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.