Our PSD-95-eGFP experiments showed that this recombinant protein is a good measure of synapse size. This tempted us to use the protein to monitor synapse size changes as part of synaptic potentiation and depression (LTP and LTD respectively) or other types of plasticity events. Some of the clear-est examples of plasticity-related synapse size changes have been provided by LTP experiments in vitro (Matsuzaki et al., 2004; Kopec et al., 2006;
Harvey, Svoboda, 2007). These studies showed that the size and molecular constituents of spines that undergo LTP display signicant alterations. It is tempting to speculate that the PSD-95-eGFP uctuations that we observed in naïve animals reect LTP and LTD-like processes. However, we had no direct access in these mice to measure whether a single cell was indeed po-tentiated or depressed. In addition, LTP and LTD may happen at dierent synapses on the same dendrite and thereby cancel out one another at the level of the whole cell. It is likely that experience-dependent plasticity paradigms increase LTP and LTD events. However, in these paradigms one faces the same temporal and spatial uncertainties. The best way to monitor synapse size changes in vivo in relation to LTP would be through the application of a robust LTP paradigm. In vitro, LTP is usually induced by high-frequency
Figure 3.2: Left, whisker deection evoked PSP peaks over time. Top right, average whisker deection evoked PSP traces before and after 8 Hz whisker stimulation. Bottom right, average whisker deection evoked PSP peaks per cell (n = 11; **, p < 0.01, t-test).
aerent stimulation, by combining presynaptic input with backpropagating action potentials, or chemically. This requires electrode-based stimulation or pharmacological access. However, one cannot combine such invasive tech-niques with chronic in vivo imaging. Previous experiments have shown that rhythmic whisker stimulation can induce the potentiation of whisker-evoked local eld potentials (Megevand et al., 2009). My colleague Frédéric Gambino veried this eect using whole cell recordings in vivo (Gambino-Holtmaat ab-stract SFN 2013). He showed that a 1 minute 8 Hz whisker stimulus increased subsequent whisker-induced post synaptic potentials (Fig. 3.2).
Based on these ndings we set out experiments to measure spine size changes upon LTP induction in vivo (as a pilot to measuring LTP-related PSD-95-eGFP uctuations). To asses if 8 Hz whisker stimulation had a structural eect on L2/3 pyramidal cells, labeling was performed with a GFP
Figure 3.3: In utero electroporation of cortical region with low density label-ing. A single cell with its own dendritic processes is distinctly visible. Scale bar 50 µm.
plasmid (pGAG-GFP-WPRE) introduced by in utero electroporation as pre-viously described. Given our earlier experience with the confounding eect of dense expression patterns, we wanted to reduce the number of labeled cells. By increasing the time between plasmid injection in the ventricle and the actual delivery of the electrical train pulse, we were able to reduce the number of transfected cells. In contrast to earlier experiments we were not concerned by lack of co-expression. Therefore, we could reduce the number of transfected cells without repercussions. After cranial window implantation the region was screened for suciently isolated cells where the originating cell body and identity of the dendritic processes was unambiguous (Fig. 3.3).
If such a cell was found the barrel cortex was mapped using intrinsic optical imaging (for details see below). This allowed the identication of the home column of the cell of interest. High magnication images were then acquired
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Time [min] Time [min] Time [min] Time [min] Time [min] Time [min]
10 min 8 Hz
Whisker stimulation Control
Figure 3.4: Relative volume changes between the 4 imaging sessions. Whisker stimulation occurred just before timepoint 3 in experimental animals (right).
Red lines represent the means of volume changes.
every hour for 4 hours (4 imaging time points). The principal whisker was stimulated for 10 minutes at 8 Hz (50 ms deections) just before the third imaging session. Spine uorescence intensities were measured as previously described, and volume changes were compared before and after whisker stim-ulation as well as to control animals (Fig. 3.4).
No major dierences in volume changes were observed after whisker stim-ulation. The lack of detectable changes could be explained by the relative low number of spines that are likely to be involved in such a process. Since we could only image a limited number of spines per cell at high resolution (100-500 out of a total of thousands of spines per neuron), we could have eas-ily missed the few spines that were potentiated. Even if LTP had occurred at many spines, it is likely that the changes were relatively small as compared to the in vitro experiments, and be at a level similar to the natural uctua-tions that can be observed in vivo (the signal-to-noise ratio is too low). In
addition, in vivo imaging comes with considerable movement noise that may have blurred the eects on spine size.
These experiments were complicated by the following issues:
1. Low control over the density of labeling. Some mice had a relatively high density of labeling which resulted in diculties to identify the cell body to which the imaged dendrites belonged. Therefore, we could not unambigu-ously identify the home barrel column of each cell.
2. In animals where the labeling was suciently sparse, the position of the neuron in the barrel eld was highly variable. Therefore the home barrel column diered between animals. This potentially reduced reproducibility.
3. Point 1 and 2 would complicate even further the experiments using PSD-95-eGFP as a marker for synaptic potentiation (which was what we aimed for), since we would have to rely on co-expression of proteins.
Further experiments were put on hold until the 8 Hz whisker stimulation LTP protocol was better characterized potentially revealing hot spots for LTP. In the mean time we worked on the development of a more suitable labeling technique (see below).