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. 2016 Sep 5;10:207. doi: 10.3389/fncel.2016.00207

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Copyright © 2016 Zhang, Hannan, Stapper, Kern, Jahn and Rasse.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor 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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Characterization of impaired transport at the NMJ and ventral nerve cord (VNC) in unc-104^bris mutants. (A) Representative confocal images of NMJs from control and unc-104^bris mutant larvae stained with antibody against vescicular glutamate transporter (VGlut, green in A). Neuronal membranes were visualized using horseradish peroxidase (HRP) staining (magenta in A). (B) Quantification of VGlut at the NMJs shown in (A), per HRP area (VGlut density; control: 54.70 ± 2.75 a.u.; unc-104^bris: 8.92 ± 1.04 a.u., p < 0.001). Experiments were performed at 29°C. (C) Representative confocal images showing abnormal accumulation of VGlut and cysteine string protein (CSP) in the cell body (CB) region of the larval VNC in unc-104^bris compared to control larvae. (D,E) Quantifications of the intensities of VGlut and CSP (in a.u.) in the CB and neuropil (NP) region of the VNC in control and unc-104^bris larvae. VGlut intensity at the CB (control: 7.37 ± 1.40 a.u.; unc-104^bris: 9.017 ± 1.71 a.u., p < 0.05), CSP intensity at the CB (control: 16.91 ± 2.46 a.u.; unc-104^bris: 29.117 ± 4.80 a.u., p < 0.001), VGlut intensity at the NP (control: 15.61 ± 2.25 a.u.; unc-104^bris: 11.79 ± 3.46 a.u., p < 0.01), CSP intensity at the NP (control: 32.85 ± 4.77 a.u.; unc-104^bris: 21.865 ± 6.58 a.u., p < 0.001). (F,G) Representative electron micrographs showing the NP region of VNCs from control and unc-104^bris larvae (F) and two representative T-bars from control and unc-104^bris mutant larval VNC each (G). (H–J) Quantifications of synaptic vesicles (SVs; red arrowheads), AZs (orange arrowheads) and mitochondria (yellow arrowheads) at the VNCs in control and unc-104^bris larvae in (F). n > 40, n represents number of cells analyzed. (H′–J′) Quantifications of SVs, AZs and mitochondria at the VNCs in control and unc-104^bris larvae with analysis only limited to boutons with the presence of a synapse. n > 40, n represents number of cells analyzed. Genotypes: control (w¹¹¹⁸), unc-104^bris (;unc-104^bris/unc-104^d11024;). Scale bar in (A): 10 μm, (C): 10 μm, (F): 0.3 μm, (G): 0.1 μm. Experiments were performed at 25°C. Statistical test: Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. p > 0.05. Error bars indicate the SEM.