HVC synapse density, including the density of asymmetric and symmetric synapse subtypes, can be measured directly with FIB-SEM datasets. To further compare the FIB-SEM and ssSEM datasets, the synapse density of the same sample that had been measured with the ssSEM dataset and dissector method (see Method section 2.2.5) in Experiment I, was measured again with the FIB-SEM datasets.
For any given brain sample, the total number of synapses (or the total number of asymmetric synapses or symmetric synapses) N was obtained from the FIB-SEM dataset. The physical volume of the FIB-SEM dataset V was easily calculated with the known voxel size and overall size of the FIB-SEM datasets. The density of synapse NV, was thus calculated with the same equation as Equation 2. 2.
The resulting synapse density of each bird is provided as a single data point. The mean group synapse density value was calculated by averaging the values of all the birds belonging to the same group. The HVC synapse density results that were obtained with the FIB-SEM datasets of each bird and group are listed in Table S. 9, and illustrated in Figure S. 16, Figure S. 17, and Figure S. 18, for the total synapses, asymmetric synapses, and symmetric synapses, respectively. Difference in the densities among groups were evaluated with Wilcoxon rank-sum test, and differences with p value less than 0.05 were considered significant.
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Table S. 5: HVC synapse densities that were obtained with the FIB-SEM datasets from the brain sample blocks for all the birds used in Experiment I. The overall synapse density and densities of the asymmetric and symmetric synapse subtypes are listed. The number of synapses counted and classified and the sampling volume of the FIB-SEM datasets (after calibration) for each bird are listed. The calculated HVC synapse densities, including the synapse subtypes, are presented with SE for each bird and group.
The total number of synapses counted and classified with the FIB-SEM datasets for each bird were around 400, with sampling volumes roughly around 500 µm3.
Figure S. 16: The HVC synapse densities, which were determined with FIB-SEM datasets from the brain sample blocks. The colored boxes represent the synapse densities of individual birds. Since for each bird the synapse density was a single data point, there was no error bar generated to show the variations within the data. The colored bars
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represent the group means. The group means ± SE are written in the colored bars. The synapse density data for each bird and group are listed in Table S. 9. No significant differences were found among the groups.
The mean group densities in HVC for total synapses were 7.98 × 108 synapses/mm3 in the ISO group; 8.00 × 108 synapses/mm3 in the SHORT group, which was almost the same as in the ISO group; and decreased to 7.50 × 108 synapses/mm3 in the LONG group.
Figure S. 17: The HVC asymmetric synapse densities, which were determined with FIB-SEM datasets from the brain sample blocks. The colored boxes represent the asymmetric synapse densities of individual birds. No error bars generated to show the variations within the data due to the single data point that representing each bird. The colored bars represent the group means. The group means ± SE are written in the colored bars. The asymmetric synapse density data for each bird and group are listed in Table S. 9. No significant differences were found among the groups.
The mean group densities in HVC for asymmetric synapses were 6.14 × 108 synapses/mm3 in the ISO group; 5.89 × 108 synapses/mm3 in the SHORT group, indicating a small decrease; and 5.79 × 108 synapses/mm3 in the LONG group, showing a larger decrease.
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Figure S. 18: The HVC symmetric synapse densities, which were determined with FIB-SEM datasets from the brain sample blocks. The colored boxes represent the symmetric synapse densities of individual birds. No error bars generated to show the variations within the data due to the single data point that representing each bird. The colored bars represent the group means. The group means ± SE are written in the colored bars. The symmetric synapse density data for each bird and group are listed in Table S. 9. *p<0.05 in a Wilcoxon rank-sum test.
The mean group densities in HVC for symmetric synapses were 2.31 × 108 synapses/mm3 in the ISO group; 2.65 × 108 synapses/mm3 in the SHORT group, indicating a small increase; and 2.28 × 108 synapses/mm3 in the LONG group, indicating a decrease.
Table S. 6: The results of the Wilcoxon rank-sum tests of the HVC total synapse densities that were obtained with FIB-SEM datasets in the groups in Experiment I. No significant differences were found among the groups.
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Table S. 7: The results of the Wilcoxon rank-sum tests of the HVC asymmetric synapse densities that were obtained with FIB-SEM datasets in the groups in Experiment I. No significant differences were found among the groups.
Table S. 8: The results of the Wilcoxon rank-sum tests of the HVC symmetric synapse densities that were obtained with FIB-SEM datasets in the groups in Experiment I. P values less than 0.05 indicating significance, are written in bold font. Significant differences were found between the ISO and SHORT groups, and the SHORT and LONG groups.
The HVC synapse densities that were obtained with the FIB-SEM datasets resulted in higher values compared with the densities obtained with the ssSEM datasets (see Result section 2.3.2, 2.3.3, and 2.3.4). However, the relative differences between the groups in the results obtained with the two methods were similar. Similar patterns of decreased densities were observed in HVC total synapse density and HVC asymmetric synapse density. In addition, the same higher density was observed only in the SHORT group with the HVC symmetric synapse density results. The differences among groups were not significant except for the SHORT group that had a significantly higher symmetric synapse density in HVC than the other two groups.
In summary, the HVC synapse density measurements were systematically higher in the results obtained with the FIB-SEM dataset compared with the results obtained with the ssSEM dataset and dissector method. Similar group-wise comparisons were observed with both methods, but the statistics were different. The change in the asymmetric and symmetric synapse densities in HVC among the ISO, SHORT and LONG groups were confirmed with both methods.
The comparison and properties of the two different type of EM datasets, together with the quantification methods are discussed in the end of chapter 2 (see Discussion section 2.4.1).