2.4. Discussion
3.3.2. Volume of HVC
The volumes of HVC in each bird and the group means are listed in Table 3. 3, and illustrated in Figure 3. 11.
Table 3. 3: The volumes of HVC. The measure in the left and right hemispheres were averaged for each bird (3rd column). The mean ± SE HVC volume is shown for each bird and group.
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Figure 3. 11: The HVC volume. The boxes represent the volumes of individual birds.
The error bars indicate the SE which representing the intra-hemisphere variance of the HVC volume in a given bird. The colored bars represent the group means, with the group means ± SE written in them. The colors in the boxes and bars indicate their group (red: ISO_30; brown: ISO_60; dark blue: TUT_60; yellow: ISO_90; light blue:
TUT_90). *p < 0.05 in a Wilcoxon rank-sum test.
All of the subsequent figures for Experiment II are plotted similarly, if not specified otherwise.
The data from two hemispheres were averaged for each bird, and data from 4 birds were averaged for each group.
The ISO_30 group had the lowest mean volume of HVC, 0.34 mm3, and the volumes were increased at 60 dph (ISO_60), 0.54 mm3, and decreased at 90 dph (ISO_90), 0.46 mm3, in the song-isolated groups. In the song-tutored groups, the HVC volumes were increased at 60 dph (TUT_60), 0.45 mm3, and increased more at 90 dph (TUT_90), 0.49 mm3.
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Table 3. 4: The results of the pairwise Wilcoxon rank-sum test of the HVC volumes. P values less than 0.05, which indicate significance, are written in bold font. A significant difference was found for the comparison of the ISO_30 and ISO_60 experimental groups.
The mean group HVC volume was significantly smaller in the ISO_30 group compared with the volume of the ISO_60 group. No significant differences were found from any of the other comparisons (Table 3. 4).
In summary, these results suggested that, when the birds were deprived of sensory exposure to the tutor song, the HVC volume significantly increased by 59% between 30 dph and 60 dph. It then slightly decreased by 9% between 60 dph and 90 dph, which was not significant. When the birds were given the proper sensory exposure to the tutor song, the HVC volume still increased by 35% between 30 dph and 60 dph and increased slightly more by 7% between 60 dph and 90 dph, but both were not significant changes. Comparisons between age-matched song-isolated and song-tutored groups did not reveal any significant differences in HVC volume. In general, HVC grows between 30 dph and 60 dph and then remains stable between 60 dph and 90 dph, and the volume is not influenced by song experience.
3.3.3. Synapse density
The results for synapse density, including asymmetric and symmetric synapse subtypes, in HVC of each bird and group are listed in Table 3. 5, and illustrated in Figure 3. 12, Figure 3. 13, and Figure 3. 14.
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Table 3. 5: The synapse densities of the total synapses, asymmetric synapses, and symmetric synapses in the HVC. The dissectedsynapses were first identified and then classified into asymmetric or symmetric subtypes. The number of dissected synapses, number of dissectors, and summed dissector volume (after correcting for tissue deformation) in the ssSEM dataset for each bird are listed. The calculated values of the mean ± SE synapse densities in the HVC are shown for each bird and group. *Table 3. 12 & Table 3. 15 are organized similarly, if not specified otherwise.
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Figure 3. 12: Synapse densities in HVC (for data, see Table 3. 5), as determined with ssSEM datasets. See Figure 3. 11 legend for additional information. Wilcoxon rank-sum tests were performed between the groups. The significant differences between the groups are not shown on the figure for simplicity (for the test results, see Table 3. 7).
The total dissector counts for each bird were around 100, with the resulting sampling volume ranging approximately from 400 to 500 µm3. The overall synapse density in HVC at 30 dph (ISO_30) was 4.55 × 108 synapses/mm3, it increased to 5.43 × 108 synapses/mm3 at 60 dph (ISO_60), and then decreased to 4.10 × 108 synapses/mm3 at 90 dph (ISO_90) in the song-isolated groups. For the song-tutored groups, the density decreased to 2.75 × 108 synapses/mm3 at 60 dph (TUT_60) and then increased to 3.32 × 108 synapses/mm3 at 90 dph (TUT_90). For the age-matched comparisons between the song-isolated and song-tutored groups, the synapse density in HVC was always lower in the song-tutored groups.
Table 3. 6: The parameters and statistics of the linear mixed-effects model that was fit to the synapse density data as measured from ssSEM datasets. The fitted equations are Equation 3. 11 – Equation 3. 15 for the five experiment groups. For the ISO_30 group, the p value indicates the significance of the estimated group mean compared to zero. For the ISO_60 and ISO_90 groups, the Estimates represent the deviations of their
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estimated values from the values of the ISO_30 and ISO_60 groups, respectively. The p values indicate the significance of the deviations of the estimates for the ISO_60 and ISO_90 groups over the 30-day intervals, which represents the significance of the contribution of the fixed group effects (development) to synapse density in HVC. For the TUT_60 and TUT_90 groups, the Estimates represent the deviations of their estimated values from the values of the ISO_60 group and ISO_90 groups, respectively.
The p values indicate the significance of the deviations of the estimates for the tutored groups from their age-matched song-isolated groups, which represents the significance of the fixed group effects (tutoring) to HVC synapse density.
*Table 3. 8, Table 3. 10, and Table 3. 13 are similarly organized, if not specified otherwise.
The linear model fit all 2084 dissectors of the 20 birds from the 5 groups. The model-fitting statistics are listed in Table 3. 6.
The model revealed good estimates of the group means (ISO_30: 4.56 × 108 synapses/mm3, ISO_60: 4.56 + 0.87 = 5.43 × 108 synapses/mm3, TUT_60: 5.43 – 2.69 = 2.74 × 108 synapses/mm3, ISO_90: 5.43 – 1.30 = 4.13 × 108 synapses/mm3, and TUT_90: 4.13 – 0.81 = 3.32 × 108 synapses/mm3) compared with the calculated group means listed in Table 3. 5. The estimates of the means of the song-isolated groups ISO_60 and ISO_90 were calculated according to their deviations from the mean of the ISO_30 and ISO_60 groups, respectively. These estimated deviations reflected potential developmental effects, in a 30-day interval, on synapse density in HVC. The estimated deviations were significant in both groups, indicating the potential significant differences due to development in the song-isolated groups. The estimates of the means of the song-tutored groups TUT_60 and TUT_90 were calculated according to their deviations from the mean of their age-matched song-isolated groups, ISO_60 and ISO_90, respectively. These deviations from the means of the song-isolated groups reflected potential effects of tutoring on the synapse density in HVC at different developmental stages (60 dph and 90 dph).
These deviations were very significant in the TUT_60 and TUT_90 groups, indicating the potential significant differences due to tutoring between the age-matched song-isolated and song-tutored groups.
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Table 3. 7: The results of the pairwise Wilcoxon rank-sum test on the synapse density in HVC. P values less than 0.05, which indicate significance, are written in bold font.
Significant differences were found in all of the comparisons except between the ISO_30 and ISO_90 experimental groups.
With 4 birds as the 4 data points in each group, the group-wise rank-sum test results are listed in Table 3. 7. Significant differences were found between each pair except between the ISO_30 and ISO_90 groups.
In summary, these results suggested that, when the juvenile birds were deprived of sensory exposure to the tutor song, the synapse density in HVC increased by 19%
from 30 dph to 60 dph and then decreased by 32% from 60 dph to 90 dph. Both the initial increase and later decrease in density were significant. When the juveniles were provided song tutoring, the HVC synapse density decreased drastically by 40%
from 30 dph to 60 dph and then increased by 21% from 60 dph to 90 dph. Both the initial decrease and later increase in density were significant. When the age-matched groups were compared, both tutored groups show significantly decreased HVC synapse densities compared to the age-matched song-isolated groups.
Comparisons between groups that differed in more than one condition, such as developmental stage and song experience, will not be discussed. It is too difficult to make conclusions from such comparisons. The differences could arise from development, or song experience, or both.
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Figure 3. 13: The asymmetric synapse densities in HVC (for data, see Table 3. 5) , as determined with ssSEM datasets. See Figure 3. 11 legend for additional information.
In HVC, the asymmetric synapse density at 30 dph (ISO_30) was 4.23 × 108 synapses/mm3, it increased to 4.34 × 108 synapses/mm3 at 60 dph (ISO_60), and then decreased to 3.30 × 108 synapses/mm3 at 90 dph (ISO_90) in the song-isolated groups. For the song-tutored groups, the density decreased to 2.12 × 108 synapses/mm3 at 60 dph (TUT_60) and then increased to 2.87 × 108 synapses/mm3 at 90 dph (TUT_90). For the age-matched groups, the asymmetric synapse density in HVC was always lower in the song-tutored groups compared to the song-isolated groups.
Table 3. 8: The parameters and statistics of the linear mixed-effects model that was fit to the asymmetric synapse density data as measured from ssSEM datasets. This table shows that the group effects (development and tutoring) contributed significantly to the asymmetric synapse density differences in HVC in all of the groups except the ISO_60 group.
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The model-fitting resulted in good estimates of the group means (ISO_30: 4.23 × 108 synapses/mm3, ISO_60: 4.23 + 0.11 = 4.34 × 108 synapses/mm3, TUT_60: 4.34 – 2.23 = 2.11 × 108 synapses/mm3, ISO_90: 4.34 – 1.02 = 3.32 × 108 synapses/mm3, and TUT_90: 3.32 – 0.46 = 2.86 × 108 synapses/mm3) compared to the calculated group means listed in Table 3. 5. The estimated mean of the ISO_60 group did not significantly deviate from the group mean of ISO_30 (p = 0.58), while the deviation of the ISO_90 mean from ISO_60 was significant. The estimated means of the song-tutored groups were both significantly lower than those of their age-matched song-isolated groups. These results suggested that there might be a significant developmental effect between 60 dph and 90 dph on HVC asymmetric synapse density, while tutoring had significant effects on this density at both ages.
Table 3. 9: The results of the pairwise Wilcoxon rank-sum tests of the asymmetric synapse density in HVC. P values less than 0.05, which indicate significance, are written in bold font. Significant differences were found in all of the comparisons except between the ISO_30 and ISO_60, and ISO_90 and TUT_90 experimental groups.
The group-wise rank-sum test results are listed in Table 3. 9. Significant differences were found for each pair of comparisons except between the 30 dph and 60 dph song-isolated groups and the song-isolated and song-tutored groups at 90 dph.
In summary, the asymmetric synapse density followed a similar pattern of development as the total synapse density in HVC. When the juvenile birds were deprived of sensory exposure to the tutor song, the HVC asymmetric synapse density increased, although not significantly, by 3% from 30 dph to 60 dph and then decreases significantly by 24% from 60 dph to 90 dph. When the juvenile birds were provided the proper sensory experience of the tutor song, the HVC asymmetric synapse density of the juvenile birds decreased significantly by 50% from 30 dph to 60 dph and then increased significantly by 35% from 60 to 90 dph. At 60 dph, the song-isolated groups had significantly higher (more than twice) HVC asymmetric
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synapse densities compared to those in the song-tutored groups. At 90 dph, the song-isolated groups had higher HVC asymmetric synapse densities compared with the song-tutored group, but the difference was not significant (p = 0.06).
Figure 3. 14: The symmetric synapse densities in HVC (for data, see Table 3. 5) , as determined with ssSEM datasets. See Figure 3. 11 legend for additional information.
In HVC, the symmetric synapse densities were much lower compared to the asymmetric synapse densities in all of the groups. In the song-isolated groups, the HVC symmetric synapse density was 0.32 × 108 synapses/mm3 at 30 dph (ISO_30), it increased drastically to 1.08 × 108 synapses/mm3 at 60 dph (ISO_60), and then decreased to 0.80 × 108 synapses/mm3 at 90 dph (ISO_90). For the song-tutored groups, at 60 dph (TUT_60), the density value increased a lot to 0.63 × 108 synapses/mm3 and then decreased to 0.46 × 108 synapses/mm3 at 90 dph (TUT_90).
For the age-matched comparisons between the song-isolated and song-tutored groups, the asymmetric synapse density in HVC was always lower in the song-tutored groups.
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Table 3. 10: The parameters and statistics of the linear mixed-effects model that was fit to the symmetric synapse density data as measured from ssSEM datasets. This table shows that the group effects (development and tutoring) contributed significantly to the HVC symmetric synapse density differences in all of the groups.
The model-fitting statistics of the symmetric synapse density in HVC are listed in Table 3. 10. The model-fitting resulted in good estimates of the means of the groups (ISO_30: 0.32 × 108 synapses/mm3, ISO_60: 0.32 + 0.76 = 1.08 × 108 synapses/mm3, TUT_60: 1.08 – 0.46 = 0.62 × 108 synapses/mm3, ISO_90: 1.08 – 0.28 = 0.80 × 108 synapses/mm3, and TUT_90: 0.80 – 0.35 = 0.45 × 108 synapses/mm3) when compared to the calculated group means listed in Table 3. 5.
The estimated deviations of the means of all of the groups were significant with the model-fitting statistics. These results suggested that there might be significant developmental effects on HVC symmetric synapse density at 60 dph and 90 dph compared to their 30-day younger song-isolated groups, ISO_30 and ISO60, respectively. In addition, it also suggested tutoring significantly affected the HVC symmetric synapse densities at both ages.
Table 3. 11: The results of the pairwise Wilcoxon rank-sum tests of the symmetric synapse densities in HVC. P values less than 0.05, which indicate significance, are written in bold font. Significant differences were found in all of the comparisons except between the ISO_30 and TUT_90, TUT_60 and ISO_90, and TUT_60 and TUT_90 experimental groups.
The group-wise rank-sum test results are listed in Table 3. 11. Significant differences were found between each pair of comparisons except between the 60 dph and 90 dph song-tutored groups. The other two nonsignificant test results were between groups that differed in both age and tutoring experience and were therefore ignored.
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In summary, the symmetric synapse density in HVC followed an obviously different developmental curve compared with the curve found for the HVC asymmetric synapse density. When juvenile birds were deprived of sensory exposure to the tutor song, their HVC symmetric synapse density increased significantly more than three times from 30 dph to 60 dph and then decreased significantly by 26% from 60 dph to 90 dph. When the juvenile birds were provided the proper sensory experience of the tutor song, their HVC symmetric synapse density increased significantly almost one time from 30 dph to 60 dph and then decreased not very significantly by 27%
from 60 dph to 90 dph. As was observed for total synapse density and asymmetric synapse density in HVC, the song-tutored groups also showed significantly decreased symmetric synapse densities in HVC compared to the age-matched song-isolated groups.
3.3.4. Percentage of symmetric synapses
The percentage of the symmetric synapses in HVC estimated for each bird and group are listed in Table 3. 12 and illustrated in Figure 3. 15.
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Table 3. 12: The percentage of the symmetric synaspes in HVC. The percentage of the symmetric synapses was jackknife resampled from the dissector data. The resulting average percentage value for each bird, is shown with the standard deviation in the 5th column. The group mean values are shown with SE in the last column.
Figure 3. 15: The percentage of symmetric synapses in HVC (for data, see Table 3. 12) , as determined with ssSEM datasets. See Figure 3. 11 legend for additional information.
The error bars indicated the standard deviations of the jackknife resampling. The Wilcoxon rank-sum tests were performed between the groups. The significant differences between the groups are not shown on the figure for simplicity, and the test results are provided in Table 3. 14.
The percentages were determined with jackknife resampling of the dissector data (as described in section 3.2.6), and each single resampled symmetric synapse percentage was calculated from a given dissector with Equation 3. 4. The number of samplings and the sample distribution were therefore the same as the density quantification results (see Table 3. 12, 4th column and Figure S. 13).
In the song-isolated groups, the group mean percentage of symmetric synapses in HVC was 6.98% at 30 dph (ISO_30 group), and it increased at 60 dph (ISO_60 group) to reach 19.99% and stayed at almost the same level at 90 dph (ISO_90 group) with a little drop to 19.56%. For the song-tutored groups, the symmetric synapse percentage in HVC increased to 22.91% at 60 dph (TUT_60 group) and then decreased a lot to 13.64% at 90 dph (TUT_90). For the age-matched comparisons, the percentage of the symmetric synapses in HVC was higher in the song-tutored group at 60 dph but lower at 90 dph compared to song-isolated groups.
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Table 3. 13: The parameters and statistics of the linear mixed-effects model that was fit to the jackknife resampled symmetric synapse percentage data as measured from ssSEM datasets. This table shows that the group effects (development and tutoring) contributed significantly to the symmetric synapse percentage differences in HVC in all of the groups except ISO_90 group.
The linear model, which fit all 2084 jackknife resampled dissector data of the 20 birds from the 5 groups, resulted in the model-fitting statistics listed in Table 3. 13.
The model resulted in good estimates of the mean group percentages (ISO_30:
6.98%, ISO_60: 6.98% + 13.00% = 19.98%, TUT_60: 19.98% + 2.92% = 22.90%, ISO_90: 19.98% – 0.43% = 19.55%, and TUT_90: 19.55% – 5.91% = 13.64%) compared to the calculated group means listed in Table 3. 12. The estimated deviations were significant in all of the groups except group ISO_90. These results suggested that the developmental effects significantly affected the HVC symmetric synapse percentages at 60 dph compared to 30 dph, but not at 90 dph compared to 60 dph. In addition, tutoring also significantly affected the HVC symmetric synapse percentage at both ages.
Table 3. 14: The results of the pairwise Wilcoxon rank-sum tests of the symmetric synapse percentages in HVC. P values less than 0.05, which indicate significance, are
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written in bold font. Significant differences were found in all of the comparisons except between the ISO_60 and ISO_90 experimental groups.
The group-wise rank-sum test results are listed in Table 3. 14. Significant differences were found in each pair of comparisons except between the 60 dph and 90 dph song-isolated groups.
In summary, the percentage of the HVC symmetric synapses was very low (6.98%) at 30 dph, which mainly due to the very low symmetric synapse density at this early development stage (see Table 3. 5 and Figure 3. 14, ISO_30 group). When juvenile birds were deprived of sensory exposure to the tutor song, the percentage of the HVC symmetric synapses significantly increased almost two times from 30 dph to 60 dph and then insignificantly decreased 2% to 19.56% at 90 dph. When the juvenile birds were provided tutoring, the percentage of the HVC symmetric synapses significantly increased more than two times from 30 to 60 dph and then significantly decreased by 40% from 60 to 90 dph. The age-matched comparisons between the song-isolated and song-tutored groups showed significantly higher percentage of symmetric synapses in HVC in the song-tutored group at 60 dph.
However, at 90 dph, the symmetric synapse percentage was significantly lower in the song-tutored group.