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. 2019 Mar 27;39(13):2416–2429. doi: 10.1523/JNEUROSCI.3068-18.2019

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Copyright © 2019 Gratz et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 4. — Endogenous Cac is differentially regulated in chronic PHP and PHD. A–F, Chronic expression of PHP and PHD occurs normally in cac^sfGFP-N animals lacking GluRIIA (GluRIIA^SP16) or overexpressing vGlut (vGlut-OE; OK371-Gal4>UAS-vGlut), respectively. A–C, Representative traces of EJPs and mEJPs recorded in 0.4 mm Ca²⁺ at cac^sfGFP-N (A), cac^sfGFP-N; GluRIIA NMJs (B), and cac^sfGFP-N; vGlut-OE (C). D, mEJP amplitude is decreased in cac^sfGFP-N; GluRIIA and increased in cac^sfGFP-N; vGlut-OE as expected (cac^sfGFP-N, 1.0 ± 0.08, n = 8 NMJs from 4 larvae; cac^sfGFP-N; GluRIIA, 0.48 ± 0.02, n = 7 NMJs from 4 larvae, p = 0.0001, t test with Welch's correction; cac^sfGFP-N; vGlut-OE, 1.5 ± 0.08, n = 10 NMJs from 4 larvae, p = 0.0017, Student's t test). E, EJP amplitude is unchanged between cac^sfGFP-N, cac^sfGFP-N; GluRIIA and cac^sfGFP-N; vGlut-OE NMJs (cac^sfGFP-N, 30.3 ± 1.70, n = 8 NMJs from 4 larvae; cac^sfGFP-N; GluRIIA, 27.1 ± 0.60, n = 7 NMJs from 5 larvae, p = 0.1115, t test with Welch's correction; cac^sfGFP-N; vGlut-OE, 29.8 ± 0.81, n = 10 NMJs from 4 larvae, p = 0.80, Student's t test), as expected. F, Quantal content is increased in cac^sfGFP-N; GluRIIA and decreased in cac^sfGFP-N; vGlut-OE as expected (cac^sfGFP-N, 30.7 ± 3.0, n = 8 NMJs from 4 larvae; cac^sfGFP-N; GluRIIA, 56.6 ± 2.4, n = 7 NMJs from 4 larvae, p < 0.0001, Student's t test; cac^sfGFP-N; vGlut-OE, 21.0 ± 1.3, n = 10 NMJs from 4 larvae, p = 0.0145, t test with Welch's correction). G, Confocal Z-projections of Cac and Brp in cac^sfGFP-N, in cac^sfGFP-N; GluRIIA and in cac^sfGFP-N; vGlut-OE motorneuron boutons colabeled with antibodies against GFP and Brp. H, Normalized intensity of Cac and Brp puncta averaged for each NMJ in control, GluRIIA and vGlut-OE animals (Cac levels: cac^sfGFP-N, 1.00 ± 0.05, n = 22 NMJs from 9 larvae; cac^sfGFP-N; GluRIIA, 1.36 ± 0.06, n = 14 NMJs from 5 larvae, p < 0.0001, Student's t test; and cac^sfGFP-N; vGlut-OE, 1.07 ± 0.06, n = 11 NMJs from 5 larvae, p = 0.43, Student's t test. Brp levels: cac^sfGFP-N, 1.00 ± 0.05, n = 22 NMJs from 4 larvae; cac^sfGFP-N; GluRIIA, 1.44 ± 0.04, n = 14 NMJs from 5 larvae, p < 0.0001, Student's t test; cac^sfGFP-N; vGlut-OE, 1.08 ± 0.06, n = 11 NMJs from 5 larvae, p = 0.19, Student's t test). I, Cumulative probability distributions of Cac and Brp puncta intensities demonstrate an increase in both Brp and Cac levels across AZs of GluRIIA animals [Cac levels: cac^sfGFP-N, n = 1475 AZs; cac^sfGFP-N; GluRIIA, n = 1475; (3 data points are outside the x-axis limits shown); Brp levels: cac^sfGFP-N, n = 1475 AZs; cac^sfGFP-N; GluRIIA, n = 1475 AZs]. J, Cumulative probability distributions of Cac and Brp puncta intensities show no decrease in Brp or Cac levels across AZs of vGlut-OE animals [Cac levels: cac^sfGFP-N, n = 1475 AZs; cac^sfGFP-N; vGlut-OE, n = 1443 (7 data points are outside the x-axis limits shown); Brp levels: cac^sfGFP-N, n = 1475 AZs; cac^sfGFP-N; vGlut-OE, n = 1443 AZs]. Not significant (ns), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.