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. 2010 Apr 26;107(19):8836–8841. doi: 10.1073/pnas.0906087107

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Fig. 4. — Changes in active zone size lead to corresponding alterations in RRP size and p_r. Neurons transfected with sypH2 and tdT-Bsn were used in a time lapse experiment to identify synapses at which p_r, RRP, and/or active zone cytomatrix sizes changed over the course of 6 h. Cells were then fixed and stained with anti-MAP2 antibodies and Cy5-conjugated phalloidin to visualize dendritic tubulin (blue) and actin (green), respectively, allowing the identification of axodendritic synapses. Only active zone cytomatrices within 1 μm of a MAP2-positive dendritic shaft that were colocalized with a phalloidin-visualized spine were included in the subsequent analysis. (A) Responses to train stimulation (RRP) and average responses to isolated stimuli at 0.2 Hz (p_r) and tdT-Bassoon fluorescence (Bsn) at several synapses at the beginning of the experiment (Left) and after 6 h (Right). Filled or open arrowheads denote synapses at which tdT-Bsn fluorescence as well as RRP and p_r measures increase or decrease, respectively. (Bottom Right) The same field after visualization of actin and MAP2 (MAP2/actin). (Scale bar, 4 μm.) (B) At the synapses denoted with 1 and 2 in (A), sypH2 responses stimulus trains increased or decreased significantly (P < 0.001) in parallel with changes in tdT-Bsn fluorescence (P < 10⁻⁴ and P < 0.01, E). (C) At the same synapses, a significant enhancement or attenuation, respectively, in the averaged response to isolated stimuli at 0.2 Hz was observed (P < 0.001). (D) Correlation between changes in the sypH2 response to stimulus trains delivered at the onset of the experiment (ΔF₁) and after 6 h (ΔF₂), and changes in tdT-Bsn fluorescence for 45 synapses in this experiment (r = 0.697, P < 10⁻⁶, Pearson product moment correlation). Synapses 1 and 2 are highlighted in red and blue, respectively. (E) Correlation between changes in the average sypH2 response to isolated stimuli delivered at the onset (ΔF₁) and end of the experiment (ΔF₂), respectively, and changes in tdT-Bsn fluorescence (r = 0.544, P < 0.001). sypH2 fluorescence changes were converted into estimates of changes in RRP or p_r as outlined in the legend to Fig. 2. (F) Changes in tdT-Bassoon fluorescence that occurred at a total of 122 synapses in three experiments that were binned according to the change in RRP size occurring during a 5- to 6-h experiment. (G) Changes in tdT-Bassoon fluorescence at the same 122 synapses, binned for changes in p_r.