Structural changes in the brain have been observed to be tightly associated with brain functional changes, such as learning (Draganski et al. 2006), memory formation (Bailey and Kandel 1993), and pathological changes (Grutzendler and
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Gan 2006). In particular, during early postnatal development, structural changes often coincide with the emergence of brain functions such as binocular vision
To characterize the brain structural changes during a critical period is of particular interests to neuroscientists, since brain structure is not merely shaped by the regular developmental processes, but also influenced by external experience (Hensch 2005).
In the primary visual cortex of cats and rodents, normal binocular vision can only be established during a short postnatal period. The permanent loss of normal binocular vision and the anatomical remodeling within the visual cortex following monocular deprivation (MD) are classic examples of critical period development (Morishita and Hensch 2008). During the normal maturation of visual cortex in cat, synapse density rapidly increases (up to 70 days postnatal) and then decreases significantly toward the end of the critical period. Excitatory and inhibitory synapses exhibit different developmental patterns, in that the former increases at a declining rate to a stable level at about 70 days and the latter increases approximately linearly to reach a near adult value at around 110 days (Winfield 1981). Such difference would result in an increase in the level of inhibition in the visual cortex near the end of the critical period, which has been demonstrated as a crucial regulating factor of brain plasticity changes (Chen et al. 2011; Fagiolini and Hensch 2000; Iwai et al. 2003).
With MD, the spine density in V1 rapidly decreases, which accurately reflects the competitive interactions between the neural inputs from the two eyes because it does not occur when both eyes are sutured (Majewska and Sur 2003). Spine density largely recovers after prolonged MD, indicating that this competing-input-triggered pruning is mostly transient (Mataga, Mizuguchi, and Hensch 2004).
Similarly, in the rodent barrel cortex, whisker deprivation during the critical period can affect the normal sensory map formation, and produces profoundly long-lasting abnormal receptive fields (Fox 1992; Stern, Maravall, and Svoboda 2001). The cortical circuits of barrel cortex are highly dynamic and regulated by sensory experience during both development and adulthood. During development, whisker sensory deprivation reduces spine protrusive motility (not to be confused with spine turnover) in the deprived region of the barrel cortex (Lendvai et al. 2000). In the adult, the dendritic spines of the barrel cortex are still highly dynamic and can be stabilized by novel sensory experiences (Holtmaat et al. 2006). The sprouting and retraction of these dendritic spines are tightly associated with synapse formation and elimination, which further suggests an adaptive synaptic remodeling mechanism underlying the sensory experience-dependent brain plasticity changes (Trachtenberg et al. 2002). Altering sensory experience by trimming whiskers in adult animals
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induces targeted rewiring of the local excitatory connections (Cheetham et al. 2008).
Persistent whisker stimulation resulted in a significant increase in the density of the inhibitory synapses in the corresponding cortical barrel, and persists after 4 days (Knott et al. 2002).
Not surprisingly, trends of similar structural remodeling are also observed in HVC of songbirds. In general, the neurostructural features, such as volume, neuron number, neuron size, and synapse density increase steadily in HVC between 20 and 90 dph, an age range that coincides with the behavioral development of birdsong (Bottjer, Glaessner, and Arnold 1985; Herrmann and Bischof 1986; Nordeen and Nordeen 1988). Sensory deprivation of tutor song by means of song-isolation or deafening lead to a significant increase in HVC synapse density in juvenile Bengalese finches (Peng et al. 2012). In this study, alternatively, the lower synapse count in the normal reared (song exposed) birds could be explained by experience-dependent synapse pruning. Manipulation of inhibitory synaptic transmission in HVC during the song learning period prematurely closed the critical period plasticity window (Yazaki-Sugiyama et al. 2007, 2009). The experience-dependent synapse pruning, and brain plasticity changes regulated by inhibitory synaptic transmission, are also observed in the visual and somatosensory cortex during critical period development, which represents a common phenomenon across many different critical period learning systems (Hensch 2004). The song experience dependent structural remodeling of HVC was also confirmed in a two-photon in-vivo imaging study, which revealed that enhanced spine dynamics in HVC correlated with behavioral song learning and that the initial exposure to the tutor song was the critical instructive experience that rapidly stabilized and strengthened the dynamic spines in HVC (Roberts et al. 2010).
The two-photon microscopy-based techniques are limited by their spatial resolution and cannot resolve the HVC synapse changes during song development. Subsequent EM examination revealed the dendritic spines previously defined at the fluorescent microscopy level do not always exhibit synapses (Blazquez-Llorca et al. 2015), which makes EM identification almost indispensable for synapse centered structural studies. Besides, the structural remodeling of the HVC inhibitory circuits are not accessible with two-photon microscopy, since a great deal of the inhibitory synapses are aspinous (Mooney 2000). Nevertheless the HVC inhibitory circuit is actively involved in the song learning process, as demonstrated in electrophysiology (Vallentin et al. 2016) and pharmacology (Yazaki-Sugiyama et al. 2009) studies.
These facts suggest that EM-based studies are essential to fully explore the developmental and experience-dependent structural synapse remodeling in HVC.
In the next section, I will elaborate on the current approaches used in neuroanatomy research, including different microscopy techniques and their applications. At the end, I will provide the rationale of the techniques of choice, justified by the specific biological question of the current study.