Synapse size

The distributions of the synapse volume data are presented in Figure 2. 24. The synapse volume data were log-transformed, and then pooled over all birds belonging to the same group. Generally, the synapse volume data of the different groups and different subtypes all appeared to have a log-normal distribution. This distribution pattern was in agreement with reported distributions of synapse sizes in rodent cortex from in-vivo (Loewenstein, Kuras, and Rumpel 2011) and in-vitro (Merchán-Pérez et al. 2014) studies.

Figure 2. 24: Histograms of the distributions of the synapse volume data. All segmented synapses of the birds in each experimental group were pooled together to generate the histograms (for data, see Table 2. 14 and Table 2. 15). The data were log-transformed. A Gaussian distribution was fit to the histograms as shown by the red curves.

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The asymmetric and symmetric synapse segmentations were classified from the overall synapse segmentation results. Thus, the number of samples were smaller in the two synapse subtypes. However, even for the symmetric synapses, which had the smallest sample size (Figure 2. 24, 3^rd column; Table 2. 14, 3^rd column-symmetric synapses), the goodness-of-fit check (Kolmogorov-Smirnov test) still revealed the group sum fit a Gaussian distribution significantly well (see Table 2. 14, 4^th column-symmetric synapses, rows of group sums: ISO: p = 3.55×10^-5, SHORT: p = 4.46×10^-6, LONG: p = 2.72×10^-5). The use of parametric statistical tests, such as two-tailed two-sample t-tests, to compare the group means was therefore valid.

Table 2. 14: The results of the one-sample Kolmogorov-Smirnov tests of the synapse size data. P values less than 0.05, which indicate significance, are written in bold font and represent significantly good fits to the Gaussian distribution.

The synapse size measurements of each bird and group are listed in Table 2. 15, which includes the sizes of the asymmetric and symmetric synapse subtypes in HVC.

The results are shown in Figure 2. 25, Figure 2. 26, and Figure 2. 27 for the total synapses, asymmetric synapses, and symmetric synapses respectively.

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Table 2. 15: The synapse size in HVC, including the data of the asymmetric and symmetric subtypes. The numbers of segmented synapses, the calculated mean ± SE synapse size in HVC are listed for each bird and group.

Figure 2. 25: The synapse sizes in HVC (for data, see Table 2. 15), as determined with FIB-SEM datasets. See Figure 2. 20 legend for additional information.

The total number of the segmented synapses in HVC for each bird were around 300 (Table 2. 15, 3^rd column). The mean synapse size was 3.49 × 10⁶ nm³ in the ISO group. The mean sizes were increased in the SHORT group (4.86 × 10⁶ nm³) and decreased in the LONG group (4.23 × 10⁶ nm³).

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Figure 2. 26: The asymmetric synapse sizes in HVC (for data, see Table 2. 15), as determined with FIB-SEM datasets. See Figure 2. 20 legend for additional information.

The total segmented asymmetric synapses in HVC of each bird were around 200 (Table 2. 15, 3^rd column). The mean synapse size was 4.01 × 10⁶ nm³ in the ISO group. The size was increased almost 40% (5.67 × 10⁶ nm³) in the SHORT group and decreased to 4.79 × 10⁶ nm³ in the LONG group.

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Figure 2. 27: The symmetric synapse sizes in HVC (for data, see Table 2. 15), as determined with FIB-SEM datasets. See Figure 2. 20 legend for additional information.

The total numbers of segmented symmetric synapses in HVC of each bird were around 80 (Table 2. 15, 3^rd column). The mean synapse size was 2.46 × 10⁶ nm³ in the ISO group. The mean size was increased to 3.37 × 10⁶ nm³ in the SHORT group and decreased to 3.10 × 10⁶ nm³ in the LONG group.

Table 2. 16: The parameters and statistics of the linear mixed-effects model fit to the synapse size data as measured from FIB-SEM datasets. The group effects (tutoring) did not significantly contribute to the synapse size differences in HVC, except for the asymmetric synapse size difference in the SHORT group. See Table 2. 4 for additional information.

The statistics of the linear models fit to the overall synapse size, asymmetric synapse size, and symmetric synapse size in HVC are all listed in Table 2. 16. The models either fit in total 3821 segmented synapses, or alternatively fit 2884 segmented asymmetric synapses, or fit 875 segmented symmetric synapses, in the 12 birds from the 3 groups.

The models resulted in good estimates of the group means for all three modeled parameters (Table 2. 16, 1^st column) compared to the measured values in Table 2. 15.

No significant deviations were found, as indicated by the p values in Table 2. 16, except for the asymmetric synapse size estimation in the SHORT group, which had a p value of 0.05.

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Table 2. 17: The results of the pairwise two-sample t-tests of the log-transformed synapse sizes in HVC. No significant differences were found among the experimental groups.

The results of the group-wise two-sample t-tests of synapse size in HVC are listed in Table 2. 17. No significant differences were observed between any of the groups.

The synapses in HVC were smallest in the ISO group, which had a mean size of 3.49 × 10⁶ nm³. The mean size was increased (4.86 × 10⁶ nm³) in the SHORT group and decreased (4.23 × 10⁶ nm³) in the LONG group. No significant differences were observed among the three groups.

Table 2. 18: The results of the pairwise two-sample t-tests of log-transformed asymmetric synapse size in HVC. No significant differences were found among the experimental groups, except for a slight trend of significance (p = 0.07) between the ISO and SHORT groups.

The results of the group-wise two-sample t-tests of asymmetric synapse size in HVC are listed in Table 2. 18. No significant differences were found except a slight trend of significance observed between the ISO and SHORT groups, as suggested by both the linear model (p = 0.05) and the two-sample t-test (p = 0.07).

The asymmetric synapses in HVC were smallest in the ISO group (mean size, 4.01

× 10⁶ nm³). Their sizes increased by 41% (5.67 × 10⁶ nm³) in the SHORT group and then decreased (4.79 × 10⁶ nm³) in the LONG group.

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Table 2. 19: The results of the pairwise two-sample t-tests of the log-transformed symmetric synapse sizes in HVC. No significant differences were found among the experimental groups.

The results of the group-wise two-sample t-tests of the symmetric synapse size in HVC are listed in Table 2. 19. No significant differences were observed between any of the groups.

The symmetric synapses in HVC were also smallest in the ISO group (mean size, 2.46 × 10⁶ nm³). Their sizes slightly increased (3.37 × 10⁶ nm³) in the SHORT group and then decreased (3.10 × 10⁶ nm³) in the LONG group. No significant differences were observed among the three groups.

To summarize, the average size of synapses in HVC is smallest in the ISO group, as well as the asymmetric and symmetric subtypes. In the SHORT group which the juvenile birds received 1 day sensory exposure to the tutor song, the sizes of synapses on average are bigger compared to the ISO group. This was observed in both asymmetric and symmetric synapses in HVC. However none of the differences are significant in the two-sample t-test. A slight trend of significance (p = 0.07) was observed in the asymmetric synapse sizes between the ISO and SHORT group.

These results indicate initial sensory exposure to tutor song might had a potential acute effect that resulted in size increasing in the HVC synapses, especially in the asymmetric synapses. After longer exposure to tutor song, the HVC average synapse size decreases a little, however is not significantly different from either the short-tutored (SHORT) group or the non-tutored (ISO) group (see Table 2. 17).