In the previous sections I summarized and concluded the findings of the two experiments, in addition I compared the findings among different learning conditions for the excitatory and inhibitory synapses in HVC. In the previous chapters I cited many related studies, on developmental, or experience-dependent brain structural changes. Here in this conclusion section, I will provide a systematic comparison of my current findings on the songbird HVC structural synapse changes during song learning, with previous findings summarized from these literatures. I will extract common observations from different animals in order to explain my findings, and to propose more general neural structural regulatory principles during behavior development. In the end, based on my findings and discussions, I will describe a very simple scenario of a series of events occur in HVC during the song learning in juvenile zebra finches.
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First, I found an experience-dependent synapse pruning that sensory exposure to the tutor song always yield lower synapse count in HVC compared to age-matched song isolated groups, independent of short or long, good or bad subsequent song imitations. The same was observed in Bengalese finches, that song deprivation lead to significant higher synapse count in HVC. Similarly, increased spine density was also observed in young adult mouse visual cortex (Hofer et al. 2009) and somatosensory cortex (Zuo et al. 2005) after corresponding sensory deprivations.
Taken together, the observed experience-dependent pruning of synapses reflects a rapid reacting and long lasting selection process that those synapses not involving in the corresponding sensory information processing are kept getting pruned away.
Second, I observed a potential excitatory synapse enlargement shortly after initial exposure to the tutor song. Although not significant, this was in agreement with previous reported synaptic enhancement shortly following initial tutoring (Roberts et al. 2010). Similarly, following whisker trimming (as sensory deprivation), the synaptic enlargement was also observe in excitatory synapses in the spared barrel cortex of adult rat (Cheetham et al. 2014). This in general reflected experience-dependent strengthening of local excitatory connections.
Third, I observed multiple times a peak-decline developmental pattern in multiple neurostructural parameters, including the volume of HVC, synapse density, and total synapse number in HVC. This pattern was described in rabbit and cat visual cortex development (Murphy and Magness 1984; Winfield 1981), synaptogenesis in the rat barrel cortex (Micheva and Beaulieu 1996), as well as an early study on the postnatal brain development in the zebra finches (Herrmann and Bischof 1986). The peak was suggested to represent the overproduction of neuronal connections that also reflecting elevated brain structural plasticity at the onset of behavior development. And I combined with my current findings to further elaborate on the subsequent decline pattern, as a result of natural maturation process that normally regulated by critical sensory experience during postnatal development.
Fourth, I observed an increase in the density and number of excitatory synapses in HVC between 60 dph and 90 dph, in the song-tutored birds. I hypothesized this newly formation of excitatory synapses is a consequence of song motor practice, similar to the observation in juvenile and adult mouse motor cortex following motor learning (Fu et al. 2012; Xu et al. 2009).
Fifth, I found an interesting rapid formation of inhibitory synapses induced by initial sensory exposure to the tutor song. In the adult mouse barrel cortex, it was reported persisted whisker stimulation induces rapid inhibitory synapse formation (Knott et al.
2002), which is quite similar to my finding. The author hypothesized a role of the inhibitory synapse dynamics in the regulation of local circuit plasticity. Together with my finding, the regulatory role of the inhibitory circuit could be expanded to be exist during both postnatal development and adulthood.
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Lastly, I observed the developmental formation of inhibitory synapses in HVC occured mostly between 30 dph and 60 dph, reflecting a delayed development pattern compared with the excitatory synapses. Similar result was reported at early stage of postnatal development (~ 2 weeks) in the rat barrel cortex, that the maturation of inhibitory circuit lags behind that of excitatory circuit (Micheva and Beaulieu 1996). The author suggested that the inhibitory circuit plays an important trophic role in regulating the development of cortical circuitry, which is in agreement with the previous proposed plasticity regulatory role of the inhibitory circuits. This hypothesis was further supported with observed high dynamics of inhibitory synapses that clearly different from the excitatory synapses innervating the same postsynaptic target in the developing mouse visual cortex (Villa et al.
2016). Related to the previous discussed experience-dependent pruning, the GABAergic inhibition also suggested to regulate the developmental synapse eliminations (Nakayama et al. 2012). Moreover, I observed the percentage of inhibitory synapses in HVC changes with different learning condition constantly together, and changes also with different developmental stages. I hypothesize the changes in the percentage of the inhibitory synapses therefore reflect a shift of the network E/I balance in HVC. The shift of E/I balance is always accompanied with brain plasticity changes during the critical period (Hensch 2004), and therefore studied intensively in the cat and rodent visual cortex development (Hensch and Fagiolini 2004). In summary the local GABAergic circuit regulates the plasticity of the experience-dependent development of the visual cortex (Hensch 1998), and also suggested to regulate the brain plasticity changes during song learning in zebra finches (Yazaki-Sugiyama et al. 2009).
Taken together, these results suggest that a sequence of events occurs in the beginning of song learning. First, sensory exposure to the tutor song drives a shift in the HVC E/I balance, which might be predominately caused by the fast maturation of parvalbumin-positive inhibitory interneurons in HVC (Cummings 2016;
Trachtenberg 2015). Second, the inhibitory circuit plays regulatory role, that facilitates the strengthening of the tutor song information processing in HVC and protects the learned portion of the song (Vallentin et al. 2016). Regulated by the inhibitory circuit, the HVC excitatory circuit undergoes tutor song sensory experience-dependent remodeling that selectively stabilize and strengthening a subset of connections that involved in the processing of the sensory information of the tutor song, while pruning away other connections that fail to function during this process. Last, vocal practice promotes and stabilizes the excitatory premotor synapses in HVC around 90 dph, while the inhibitory synapses are further developmentally pruned to a lower level. These two effects together result in a low percentage of inhibitory synapses in HVC and another shift in the network E/I balance, which might lead to a subsequent decrease of plasticity when approaching the end of the song learning window (Feldman 2000).