Article Figures & Data
Figures
- Figure 1.
Development of excitatory SBB morphology. Spinules (blue) were observed within cortical boutons (purple) at every postnatal day age group examined. In 2D TEM sections, spinules were occasionally observed invaginating into SBB profiles (e.g., p21–p28); however, most spinules appeared as ovoid or circular double membrane-bound structures encapsulated within their “host” bouton. PSDs (green) at SBB synapses sometimes contained perforations (e.g., p60–p66 and >p90 panels). Note that SBBs at times displayed multiple spinule cross-sections (i.e., 2D profiles of putative spinules), yet in our 2D TEM analyses, it was not possible to attribute these to single spinules with complex morphologies, or to multiple spinules protruding into a single SBB. s = postsynaptic spine; d = postsynaptic dendrite. Scale bars = 0.5 μm.
- Figure 2.
Excitatory bouton areas and spinule prevalence increase over development. A, 2D synapse profile length, as defined by the PSD, remains relatively stable from before eye-opening (p28) until the end of the critical period for plasticity in ferret visual cortex (p60). However, PSD length increases substantially from the end of the critical period until at least the cusp of maturity (>p90 ages). B, Excitatory presynaptic bouton areas are largest at a time when most boutons appear to have elongated en passant morphology (p21–p28). Bouton size decreases significantly by p46–p47 and then shows a steady increase until at least p90. C, Average spinule profile areas do not appreciable change across the postnatal ages examined. Note that spinule areas show relatively large interanimal variation attributed to the variation in spinule sizes from distinct parent neurite/glia origins. D, Excitatory SBBs are most prevalent in late adolescence (>p90) versus at the height (p46–p47) or end (p46–p47) of the critical period for plasticity in ferret V1. A trend toward increased SBB prevalence is also seen between p46–p47 and p60 ages. For A–D, statistical comparisons were performed between p21–p28 and p46–p47, p46–p47, and p60–p66, and p60–p66, and >p90 (for details, see Table 3). Gray circles = individual animal means; Purple circles = group means ± SEM; **p < 0.015, ***p < 0.001.
- Figure 3.
FIBSEM images of SBBs in p21 and p46 V1. A, Adjacent FIBSEM images of an SBB in L4 of V1 from a p21 ferret. FIBSEM images are ∼25 nm apart in z (depth) on average, picked to display the progression of spinule invagination and engulfment by the SBB. Top panel, Raw FIBSEM images. Bottom panel, Pseudo-colored to highlight SBB (purple), adjacent axon projecting a spinule (orange), and postsynaptic dendrite (red). Left, Full reconstruction of this SBB showing transparent bouton with engulfed adjacent axon spinule and postsynaptic dendrite. Note the macular shaped PSD (green) formed between the SBB and the red dendrite. Identical SBB as shown in Figure 5A,A1. B, Adjacent FIBSEM images of an SBB in L4 of V1 from a p46 ferret. Top and bottom panels arranged and colored as in A, showing a postsynaptic spine (red) projecting a spinule into its presynaptic SBB partner (purple). Left, Full reconstruction of this p46 SBB showing postsynaptic spine spinule enveloped by its presynaptic bouton. Note the perforated complex shaped PSD (green). Identical SBB as shown in Figure 5D,D1. Scale bars = 0.5 μm; 3D scale cubes = 0.5 μm3.
- Figure 4.
FIBSEM images of SBBs in p60 and >p90 V1. A, Adjacent FIBSEM images of an SBB in L4 of V1 from a p60 ferret. FIBSEM images are ∼25 nm apart in z (depth) on average, picked to display the progression of spinule invagination and engulfment into the SBB. Top panel, Raw FIBSEM images. Bottom panel, Pseudo-colored to highlight SBB (purple), adjacent axon/bouton (orange), and postsynaptic spine (red). Note the adjacent bouton (orange) with a synapse onto a spine that protrudes a spinule into this SBB at the bottom left of the first image in the series, and the spinule from the postsynaptic spine that invaginates into this SBB across the middle of its perforated PSD. Left, Full reconstruction of this SBB showing transparent bouton (purple) with engulfed postsynaptic spine (red) and adjacent bouton (orange) spinules. Note the horseshoe-shaped perforated PSD. Identical SBB as shown in Figure 6C,C1. B, Adjacent FIBSEM images of an SBB in L4 of V1 from a >p90 ferret. Top and bottom panels arranged and colored as in A. Note the postsynaptic spine (red) that sends its spinule into its SBB (purple) partner from the edge of the PSD. Left, Full reconstruction of this SBB (purple), made transparent to show the engulfed anchor-like spinule from its postsynaptic spine partner. Macular-shaped PSD (green) appears yellow within spine. Identical SBB as shown in Figure 7D,D1. 2D scale bars = 0.5 μm; 3D scale cubes = 0.5 μm3.
- Figure 5.
3D Reconstructions of p21 and p46 SBBs. Focused ion beam serial electron microscopy 3D reconstructions of SBBs (purple) and their spinules from axon/boutons (yellow), and spines/dendrites (gray). Object color scheme shown at top. A–C, 3D reconstructions from a p21 ferret showing spinules from an adjacent axon (A, A1), postsynaptic dendrite (B, B1), and adjacent dendrite (C, C1) projecting into L4 SBBs. Note that B1 shows the spinule emerging from the edge (below and left) of the PSD. Reconstruction shown in A is the identical SBB shown in Figure 3A. D–F, 3D reconstructions from a p46 ferret showing hook-like spinule from a postsynaptic spine (D, D1), and spinules from adjacent axons and adjacent dendrites (E, E1, F, F1) projecting into L4 SBBs. Note the complex perforated PSD in D1, the identical SBB shown in Figure 3B. In F, F1, an SBB with synapses (green) onto two postsynaptic spines receives a large adjacent dendrite spinule (F) and a large adjacent axon spinule (F1). A1–F1, Identical SBBs as shown to the left in A–F, but with transparent postsynaptic neurites and/or SBBs to highlight the morphology and locations of the PSDs (green) or spinules. 3D scale cubes = 0.5 μm3.
- Figure 6.
3D Reconstructions of p60 SBBs. Focused ion beam serial electron microscopy 3D reconstructions of SBBs (purple) and their spinules from axon/boutons (yellow), and spines/dendrites (gray). Object color scheme as shown in Figure 5. A–C, Reconstructions of a postsynaptic spine head (A) and postsynaptic spine anchor-like spinules (B, C) projecting into L4 p60 SBBs. Note that in C, a second spinule from a synaptic bouton (orange) projects into the “upper” portion of the SBB (same bouton shown in Fig. 4A). C–G, Reconstructions showing axons/bouton spinules of various sizes engulfed by p60 SBBs. Note that in E, a presynaptic bouton (orange) receives a spinule from its postsynaptic spine partner and a portion of this bouton with its engulfed spinule protrude into a large adjacent SBB (purple) with synapses (green) onto three postsynaptic spines. H, An SBB with a synapse onto a postsynaptic dendrite (top) receives a spinule from an adjacent dendrite (bottom). A1–H1, Identical SBBs as shown to the left in A–H, but with transparent postsynaptic neurites and/or SBBs to highlight the morphology and locations of the PSDs (green) or spinules. 3D scale cubes = 0.5 μm3.
- Figure 7.
3D Reconstructions of >p90 SBBs. Focused ion beam serial electron microscopy 3D reconstructions of SBBs (purple) and their spinules from axon/boutons (yellow), spines/dendrites (gray), and glia (pink) from L4 of V1 of a >p90 ferret. Object color scheme as shown in Figure 5. A, B, Reconstructions showing adjacent axons/bouton spinules engulfed by SBBs with synapses onto postsynaptic spines. C–F, Reconstructions of postsynaptic spine heads (C, F), and anchor-like spinules (D, E) projecting into their SBB partners. Note the horseshoe-shaped perforated PSD (green) in C1, and the SBB shown in F, F1 enveloping approximately two-thirds of its postsynaptic spine partner. Reconstruction shown in D is the identical SBB shown in Figure 4B. G, An SBB with a synapse onto a postsynaptic spine receives a spinule from an adjacent glia. H, An SBB with a synapse onto a postsynaptic dendrite engulfs a spinule from an adjacent dendrite. A1–H1, Identical SBBs as shown to the left in A–H, but with transparent postsynaptic neurites and/or SBBs to highlight the morphology and locations of the PSDs (green) or spinules. 3D scale cubes = 0.5 μm3.
- Figure 8.
SBBs progressively engulf spinules as plasticity wanes in V1. A, Percentages of excitatory SBBs across the developmental ages examined in 2D TEM and FIBSEM (3D) images. 3D FIBSEM data at p21, p46, p60, and >p90 are represented by red diamonds, 2D data as shown in Figure 2D. Statistical comparisons were performed between p21 and p46, p46 and p60, and p60 and >p90 (for details, see Table 3). B, FIBSEM images showing an SBB with a synapse onto a postsynaptic spine containing a perforated PSD (top panel), and an SBB with a with a synapse onto a spine displaying a macular PSD (bottom panel). 3D reconstructions of each SBB and their corresponding PSDs are shown to the right. SBBs shown in purple, PSDs shown in green, postsynaptic spines are made transparent to visualize PSDs. Identical SBBs as shown in Figure 7C,D. C, FIBSEM developmental analysis showing the average percentages of excitatory presynaptic boutons with perforated PSDs in L4. D, FIBSEM analysis showing the percentages of SBBs versus Non-SBBs (i.e., excitatory presynaptic boutons without spinules) with perforated PSDs at p60 and >p90. E, FIBSEM analysis showing the percentages of p60 and >p90 SBBs containing at least one spinule from a defined neurite or glial origin. For example, ∼56% of SBBs at p60 contain at least one spinule from an adjacent axon. Scale bars = 0.5 μm for FIBSEM images in B; 3D scale cubes = 0.5 μm3 for FIBSEM reconstructions in B; *p < 0.05, **p < 0.015.
Tables
Age Total boutons Total synapses PSD length (μm) Bouton area (μm2) % SBBs Spinule area (μm2) Spinule:bouton area (%) p21 70 74 0.29 0.34 4% 0.04 12% p28 71 78 0.27 0.42 7% 0.02 6% Group sum
Group mean ± SEM (n = animals)141 152 0.28 ± 0.01 0.38 ± 0.03 6 ± 1.4% 0.03 ± 0.01 9 ± 3.1% Cumulative mean ± SEM (n = sampled structures) 0.28 ± 0.01 0.38 ± 0.03 6 ± 1.4% 0.03 ± 0.01 6 ± 1.5% p46 102 102 0.28 0.19 6% 0.02 6% p47 94 94 0.29 0.21 6% 0.03 12% Group sum
Group mean ± SEM (n = animals)196 196 0.29 ± 0.01 0.20 ± 0.01 6 ± 0.3% 0.03 ± 0.001 9 ± 3.2% Cumulative mean ± SEM (n = sampled structures) 0.29 ± 0.01 0.20 ± 0.01 6 ± 0.3% 0.02 ± 0.004 8 ± 3.2% p60_1 25 29 0.28 0.21 10% 0.04 14% p60_2 111 113 0.29 0.28 13% 0.01 4% p66_1 118 124 0.31 0.28 11% 0.04 14% Group sum
Group mean ± SEM (n = animals)254 266 0.30 ± 0.01 0.26 ± 0.02 11 ± 0.8% 0.03 ± 0.01 11 ± 3.4% Cumulative mean ± SEM (n = sampled structures) 0.30 ± 0.01 0.27 ± 0.01 11 ± 0.8% 0.03 ± 0.01 10 ± 1.7 >p90_1 61 61 0.32 0.29 16% 0.06 16% >p90_2 149 150 0.36 0.33 15% 0.02 5% >p90_3 109 112 0.34 0.35 22% 0.04 4% Group sum
Group mean ± SEM (n = animals)319 323 0.35 ± 0.01 0.32 ± 0.01 18 ± 2.1% 0.04 ± 0.01 8 ± 4% Cumulative mean ± SEM (n = sampled structures) 0.35 ± 0.01 0.33 ± 0.01 18 ± 2.1% 0.04 ± 0.01 8 ± 1% Means and SEM for individual animals and developmental age groups are listed for each 2D TEM analysis measurement (column titles). Population sizes are listed for individual animals and age groups under total boutons and total synapses columns. % SBBs = percentage of sampled excitatory presynaptic boutons that contained spinules. Spinule: bouton area % = percentage of bouton area occupied by spinule area.
- Table 2
Percentages of SBBs, perforated PSDs, spinule origins, and sampled structures for FIBSEM analyses
FIBSEM Age Total boutons Total synapses % SBBs % Perforated PSDs % PS spine % Adj axon % Adj spine % Adj dendrite % Adj glia % Unknown >p90 223 237 24.2 29.5 37.0 35.2 7.4 14.8 5.6 3.7 p60 235 256 14.5 18.4 14.7 55.9 8.8 17.7 0.0 0.0 p46 157 163 6.4 16.6 10.0 50.0 0.0 40.0 0.0 0.0 p21 61 62 6.6 16.1 25.0 25.0 0.0 25.0 0.0 25.0 Total sampled boutons and synapses for each age are listed under total boutons and total synapses columns for FIBSEM analyses at each age examined. Percentages of SBBs and perforated PSDs in FIBSEM analyses are listed in their respective columns for each age examined. Percentages of SBBs containing at least one spinule from a neurite/glial source are listed in their respective columns for each age examined. PS = postsynaptic; adj = adjacent (i.e., not synaptic); unknown = spinules whose origin could not be determined (for details, see Materials and Methods). Since some SBBs contained spinules from multiple sources, percentages of spinules from all origins may sum to greater than 100%.
- Table 3
Statistical Comparisons for 2D Transmission Electron Microscopy (TEM) and 3D Focused Ion Beam Scanning Electron Microscopy (FIBSEM) Analyses.
Comparison Test n (animals, total sampled structures) % Change Power(post-hoc, two-tailed) p value Synapse Length All Ages ANOVA, one-way 6.7 x 10-7* >p90 vs. p60-66 Bonferroni post hoc t Test, two-tailed >p90 (3, 323); p60-66 (3, 266) 16% 0.96 2.1 x 10-4* p60-66 vs. p46-47 Bonferroni post hoc t Test, two-tailed p60-66 (3, 266); p46-47 (2, 196) 5% 0.14 0.327 p46-47 vs. p21-28 Bonferroni post hoc t Test, two-tailed p46-47 (2, 196); p21-28 (2, 152) 2% 0.07 0.669 Bouton Area All Ages ANOVA, one-way 6.0 x 10-13* >p90 vs. p60-66 Bonferroni post hoc t Test, two-tailed >p90 (3, 319); p60-66 (3, 254) 20% 0.82 0.005* p60-66 vs. p46-47 Bonferroni post hoc t Test, two-tailed p60-66 (3, 254); p46-47 (2, 196) 39% 0.98 6.1 x 10-5* p46-47 vs. p21-28 Bonferroni post hoc t Test, two-tailed p46-47 (2, 196); p21-28 (2, 141) 93% 0.99 1.34 x 10-11* Spinule Area All Ages ANOVA, one-way 0.671 Spinule-Bearing Bouton Area >p90 SBBs vs. No Spinules Mann-Whitney U test >p90 SBBs (3, 57); >p90 NoSpin (3, 266) 88% 0.99 2.69 x 10-9* % Spinule-Bearing Boutons 2D All Ages (4 x 2 table) X2 Test, two-tailed, Bonferroni corrected 5.3 x 10-5* >p90 vs. p60-66 X2 Test, two-tailed, Bonferroni corrected >p90 (3, 319); p60-66 (3, 254) 68% 0.73 0.009* p60-66 vs. p46-47 X2 Test, two-tailed, Bonferroni corrected p60-66 (3, 254); p46-47 (2, 196) 92% 0.52 0.041 p46-47 vs. p21 X2 Test, two-tailed, Bonferroni corrected p46-47 (2, 196); p21-28 (2, 141) 8% 0.04 0.863 FIBSEM All Ages (4 x 2 table) < 1.0 x 10-5* >p90 vs. p60 X2 Test, two-tailed, Bonferroni corrected >p90 (1, 223); p60 (1, 235) 67% 0.81 0.008* p60 vs. p46 X2 Test, two-tailed, Bonferroni corrected p60 (1, 235); p46 (1, 157) 127% 0.70 0.014* p46 vs. p21 X2 Test, two-tailed, Bonferroni corrected p46 (1, 157); p21 (1, 61) 3% 0.03 0.959 % Perforated PSD FIBSEM: All Boutons All Ages (4 x 2 table) X2 Test, two-tailed, Bonferroni corrected 0.003* >p90 vs. p60 X2 Test, two-tailed, Bonferroni corrected >p90 (1, 237); p60 (1, 256) 61% 0.82 0.004* p60 vs. p46 X2 Test, two-tailed, Bonferroni corrected p60 (1, 256); p46 (1, 163) 11% 0.06 0.639 p46 vs. p21 X2 Test, two-tailed, Bonferroni corrected p46 (1, 163); p21 (1, 62) 3% 0.04 0.937 FIBSEM: SBBs vs. Non-SBBs >p90 SBBs vs. Non-SBBs X2 Test, two-tailed, Bonferroni corrected >p90 SBBs (1,58); >p90 Non-SBBs (1,179) 87% 0.83 0.001* p60 SBBs vs. Non-SBBs X2 Test, two-tailed, Bonferroni corrected p60 SBBs (1,40); p60 Non-SBBs (1,216) 129% 0.77 0.003* Spinule Origins: >p90 vs. p60-66 % Postsynaptic Spine (>p90 vs. p60) X2 Test, two-tailed, Bonferroni corrected >p90 (1, 54); p60 (1, 34) 152% 0.63 0.024* % Adjacent Axon (>p90 vs. p60) X2 Test, two-tailed, Bonferroni corrected >p90 (1, 54); p60 (1, 34) 37% 0.48 0.030* % Adjacent Dendrite (>p90 vs. p60) X2 Test, two-tailed, Bonferroni corrected >p90 (1, 54); p60 (1, 34) 16% 0.06 0.724 % Adjacent Spine (>p90 vs. p60) X2 Test, two-tailed, Bonferroni corrected >p90 (1, 54); p60 (1, 34) 6% 0.05 0.271 % Adjacent Glia (>p90 vs. p60) X2 Test, two-tailed, Bonferroni corrected >p90 (1, 54); p60 (1, 34) n/a 0.24 0.161 Statistical tests used for 2D TEM and 3D FIBSEM analyses are listed, grouped by each quantified morphology category (left most column) and age group comparison (“Comparison” column). Percent change between means are calculated as:
, where Age2 = Mean of older age group, and Age1 = Mean of younger age group. Power is the post-hoc two-tailed Fisher’s exact test achieved statistical power for each comparison, given the two proportions and sample sizes (n), with ∝ error probability = 0.05. * = comparisons where p < 0.05 or Bonferroni corrected value. Sample sizes for SBB vs. Non-SBB perforated PSDs reflect the total number of SBB and Non-SBB synapses for each age group, and ‘n’ for ‘Spinule Origins’ comparisons reflect the total number of spinule-bearing boutons (SBBs) within each group. PSDs = Postsynaptic densities. 2D = TEM analyses. Non-SBBs = excitatory presynaptic boutons without spinules.
Extended Data Figure 2-1
Estimation statistics plots showing mean differences between developmental synapse length and bouton area comparisons. A, Gardner–Altman estimation plot (Gardner and Altman, 1986) showing the raw synapse profile length values for >p90 and p60–p66 groups (left y-axis), as well as the mean difference (black dot near right y-axis), and the bootstrap 95% confidence interval (vertical error bar from black dot). Mann–Whitney U test, p = 1.5 × 10−4. B, Estimation plot showing raw excitatory bouton areas for all developmental age groups examined. Plots marked as in A, except that mean differences between groups and confidence intervals are shown below each comparison. Mann–Whitney U test, p = 0.004 (p60–p66 vs >p90), and 2.8 × 10−14 (p21–p28 vs p46–p47). Download Figure 2-1, TIF file.
Extended Data Figure 2-2
SBBs are larger than boutons without spinules (no spinule). A, Histogram showing the distribution for the percentage of >p90 SBBs (purple) and >p90 presynaptic boutons without spinules (blue) with various sized areas. Inset, Cumulative histogram of this same comparison. B, Estimation plot showing the raw data, mean difference (black dot), and bootstrapped 95% confidence interval (lines extending from dot) for areas from >p90 SBBs and boutons without spinules. Mann–Whitney U test, p = 2.7 × 10−9; ***p < 0.001. Download Figure 2-2, TIF file.
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