2.4. Discussion
3.4.5. Summary of the results of Experiment II
It was demonstrated in Experiment I that HVC ultrastructure can be modified by song experience during song development in juvenile zebra finches. By examining HVC samples of juvenile birds at the same age but with different length of tutoring, any differences among the groups were then assumed to be restricted to the extrinsic factor, which was song experiences. Nevertheless, during critical period learning, the brain structure can be regulated by both intrinsic and extrinsic factors (Andersen 2003; Kirkwood, Lee, and Bear 1995). Thus, the co-regulation of HVC structural development that is associated with song development by age (intrinsic factor) and song experience (extrinsic factor) was further investigated and decoupled in Experiment II.
The structural development of HVC of juvenile zebra finches was tracked during song development in both song-isolated and song-tutored conditions. Three postnatal development time points, 30 dph, 60 dph, and 90 dph, were chosen to represent the early, middle, and late stages of song development in zebra finches.
Controlled tutoring was provided in between the early and middle song development stages (35 dph – 59 dph) to all juvenile birds equally in the song-tutored groups. A group in which HVC samples were extracted and examined at early development stage (ISO_30), served as the common baseline for birds in both the song-isolated and song-tutored conditions. Changes in HVC structure in the song-isolated groups at the early, middle and late developmental stages were assumed to solely result from the impact of age as the intrinsic factor of song development. With the same age increments as the song-isolated groups, the changes in the HVC structure in the song-tutored groups were assumed to result from the combined impact of age as the intrinsic factor and song experience as the extrinsic factor. Differences between the age-matched song-isolated and song-tutored groups at 60 dph and 90 dph were then assumed to result solely from the impact of song experience as the extrinsic factor on song development.
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The song-learning performances of the juvenile birds were generally expected. The birds in the later song development stage (90 dph) scored higher than birds in the middle stage (60 dph). Examination of the song-tutored birds from both Experiments I and II, demonstrated that the tutoring paradigm induced performance difference. At about 60 dph, the juvenile birds learned much better with limited daily tutor song exposure provided in Experiment II (TUT_60 group) compared with the saturated daily tutor song exposure provided in Experiment I (LONG group). The poor song learning performance of birds in Experiment I, was deduced to be resulted from the saturated sensory, not only auditory though, exposure to the tutor which have been noticed by previous studies.
Even though approached by previous studies, making a direct link between song learning and structural changes in HVC is tricky, for two reasons. First, the song learning performance was quantified by the vocal similarities of the juvenile song to the tutor song. A successful imitation of the tutor song requires a good sensory memory of the tutor song and a good motor performance of the BOS. These two processes are learned during two consecutive learning phases in the zebra finches (M S Brainard and Doupe 2000), and both require participation of HVC. Currently we have no knowledge about whether the sensory song learning and the sensorimotor song learning share the same neural substrate in HVC. In a bird exhibiting good sensory learning but poor sensorimotor learning of the tutor song and consequently scored low in the final song similarity analysis, we might still observe the sensory learning associated structural changes in HVC. However a correlation of the changes with the song learning would not be established and thus be overlooked. Second, the song learning process itself is very complex and might involve different dynamics in different individual birds (Tchernichovski 2001).
Even without a tutor, juvenile zebra finches have been shown to be able to self-tutor with their own isolated song (Fehér et al. 2017). Moreover, the vocal imitation strategy could be affected by the complexity of the acoustic features of the tutor song, the developmental stage, and arbitrary innate preferences of the juvenile bird.
Some of these factors, such as the tutor song template that is delivered and the developmental stage of the juvenile bird, can be experimentally controlled or measured. However, controlling or experimentally tracking the individual song learning trajectory of the juvenile bird would be difficult. Given that different learning strategies might recruit different neural substrates (Hayashi-Takagi et al.
2015; Lerch et al. 2011), a direct correlation between HVC structure and song-learning behavior might not always be obvious. In studies of song learning in zebra finches to date, similarity to the tutor song is still used as a golden measure of song learning performance, regardless of the learning trajectory, strategy, or innate preference of the juvenile bird. All these currently neglected factors could be nontrivial, and have impact on the song learning related HVC structural changes.
Currently for this project, song learning performance was measured and analyzed as a way to confirm song learning behavior. The song-isolated birds were deprived of song learning, the song-tutored birds learned to sing a song from the tutors, and the performances improved in general with development.
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A peak-decline neural development pattern (Murphy and Magness 1984) was observed in almost all the HVC neural structural features examined in Experiment II, in both the song-isolated an song-tutored birds. These included the volume of HVC (not for the song-tutored groups, see Results section 3.3.2), the synapse density and number including both asymmetric and symmetric subtypes, and the percentage of the symmetric synapses in HVC. Take the HVC volume for example, it first increased from 30 dph to 60 dph and then decreased from 60 to 90 dph to a lower value that roughly reflects the level in adult. This peak-decline pattern was first proposed in an early study that reported the brain volume and neuronal size exhibited the peak-decline pattern in nearly all of the brain regions examined during postnatal development in zebra finches (Herrmann and Bischof 1986). In this study the author measured the size of HVC as well as the size of HVC neurons at multiple time points during postnatal development (1, 5, 10, 20, 40, 70, and 100 dph, and > 1 year) from zebra finches housed in social conditions (so no song- or social- isolation nor controlled song tutoring, but rather close to the natural environment that juvenile birds can hear their father’s song as well as other male zebra finches’ songs). The author also measured the visual areas in the birds and found the same peak-decline development trend. Taken together their findings and similar findings in other animals (Murphy and Magness 1984) the author suggested the ‘peak’ might represent the overproduction of neuronal connections and that the later ‘decline’ to the adult value might represent the selection process that stabilizes functional synapses and eliminates connections that fail to function when critical neural input, such as the auditory information of the tutor song, reaches the related brain areas. In the song-isolated groups in Experiment II, the decline in the later stage was not experience-dependent because the song-isolated groups were completely deprived from any tutor song sensory exposure. Therefore, the declining pattern in the song-isolated groups should be better explained as the natural maturation process with which HVC plasticity decreases near the end of the critical period.
Nevertheless, the song-tutored groups also exhibited this peak-decline pattern, and always resulted in a lower value compared with the age-matched song-isolated groups (except for the HVC volume at 90 dph, and the percentage of symmetric synapses at 60 dph; for data, see Results sections 3.3.2, 3.3.3, 3.3.4 and 3.3.5).
These findings supported the original decline hypothesis that tutor song information reaches HVC and selectively maintains a subset of HVC synapses, both asymmetric and symmetric, while eliminating fail-to-function synapses. Therefore, the original peak-decline hypothesis of postnatal neural development can be further elaborated with the findings of Experiment II: the decline pattern of the neural structural features was a result of natural maturation process, and normally regulated by critical experience during the postnatal development.
In Experiment I, I found the same synapse density decrease in HVC of both the asymmetric and symmetric subtypes in the song-tutored condition compared with the song-isolated condition (except for the SHORT group which I will discuss later;
see Results sections 2.3.3 and 2.3.4, and Discussion sections 2.4.2 and 2.4.5). I
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explained these results as an experience-dependent synaptic pruning effect. The results of Experiment II further supported this conclusion. It also extended the observed experience-dependent pruning of neural connections to a later stage of song development (90 dph), and highlighted the effect of critical experience during the observed peak-decline neural structural development.
The symmetric (inhibitory) synapses in HVC make up the minority of the HVC total synapse population. The percentage of inhibitory synapses ranges from 7% at the onset of song development (30 dph) to roughly 23% at 60 dph and then eventually 14% at 90 dph. The developmental patterns exhibited by inhibitory and excitatory synapses in HVC are quite different. The majority of the inhibitory synapses were formed between 30 dph and 60 dph (see Results section 3.3.5, symmetric synapses).
As discussed in the last sections (section 3.4.3 and 3.4.4), the number and percentage of the inhibitory synapses in HVC are modulated by song experience at both 60 dph and 90 dph. Interestingly, the development of inhibitory synapses has shown to be modulated in different directions by sensory experience including both formation (Knott et al. 2002) and elimination (van Versendaal et al. 2012), probably depending on the specific role of inhibition plays in processing these sensory experiences. Aside of regulating brain excitability (Groen et al. 2014; Kosche et al.
2015), plasticity (Chen et al. 2011), and formation of receptor field (Vallentin et al.
2016; van Versendaal et al. 2012), the inhibitory circuits have also been found to regulate developmental synapse pruning (Nakayama et al. 2012). This might explain the formation of large numbers of inhibitory synapses prior to the synapse pruning of both synapse subtypes between 30 dph and 60 dph, and could actually unite with the observed sharp increase of inhibitory synapses after 1-day of tutoring (SHORT group) in Experiment I.
The findings of Experiment II suggested that the pruning effect was much more pronounced at 60 dph compared with 90 dph. At 90 dph, the differences between the song-isolated and song-tutored groups were no longer significant (see Table 3. 17 and Table 3. 18), suggesting experience-dependent synapse pruning during song learning mostly occurred during the middle of the critical period or was tightly related to the song experience (tutoring window, 35 dph – 59 dph). When the end of the critical period of song learning approached, the number of synapses of both types seemed to converge onto a similar stable level, regardless of song-learning experience. This suggested that an intrinsic homeostasis mechanism (Turrigiano and Nelson 2004) that is independent of learning experience might drive the structural maturation process in order to stabilize the HVC network and produce stereotyped behavior later.
On the structural level, the developing neural circuits maintain stable function through dynamic homeostatic adjustment in synapse number and strength, while facing potential rapid changes of external environments or stimuli, such as input of critical sensory experience. For example, by coupling the strengthening of a subset
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of excitatory synapses to a balanced overall network excitation level (weakening or pruning of the rest excitatory synapses), the homeostasis mechanism can independently introduce competition among synapses, which in turn resulted in targeted synapse pruning, formation, and potentiation (increase in synaptic weight).
Many of these listed features were observed in both Experiments I and II, such as rapid excitatory synapse enlargement and inhibitory synapse formation shortly following sensory exposure to the tutor song; developmental as well as song experience-dependent excitatory synapse pruning, in HVC during song development.
Taken together, these observations suggest that there is a homeostasis plasticity mechanism regulating the structural plasticity in HVC during song development in juvenile zebra finches, and also actively balancing the synapse activities in HVC upon song learning.
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Chapter 4
Summary and Conclusion
In the last chapter of this PhD thesis, I will summarize the scientific findings of both Experiment I and II and compare them systematically with the findings from other studies. I will first describe and illustrate the structural synapses remodeling happened in HVC, as concluded from the two experiments, in two sets of schematic figures. Then I will combine the findings of Experiment I and II, to summarize and illustrate the experience-dependent developmental patterns of the excitatory synapses and inhibitory synapses respectively. In the end, I will integrate all the findings and propose general common principles of structural synapse remodeling associated with brain functional change, while taking the song learning in juvenile zebra finches as a model system.