Article Figures & Data
Figures
- Figure 1.
During development, spine heads become smaller and spine necks become shorter and wider. A, E, Dendritic segments from CA1 (A) and L5 pyramidal cells (E) examined at P14 and P37. B–D, F–H, Kolmogorov–Smirnov tests of the cumulative frequency distributions show that for both CA1 (B–D) and L5 pyramidal cells (F–H), the distribution profiles of spine head widths (p < 0.001 for CA1; p < 0.02 for L5), neck lengths (p < 0.01) and neck widths (p < 0.001) significantly differ between the two ages. Here, whether the P value is considered significant or not is corrected for using the false discovery rate method, which controls false positives during multiple comparisons. Using two-way ANOVA with post hoc Bonferroni multiple comparisons, mean differences in these parameters also show age and brain region-specific differences in the magnitude of the effects. I, For head width, age effect: F(1,10) = 49.94, p < 0.0001; region effect: F(1,10) = 31.16, p = 0.0002; age × region interaction: F(1,10) = 8.79, p = 0.01. J, For neck length, age effect: F(1,10) = 12.25, p = 0.006; region effect: F(1,10) = 40.14, p < 0.0001. K, For neck width, age-specific effect (F(1,10) = 14.61, p = 0.003) only in CA1 (p ≤ 0.01). Shaded areas around lines show interanimal variability. In bar graphs, data are represented as mean ± SEM, where n = number of animals. Scale bar, 1 μm. *p < 0.05; **p < 0.01.
- Figure 2.
Absence of correlation between spine morphological parameters. A–F, Spine morphological parameters do not appear to strongly covary with each other in CA1 or L5 at either P14 (A–C) or P37 (D–F). At P14, there are no correlations between spine head width and neck width (A: for CA1, R2 = 0.01; for L5, R2 = 0.02) or between spine head width and neck length (C: for CA1 and L5, R2 = 0.00). Similarly, no correlations are observed between these parameters at P37 either (D: head width vs neck width for CA1, R2 = 0.06; for L5, R2 = 0.00; F: head width vs neck length for CA1, R2 = 0.00; for L5, R2 = 0.03). A weak negative correlation is observed between spine neck length and neck width in both brain regions at both ages [B: at P14, for CA1, R2 = 0.21 (p < 0.001); for L5, R2 = 0.18 (p < 0.001); E: at P37, for CA1, R2 = 0.15 (p < 0.001); for L5, R2 = 0.20 (p < 0.001)], suggesting that longer necks are likely to be narrower and vice versa. In scatter plots, the red symbols represent data from CA1 and gray symbols represent data from L5.
- Figure 3.
Loss of FMRP affects spine morphology at the nanoscale in an age and brain region-specific manner. A, E, I, M, Dendritic segments from CA1 and L5 pyramidal cells from wildtype and Fmr1KO mice at P14 (A, I) and P37 (E, M) show age and brain region-specific spine morphology changes. B, At P14, the cumulative frequency distributions of spine head widths in CA1 significantly differ between genotypes (Kolmogorov–Smirnov test: p = 0.007). C, D, No differences are observed for spine neck lengths (C) and widths (D). F–H, At P37, the distribution profiles of spine head widths (F), neck lengths (G), and neck widths (H) in CA1 are all significantly different between genotypes (Kolmogorov–Smirnov test: for head width, p = 0.03; for neck length, p = 0.02; for neck width, p = 0.04). J–P, For L5 cells, only the distribution profile of spine neck widths is significantly different at P14 (L; Kolmogorov–Smirnov test: P value = 0.004) between genotypes (J–P). Here, whether the P value is considered significant or not is corrected for using the false discovery rate method, which controls false positives during multiple comparisons. Shaded areas show the interanimal variability. Scale bar, 1 μm. *p < 0.05; **p < 0.01.
- Figure 4.
The developmental trajectory of spine morphogenesis is essentially maintained in the absence of FMRP. A–F, For both CA1 and L5, the cumulative frequency distributions of spine head width (A, D), neck length (B, E), and neck width (C, F) all significantly differ between P14 and P37 in Fmr1KO mice (Kolmogorov–Smirnov test: in CA1, for all p < 0.0001; in L5, for head width, p = 0.009; for neck length, p = 0.006; for neck width, p < 0.0001). Here, whether the P value is considered significant or not is corrected for using the false discovery rate method, which controls false positives during multiple comparisons. Using two-way ANOVA with post hoc Bonferroni multiple comparisons, mean differences in these parameters also show that the Fmr1KO mice mimic the developmental changes that occur in wildtype mice. For both regions, there is only a significant age effect of the morphological parameters. G–I, In CA1, for head width (G): F(1,11) = 73.39, p < 0.0001; for neck length (H): F(1,11) = 61.06, p < 0.0001; for neck width (I): F(1,11) = 32.62, p = 0.0001. J–L, In L5, for head width (J): F(1,12) = 12.28, p = 0.004; for neck length (K): F(1,12) = 8.64, p = 0.01; and for neck width (L): F(1,12) = 5.03, p = 0.04. Shaded areas around lines show interanimal variability. In bar graphs, data are represented as mean ± SEM, where n = number of animals. *p < 0.05; **p < 0.01.
- Figure 5.
The developmental spine morphology changes predict decreased biochemical compartmentalization with age, while loss of FMRP subtly influences this in an age and brain region-specific manner. A, D, During normal development, cumulative frequency distributions of compartmentalization factor significantly differ between ages for spines on both CA1 (A) and L5 (D) pyramidal cells (Kolmogorov–Smirnov test: p < 0.0001). The compartmentalization factor decreases with age, thus predicting decreased biochemical compartmentalization. B, C, In CA1, there are age-specific changes to the distribution of compartmentalization factor with loss of FMRP (Kolmogorov–Smirnov test: P14, p = 0.04; P37, p = 0.05) predicting decreased biochemical compartmentalization at P14 (B), but increased biochemical compartmentalization at P37 (C). E, F, Conversely, loss of FMRP does not affect the distributions of compartmentalization factor for spines on L5 pyramidal cells. Here, whether the P value is considered significant or not is corrected for using the false discovery rate method, which controls false positives during multiple comparisons. G, Using two-way ANOVA with post hoc Bonferroni multiple comparisons, mean differences in compartmentalization factor show age and brain region-specific differences in the magnitude of the effects during development, but not between the genotypes. For wildtype mice, age effect: F(1,10) = 16.37, p = 0.002; region effect: F(1,10) = 20.55, p = 0.001 with no significant interaction. For Fmr1KO mice, age effect: F(1,13) = 27.40, p = 0.0002; region effect: F(1,13) = 22.76, p = 0.0004 with no significant interaction. In bar graphs, data are represented as mean ± SEM, where n = number of animals. *p < 0.05; **p < 0.01.
Tables
Morphology parameter P14 P37 Wildtype Fmr1KO Wildtype Fmr1KO CA1 Head width Range 244–1367 nm 152–1534 nm 165–918 nm 175–1122 nm Mean ± SD per animal 598 ± 15 nm 586 ± 49 nm 393 ± 47 nm 447 ± 21 nm 95% CI on mean 584–612 nm 543–629 nm 340–446 nm 423–471 nm Median ± SD per animal 577 ± 28 nm 556 ± 63 nm 360 ± 45 nm 415 ± 28 nm Neck length Range 81–2307 nm 61–2080 nm 61–1164 nm 62–1064 nm Mean ± SD per animal 482 ± 51 nm 481 ± 52 nm 316 ± 21 nm 264 ± 45 nm 95% CI on mean 432–532 nm 435–527 nm 292–340 nm 212–315 nm Median ± SD per animal 416 ± 72 nm 376 ± 53 nm 239 ± 24 nm 211 ± 24 nm Neck width Range 66–258 nm 60–265 nm 84–465 nm 81–353 nm Mean ± SD per animal 128 ± 4 nm 132 ± 13 nm 165 ± 6 nm 150 ± 4 nm 95% CI on mean 124–132 nm 121–144 nm 159–172 nm 144–158 nm Median ± SD per animal 118 ± 2 nm 124 ± 15 nm 151 ± 8 nm 141 ± 12 nm L5 Head width Range 217–1556 nm 142–1376 nm 201–1560 nm 185–1238 nm Mean ± SD per animal 652 ± 51 nm 619 ± 39 nm 568 ± 28 nm 554 ± 39 nm 95% CI on mean 602–701 nm 588–650 nm 536–599 nm 510–598 nm Median ± SD per animal 593 ± 53 nm 590 ± 25 nm 532 ± 41 nm 535 ± 34 nm Neck length Range 62–2514 nm 81–3192 nm 61–2351 nm 82–2553 nm Mean ± SD per animal 681 ± 86 nm 688 ± 89 nm 587 ± 91 nm 523 ± 61 nm 95% CI on mean 597–765 nm 617–760 nm 484–690 nm 454–592 nm Median ± SD per animal 617 ± 80 nm 577 ± 143 nm 481 ± 124 nm 439 ± 78 nm Neck width Range 67–300 nm 65–383 nm 70–318 nm 72–369 nm Mean ± SD per animal 130 ± 21 nm 119 ± 12 nm 146 ± 12 nm 141 ± 20 nm 95% CI on mean 110–150 nm 110–129 nm 133–160 nm 119–164 nm Median ± SD per animal 124 ± 20 nm 111 ± 10 nm 134 ± 8 nm 130 ± 21 nm
