Quantitative analysis of PSD growth during synapse formation

Chapter 4.1 described the establishment of an assay which allows studying individual synapses during synapse formation in a synaptic circuit of intact Drosophila, which undergoes strengthening. After verifying that essentially all PSDs* labeled by DGluRIIA^GFP expression are part of functional synapses PSD dynamics were analyzed over time in intact larvae. Thereby, the larvae moved freely on food-containing apple agar plates in-between imaging sessions. A individual neuromuscular junction of early third instar larvae was typically imaged every 12h for 2-3 days at 16°C.

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* Studies addressing the issue of PSD remodeling so far used electron microscopy as principal technology.

Since this work uses mainly in vivo imaging no PSDs as described by electron microscopy were visualized, but the postsynaptic patches in which receptors localize. As shown in Figure 15 B these PSDs colocalize perfectly with the PSD marker DPak. Both structures, receptor field and PSD, are essentially identical. It was therefore decided to use the more common term PSD from here on, when referring to the postsynaptic patches in which receptors localize.

Fig. 17 In vivo imaging of PSD formation during the development of individual neuromuscular synapses

A-B) Confocal time series of a DGluRIIA^GFP expressing larva. Time after the start of observation is indicated.

A) In vivo imaging of an NMJ in lower magnification, scale bar 15 µm.

B) Time series of dynamic changes at identified populations of PSDs shown in higher magnification. Newly appearing PSDs are marked by arrows, a PSD present at the start of observation (t=0 h) by arrow heads.

Blue circles label an area where PSDs grow dense, white circles label an area where PSDs increase distance over time (see text). White arrows label PSDs which form distant from pre-existing PSDs. The blue arrow shows an example of two PSDs, which are so close together after outgrowth, that they can no longer be separated by confocal microscopy. Scale bar 4 µm.

Consistent with a previous report (Zito et al., 1999; Goda and Davis, 2003), it was found that the gross morphology of an individual junction is relatively stable whereas its size increases as development continues (Fig. 17 A, Fig. 17 B). Figure 17 B shows the PSDs

of a part of the junction in high magnification. We first recognized that the PSDs which were established at the beginning of a live series (Fig. 17 B, arrow) generally showed only little change during the time series. Thus, individual PSDs can be stable for at least days in intact Drosophila. These PSDs also served as “landmarks”, between which the outgrowth of new PSDs was observed. These new PSDs form predominantly distant from pre-existing PSDs suggesting that they form independent from neighboring PSDs (Fig. 17 B, white arrows). In the following, this mode of PSD formation will be referred to as de novo mode. In a few instances the formation of new PSDs in close proximity to pre-existing PSDs was observed (Fig. 17 B, white circle). Here, it was to be clarified, whether

these PSDs also formed de novo, or whether they derived from a PSD, which broke apart giving rise to two new PSDs. In the following such a potential mode of PSD formation will be referred to as splitting mode. Chapters 4.2.2-4.2.4 will address the mode of PSD formation in more detail. Here, first the outgrowth of clearly identified PSDs was quantitatively evaluated. During the in vivo imaging substantial formation of additional synapses was observed. Starting with a total of about 309 PSDs 165 new PSDs were formed within 36h.

Fig. 18 Quantification of in vivo imaging data on PSD formation

A-C) Quantification of PSD dynamics from 5 pooled imaging series. A) Size distribution of PSDs at individual imaging time points in histogram plot showing binned PSD sizes (in µm²).

The overall size distribution of PSDs is relatively stable over time. PSDs, which were first observed at the 12 h imaging time point, are shown as white parts within black bars. New PSDs are small when first observed but later approach average PSD size distribution (t=24 and 36h). B) White bars:

average PSD size calculated for different imaging time points. Average PSD size increases moderately over time. When PSDs were pooled according to their age (black bars), a strong increase in average PSD size can be observed during the first 24h. Standard error bars are shown. C) Relative growth of individual PSDs within 36h (sizet=36h/sizet=0h) plotted semi-logarithmically

The overall PSD size distribution was rather stable over time (Fig. 18 A) and the mean PSD size increased only very moderately (from 0,21 µm² to 0,24 µm² in 36h, p<0,001, see white bars Fig. 18 B). However, new PSDs, which represent the majority of all small PSDs present at any given time point, grow rapidly (shown in Fig. 18 A in white, for t=12h). Following the development of such an age-matched population (later time points see Fig. 18 A) it could be shown that, on average newly formed PSDs reach half maximal size (0,12 µm²) within 6h and 0,18 µm² within 18h of their first observation (age class 0h-12h and age class 12h-24h, see black bars in Fig. 18 B). In contrast, 24h-36h old PSDs are only slightly and non-significantly smaller (0,23 µm² to 0,26 µm², p=0,23, see black bars in Fig. 18 B) than PSDs older than 36h (black bars 18 B). Thus, after about 36h at 16°C PSDs seem to have reached their mature size. This observation is confirmed when focusing on individual PSDs. Here, most small PSDs (<0,15 µm²) grow strongly while large, mature PSDs (>0,4 µm²) change less, showing small decreases and increases to a similar degree (Fig. 18 C). It should be noted that these experiments were done at 16°C. Development of Drosophila larvae is 3 times faster at 25°C compared to 16°C (Economos and Lints, 1984).

4.2.2 Morphological imaging suggests that new PSDs form de novo, not from