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. 2022 Jul 14;11:e78648. doi: 10.7554/eLife.78648

Figure 1. Ca2+ channels are not required for active zone (AZ) seeding or scaffold accumulation.

(A) Representative images of control neuromuscular junctions (NMJs) with both Ib and Is motoneurons co-innervating muscle 1 (top left), or muscle 1 with only Ib innervation (top right), and Is and cac-flipped Ib neurons co-innervating muscle 1 (bottom left) or cac-flipped Ib neurons with no Is present (bottom right). The flip-reporter tdTomato (magenta) and HRP (neuronal membranes; purple) were imaged prior to fixation (left panels). After fixation, NMJs were immunostained for Bruchpilot (BRP) (red) and GluRIII (green) to label AZs and PSDs, respectively (right panels). (B, C) Average traces and evoked amplitudes (peak current, nA) from the indicated genotypes (co-innervated controls: orange, 42.11±1.757, n=8 NMJs from 6 animals; co-innervated CacIb-flp: green, 15.13±1.607, n=15 NMJs from 12 animals, p<0.0001; Ib-only controls: blue, 32.32±2.329, n=6 NMJs from 6 animals; Ib-only CacIb-flp: gold, 5.749±0.9544, n=5 NMJs from 5 animals, p<0.0001). (D) Neuronal (HRP-stained) area of Ib synapses (co-innervated controls: orange, 219.1±15.15, n=8 NMJs from 6 animals; co-innervated CacIb-flp: green, 190.4±21.12, 9 NMJs from 8 animals; Ib-only controls: blue, 207.8±22.4, n=6 NMJs from 6 animals; Ib-only CacIb-flp: gold, 189±24.59, n=5 NMJs from 5 animals). (E) Number of AZs formed by Ib synapses (co-innervated controls: orange, 224.3±16.88; co-innervated CacIb-flp: green, 255.1±10.13; Ib-only controls: blue, 245.8±14.73; Ib-only CacIb-flp: gold, 220.4±13.47). (F) Some NMJs lacking Cac display ectopic filopodia extending from boutons (white arrowheads). (G) Representative images of cac-flipped NMJs stained with anti-BRP (magenta) and anti-RIM-binding protein (RBP) (green). (H) Quantification of BRP abundance. Each point represents the average fluorescence intensity across the AZ population of a single NMJ (CTRL: orange, 27,736±900.2, n=12 NMJs from 6 animals; CacIb-flp: green, 25,943±843.7, n=12 NMJs from 6 animals). (I) Quantification of RBP abundance (CTRL: orange, 19,910±439.2; CacIb-flp: green, 23,593±530.6, p<0.0001). (J) Representative images of cac-flipped animals stained for syntaxin 1 (SYX1) (magenta) and complexin (CPX) (green). (K) Quantification of SYX1 abundance. Each point represents the average fluorescence intensity across the bouton population of a single NMJ (CTRL: orange, 16,028±683.5, n=9 NMJs from 5 animals; CacIb-flp: green, 17365±643.4, n=12 NMJs from 6 animals). (L) Quantification of CPX abundance (CTRL: orange, 18,810±509.4; CacIb-flp: green, 17,961±663.5).

Figure 1—source data 1. Source data for Figure 1.

Figure 1.

Figure 1—figure supplement 1. Characterization of Cac-flipping.

Figure 1—figure supplement 1.

(A) Representative ventral nerve cords (VNCs) in 1st, 2nd, and 3rd instar larvae expressing the tdTomato reporter in MN1-Ib cell bodies of neurons that have undergone a cac-flip event in abdominal segments A1-A7. White arrowheads indicate hemisegments that have not flipped. (B) Quantification of the number of MN1-Ib somas (out of 14 total neurons) expressing the tdTomato reporter in 1st, 2nd, and 3rd instar VNCs (1st: 11±0.3273, n=8 VNCs; 2nd: 12.75±0.4532, n=8 VNCs, p<0.01; 3rd: 13.5±0.2236, n=6 VNCs). (C) Representative images of MN1-Ib NMJs in the 2nd instar stage stained with Bruchpilot (BRP) and HRP. (D) Quantification of Ib active zone (AZ) number at the 2nd instar stage (orange) with 3rd instar MN1-Ib AZ number (gray) from Figure 1E for comparison (2nd: 49.75±3.155, n=8 neuromuscular junctions (NMJs); 3rd: 224.3±16.88, N=8 NMJs, p<0.0001). (E) Distribution of BRP abundance (max pixel intensity per AZ) across the AZ population in control and cac-flipped NMJs, quantified from images in Figure 1G. (F) Average AZ BRP abundance (per NMJ) for the highest BRP-enriched 50% of AZs and the least BRP-enriched 50% of AZs in control and cac-flip NMJs, quantified from images in Figure 1G (control top 50%: 36,937±1264; Flp top 50%: 34,615±1056; control bottom 50%: 18,874±647.8; Flp bottom 50%: 17,539±740.6). (G) Distribution of RIM-binding protein (RBP) abundance (max pixel intensity per AZ) across the AZ population in control and cac-flipped NMJs, quantified from images in Figure 1G. (H) Average AZ RBP abundance (per NMJ) for the highest RBP-enriched 50% of AZs and the least RBP-enriched 50% of AZs in control and cac-flipped NMJs, quantified from images in Figure 1G (control top 50%: 25,130±500.2; Flp top 50%: 31,376±654.6, p<0.01; control bottom 50%: 13,630±301.6; Flp bottom 50%: 15,853±464.9, p<0,0001). (I) Representative images of control and cac-flipped AZs stained for GluRIII and BRP, acquired from the dataset in Figure 1A–F. (J) Quantification of the number of BRP-positive AZs per NMJ lacking GluRIII signal in control and cac-flipped neurons (control: 0.8±0.2494, n=10 NMJs; Flp: 1.222±0.4648, n=9 NMJs). (K) Quantification of the number of GluRIII-positive PSDs per NMJ lacking BRP signal in control and cac-flipped neurons (control: 8.889±1.925; Flp: 11±1.354).
Figure 1—figure supplement 1—source data 1. Source data for Figure 1—figure supplement 1.