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

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Copyright © 2018 Shaib, Staudt, Harb, Klose, Shaaban, Schirra, Mohrmann, Rettig and Becherer.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CAPS promotes large dense-core vesicle (LDCV) secretion in DRG neurons. (A–D) Measurement of LDCV exocytosis. LDCVs were labeled via neuropeptide Y (NPY)-Venus overexpression and visualized using TIRFM at 10 Hz. Their exocytosis was evoked via field electrode stimulation. The protocol consisting of a 30-s pre-stimulus at 10 Hz followed by a 100 Hz stimulus is illustrated at the top of each graph. LDCV exocytosis is displayed as the average cumulative exocytosis normalized to the cell footprint area. The graphs include only the cells that responded to the stimulus with LDCV exocytosis. Error bars are SEM. (A) CAPS DKO E18 DRG neurons exhibited defective LDCV secretion compared to E18 WT neurons (N_pups = 5 for WT and 6 for CAPS DKO, n_neurons = 16 and 18 for WT and CAPS DKO, respectively). (B) Overexpression of CAPS2 rescued LDCV secretion in E18 CAPS DKO neurons (N_pups = 10 E18 embryos, n_neurons = 10, 8, and 12 for CAPS DKO, CAPS DKO expressing CAPS1, and CAPS DKO expressing CAPS2, respectively). (C) CAPS1 overexpression in adult WT neurons strongly enhanced LDCV secretion (N_mice = 5, n_neurons = 26 and 23 for WT and WT overexpressing CAPS1, respectively). (D) CAPS2 overexpression in adult WT neurons induced a robust increase in LDCV secretion (N_mice = 7, n_neurons = 18 and 15 for WT and WT transfected with CAPS2, respectively). Note that secretion in adult WT neurons (panels C,D) was lower than that in E18 WT neurons (panel A). (E) Average number of LDCVs visible in the evanescent field normalized to the cell footprint area. Error bars are SEM. (F) Box plot of LDCV release probability, which was calculated by dividing the number of fusion events by the total number of LDCVs at the footprint of the neurons presented in panels (A–D). The stippled and solid lines in the box correspond to the average and median release probability, respectively. The Mann-Whitney significance test was applied for all graphs except for (B) and the second plot in (E,F), in which an ANOVA on ranks with Dunn’s post significance test was applied; *p < 0.05, **p < 0.01, ***p < 0.001, ns (not significant) p > 0.05.