Structural synaptic plasticity can be induced optogenetically

As stated earlier, the dynamics of the structural and the functional changes in the MB188B DANs demonstrated that the activity of the MB188B DANs increased prior to the decrease in the connectivity (Figure 3.15). This situation gave rise to the possibility of activity-dependent structural change mechanisms. Thus, I questioned if an early activity increase could be a reason behind the structural changes.

78

I addressed this question by artificially depolarizing the MB188B DANs over a long period by the csChrimson and the ChR-XXL tools. Then, the GRASP fluorescence was examined. A decrease in the connectivity was expected if the connectivity decrease was long-term depolarization-dependent under hypocaloric conditions. Additionally, I artificially increased the cAMP level by the light-gated adenylate cyclase bPAC (Stierl et al, 2010) as a second mechanism and examined any potential GRASP change.

This tool was used considering the possibility of metabotropic signal transduction as a downstream of (auto)regulation mechanism based on the MB188B DANs activity increase. In these experiments in which the effect of the hypocaloric food were mimicked, I kept the flies on the isocaloric food during the long-term light stimulation. The light stimulation started when the flies were 3 days old as in the case of the onset of dietary. Prior to the onset of the light stimulation, the flies were allowed to fully develop on the standard fly food as usual.

3.8.1

To answer the question if the long-term activity increase results in the connectivity decrease, I crossed the flies expressing an optogenetic activation tool, either the UAS-csChrimson or UAS-ChR-XXL and UAS-MBGRASP with the flies containing the MB188B-Gal4 driver. Experiments were performed as described above (and also see 2.2.12). I also tested genetic control flies in which only the MBGRASP complex was expressed in the 188B DANs but not the optogenetic tool. Additionally, I subjected half of each genetic group to darkness as control of unstimulated MB188B DANS.

The relative GFP fluorescence under the light stimulation condition was calculated by normalizing the data to the mean GRASP intensity of the corresponding background under dark conditions. I aimed to eliminate the effect of the Gal4 protein dilution due to the different copy numbers of the UAS construct.

Thus, the mean of the “dark groups” for both crosses is equal to 1 in the relative reconstituted GFP fluorescence plots (Figure 3.16).

As a result, increasing the activity of the MB188B DANs by depolarizing the cells for a long period with csChrimson did not create any difference in the GRASP signal between any group (Figure 3.16A).

Similarly, activating the MB188B DANs for a long period by the ChR-XXL did not lead to any change in the GRASP signal between genotypes either (Figure 3.16B). However, I observed a bleaching effect in the GRASP signal due to the strong and long-lasting light stimulation independent of optogenetic tool

79 expression (Figure 3.16B). Therefore, I concluded that the long-term depolarization of the MB188B DANs by the cation channels based on channelrhodopsins do not mimic the decrease in the connectivity.

Figure 3.16 Induction of the structural changes in the MB188B DANs. A Quantification of the relative GRASP signal in the MB188B DANs upon the long-term activation via the csChrimson. No GRASP difference was observed between the optogenetically activated MB188B DANs and non-activated DANs. B Quantification of the relative GRASP signal in the MB188B DANs upon the long-term activation via the ChR-XXL. No difference was observed between the relative GRASP signals of the ChR-XXL expressing and non-expressing DANs. However, the light induction had bleaching effect on the GRASP signal (Statistics two-way ANOWA, p=0.0471 between the light treated groups and dark groups). C Quantification of the relative GRASP signal in the MB188B DANs upon the long-term induction of the cAMP production via the bPAC. The long-term cAMP induction created a decrease in the relative GRASP signal. The bleaching effect of the white LEDs were also observed in this experiment (Statistics two-way ANOVA followed by Tukey test, ****p<0.0001, **p=0.0072, *p=0.0127).

3.8.2

structure

In the previous part, I concluded that it is not the long-term artificial depolarization of the MB188B DANs promoting the structural induction. Next, I increased the cAMP level of the MB188B DANs for a long period to test the resulting effect on the structural changes.

Thus, I expressed the UAS-bPAC under the MB188B-Gal4 driver along with the UAS-MBGRASP.

Experimental design and data analysis were performed in the same way as in the case of the csChrimson (or the ChR-XXL). White LED lights were used to simulate bPAC.

80

Here, I also observed that light stimulation per se causes the GRASP signal to bleach independent of the fly genotype as in the case of the ChR-XXL (Figure 3.16B). However, the change in the GRASP signal due to the structural modification was still detectable since the relative GRASP fluorescence was calculated. The GRASP signal quantification showed that when the cAMP level was increased by the light stimulation in the MB188B DANs, the connectivity decreased compared to the flies that did not express the tool bPAC (Figure 3.16C).

As a result, I concluded that the effect of the hypocaloric dietary on the connectivity, which was a decrease in the GRASP signal, could be mimicked by the long-term artificial induction of the cAMP production even under isocaloric dietary. In contrast, long-term depolarization of MB188B DANs failed to create the same effect.