Synapse size and diameter in HVC

In-vivo imaging of the brain structural dynamic has generally focused on dendritic spines, whose growth always precedes synapse formation. The time course of spine

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turnover (formation and elimination) reported from in-vivo imaging studies is generally in the range of tens of minutes, way more rapid than synapse formation, which occurred on a stabilized spine older than several hours (Nagerl et al. 2007).

Spines persisting for 4 days or more are found to reliably contain synapses (Knott et al. 2006). Direct observation with GFP-tagged synaptic markers with EM has shown that the formation, elimination, and stabilization of synapses occurs over time course of 1-2 hours (Alsina, Vu, and Cohen-Cory 2001; Meyer, Bonhoeffer, and Scheuss 2014). In-vivo imaging of juvenile zebra finch HVC reported spine stabilization and enlargement within 24 hours (Roberts et al. 2010). Taken together, synapses are more stable than spines with temporal dynamic that range from hours to days, and serve as a more reliable readout of stabilized neural connections.

The sizes and Feret diameters of synapses in HVC, including both the asymmetric and symmetric synapse subtypes, showed uniformly lognormal distributions in all groups (Figure 2. 24 and Figure 2. 28). Similar distributions of synapse sizes have been observed in rat cortex and a lognormal distribution was suggested to be the best fit in a previous EM study (Merchán-Pérez et al. 2014). An in-vivo study has shown that the change in sizes of spines and synapses are proportional to their sizes, and as a result yielded also lognormal distribution in their temporal dynamics (Loewenstein et al. 2011). Simple modeling of such lognormal synaptic dynamics recaptured qualitatively the population distribution of synapse size, as well as individual synapse formation kinetics (Statman et al. 2014). Moreover, the sizes of spines or synapses are suggested to reflect the synaptic strength (Cane et al. 2014), which also follows a lognormal distribution (Song et al. 2005). Such lognormal distributed dynamics of synaptic strength of neural networks can be explained and reproduced as a consequence of self-organization in a recurrent network, combing synaptic plasticity, structural plasticity, and homeostatic plasticity rules (Miner and Triesch 2016; Teramae and Fukai 2014; Zheng, Dimitrakakis, and Triesch 2013). In summary, many functional and structural brain parameters – such as synaptic weights, the firing rates of individual neurons, the synchronous discharge of neural populations, the number of synaptic contact between neurons and the size of synapses, are lognormally distributed (Buzsáki and Mizuseki 2014), which suggests a fundamental structural and functional brain organization.

In the two song-tutored groups, in which I observed significant synapse addition and removal (see Results section 2.3.2, 2.3.3, and 2.3.4), the synapse sizes and Feret diameters still followed the lognormal distribution (see Figure 2. 24 and Figure 2.

28). Therefore, I speculate that the resulting synapse size increase in the two song-tutored groups (see Table 2. 15) is due to removal of small synapses rather than enlargement of the large synapses. The latter case do not involve synapse addition or removal, and should have resulted in a shift of the distribution towards the right side, which was not observed except for the asymmetric synapses in the SHORT group (see Figure 2. 24). Taken together, it is likely that the experience-dependent removal and addition of synapses in HVC of the two song-tutored groups happened mostly in small synapses.

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On a population level, systematic difference in synapse size are present between the asymmetric and symmetric synapses in HVC. The average sizes of HVC asymmetric synapses is roughly 70% larger than the size of symmetric synapses (in the SHORT group, this difference was two-fold). However these differences are not observed in the Feret diameters. The following apparent morphological differences between the two synaptic subtypes are easy to observe in the EM samples shown in Figure 2. 6 and Figure 2. 7 and in the reconstructed synapse segmentation shown in Figure 2.10: wider asymmetric synaptic clef, and thicker asymmetric synapse membrane specializations, especially in the PSD (Banker, Churchill, and Cotman 1974). However, the sizes of the synaptic junctions, which are approximated by the synapse Feret diameters, are not obviously different between the two synaptic subtypes. Synaptic structures have been shown to strengthen with experience and are remodeled during the long-term potentiation (LTP) process (Bosch et al. 2014).

Besides stabilization, the structural changes of synapses during LTP also include active zone expansion and synapse enlargement (Harris, Fiala, and Ostroff 2003).

While synapse enlargement can be determined from synapse size measurements, active zone expansion can be determined from synapse Feret diameter measurements. Therefore, in the current study, the systematic differences in the synaptic volumes between the asymmetric and symmetric synaptic subtypes were thought to have mainly resulted from their different synaptic protein compositions (Klemann and Roubos 2011) and not the sizes of their active zones. Therefore, in a subsequent attempt to estimate the overall excitation and inhibition in the HVC network (see Supplementary Method and Data section S.6), the Feret diameter were selected to represent the synaptic strength of a given asymmetric or symmetric synapse (Michel et al. 2015).

In general, the synapse size and Feret diameters were both larger in the song-tutored groups compared with those of the song-isolated groups, which reflected an overall strengthening of the synapses by the song experience. Especially in the 1-day (SHORT) tutored group, the asymmetric synapse sizes in HVC were significantly (see Table 2. 18) larger compared to that in the song-isolated (ISO) group. These results strongly indicated the presence of an acute effect of synaptic enhancement on the HVC excitatory synapses shortly after the initial sensory tutor song exposure.

The same comparison of synapse Feret diameter did not show any differences (see Table 2. 23), which indicated no corresponding expansion of the synapse active zone. Nevertheless, the Feret diameter of the HVC asymmetric synapses increased in the order of ISO < SHORT < LONG (see Figure 2. 30), and this might still reflect experience-dependent strengthening of synapses via expansion of the excitatory synapse active zone, but with slower temporal dynamics. Experience-dependent structural remodeling of excitatory synapses is confirmed by many studies, and found to be tightly associated with LTP in memory and learning (Bosch et al. 2014;

Cheetham et al. 2014; Lisman 2017).

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It is notable that the symmetric synapse density in the SHORT group was larger than those in the other two groups (see Figure 2. 22). The possibility of the addition of many small symmetric synapses would have resulted in a decrease in the mean symmetric synapse size. However, compared to the ISO group, the symmetric synapses in HVC in the SHORT group increased in size while decreasing in Feret diameter (see Figure 2. 27 and Figure 2. 31). Although neither of these results were significant, the decrease in Feret diameter might have resulted from the addition of many small symmetric synapses. While the increase in the mean symmetric synapse size could be explained by the enlargement of many medium-sized synapses, which was supported by the histogram of the symmetric synapses in the SHORT group in Figure 2. 24, it would have compensated for the small size of the newly formed symmetric synapses. This hypothesized enlargement in the HVC symmetric synapse thus might reflect synaptic strengthening of the HVC inhibitory network. If so, this is consistent with the rapid song experience-dependent remodeling of the HVC inhibitory networks that was proposed in the end of the last section 2.4.3