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. 2018 Sep 11;12:304. doi: 10.3389/fncel.2018.00304

Figure 4.

Figure 4

CAPS2 is the priming factor of LDCVs in DRG neurons. (A) Exemplary images of CAPS1 KO DRG neurons. The two cells visible in the bright field image (top) are either non-peptidergic (stained red with iB4-Alexa561, middle left) or peptidergic (middle right). LDCV exocytosis was measured in the NPY-Venus transfected peptidergic neuron (white arrow, total internal reflection fluorescence microscopy image, bottom). The stimulation protocol was the same as that in Figure 2. (B) Percentage of peptidergic neurons that secreted LDCVs depending on their genotype; which were WT, CAPS1 KO and CAPS2-deficient (CAPS2 KO). The number of preparations (i.e., mice) was 7, 7, and 4 for WT, CAPS1 KO and CAPS2 KO, respectively (ANOVA on ranks Holm-Sidak method: *p < 0.05). (C) Scatter dot plots of total LDCV exocytosis in the CAPS1 KO and CAPS2 KO peptidergic neurons in comparison to their respective WT controls. Orange dashes are the average LDCV exocytosis ± SEM (Nmice = 7, 7, and 4, nneurons = 32, 20 and 16 for WT, CAPS1 KO, and CAPS2 KO, respectively; ANOVA on ranks Kruskal-Wallis test: **p < 0.01). (D) Representative image of a WT non-peptidergic DRG neuron co-transfected with NPY-Venus and CAPS2-mTFP. The neuron was identified by triple staining in red for iB4-Alexa568 (top), yellow for NPY-Venus (middle), and cyan for CAPS2-mTFP (bottom). LDCV exocytosis induced by the protocol illustrated in Figure 2 was assessed in these neurons and compared to exocytosis in WT peptidergic and non-peptidergic neurons transfected with NPY-Venus only. (E) Proportion of secreting WT neurons that were non-peptidergic, peptidergic and non-peptidergic transfected with CAPS2. Note that ectopic expression of CAPS2 in non-peptidergic neurons converts these neurons to LDCV secreting cells. (F) Scatter dot plots of total LDCV secretion from WT neurons that were non-peptidergic, peptidergic and non-peptidergic overexpressing CAPS2. For panels (E,F) Nmice = 13, nneurons = 34, 13, 17 for non-peptidergic, peptidergic and non-peptidergic overexpressing CAPS2, respectively. **p < 0.01, *p < 0.05 and ns (not significant) p > 0.05, one-way ANOVA Dunn’s test.