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Figure 2 | Molecular Brain

Figure 2

From: Common strength and localization of spontaneous and evoked synaptic vesicle release sites

Figure 2

At soma-near synapses the number of spontaneously fused vesicles as well the recycling pool vesicles is higher than in distal cellular compartments. A Analysis of spontaneous and evoked release differentiated between process and soma using dual color experiments of spH transfected hippocampal neurons spontaneously labeled with αGFP-CypHer5E™. The spontaneous release (SR), recycling pool (RP), reserve pool (resP) and the total pool (TP) were determined. Scheme of experimental setup added with the mean values of spH and CypHer5E™ fluorescence used for further analysis. Boutons of spH transfected hippocampal neurons were first labeled with αGFP-CypHer5E™ by spontaneous uptake during a 120 minute period. Then a stitched image was captured to encompass the whole neuron with its processes at high resolution in both fluorescence channels (120 min). After stimulation with 40 mM K+ solution for 2 minutes in the presence of Bafilomycin an image of the same region was recorded (135 min), followed by an image after NH4+ application (140 min). B Images depicting whole neuron. Insets: Synaptic boutons at higher magnification. Scale bar, 50 μm. C Scheme of image analysis used for distinction between process and soma. D Quantification of pool sizes: RP (two-sample t-test: p = 0.001), SR (two-sample t-test: p = 0.042) and SR to RP ratio (SR/RP; two-sample t-test: p = 0.005; 11 experiments). Correlations of distinct vesicle pools differentiated for synapses located at the process or the soma: SR and RP (E; Pearson’s r process  = 0.96 ± 0.01, r soma  = 0.84 ± 0.04), SR and ResP (F; Pearson’s r process  = 0.19 ± 0.14, r soma  = 0.51 ± 0.11) and SR and TP (G; Pearson’s r process  = 0.64 ± 0.09, r soma  = 0.78 ± 0.05). Dashed line in E represents the linear fit of a subset of data putative resembling the processes running above or beneath the soma (slopeprocess = 0.55 ± 0.02, fit: y = mx + t).

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