTR and a SOD transgenic mouse showing changes in spine head size within 3 days (images are maximum intensity projections). (B) CTR mice show higher spine density than SOD mice. (C to F) Changes in spine parameter. While the head size (C) of stable spines was increased in SOD mice, no difference was observed in neck length (D), but neck width (E) was decreased in SOD mice. (F) Contour plot of head size and neck length shows a shift to larger head sizes for SOD mice (nbr, number). *P < 0.05. Data in (B) are means ± SEM and in (C to E) are median ± 95% CI. Numbers of analyzed spines are listed in table S1. Data were pooled.

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