Active Zone Maturation Controls Presynaptic Output and Release Mode and Is Regulated by Neuronal Activity

Enhanced evoked synaptic output at older PSDs. A, Representative larval M4 NMJ imaged 96 h after PC with GluR^Old PSDs individually numbered (left panel). The corresponding evoked P_r heatmap following quantal imaging with myrGCaMP7s is shown on the right, revealing AZs opposed to the oldest PSDs are among the strongest release sites. B, Correlation between PSD age, determined by GluR^Old sum fluorescent intensity of individual PSDs, and evoked P_r (Pearson's r value = 0.56). GluR^Old sum intensity was normalized for each NMJ across animals, with the max sum fluorescence PSD signal set to 1.0 for each corresponding NMJ. C, Average evoked P_r for AZs opposed to older PSDs containing any red⁺ GluR^Old compared with younger PSDs with only green⁺/red⁻ GluR^New (red⁺ PSDs, 0.193 ± 0.013; n = 174 PSDs from 5 larvae; red⁻ PSDs, 0.081 ± 0.005; n = 378 PSDs from 5 larvae; p < 0.0001). D, Representative larval M4 NMJ imaged 48 h after PC with GluR^Old PSDs individually numbered (left panel). The corresponding evoked P_r heatmap following quantal imaging with myrGCaMP7s is shown on the right. E, Correlation between PSD age determined by red⁺ GluR^Old fluorescent intensity and evoked P_r (Pearson's r value = 0.66). F, Average evoked P_r for AZs opposed to older PSDs containing any red⁺ GluR^Old compared with younger PSDs with only green⁺/red⁻ GluR^New (red⁺ PSDs, 0.174 ± 0.01; n = 179 PSDs from 5 larvae; red⁻ PSDs, 0.0397 ± 0.004; n = 192 PSDs from 5 larvae; p < 0.0001). G, Representative images of NMJ growth at M4 (left) and M6/7 (right) 4 d after PC of GluRIIE^Maple first instar larvae. Note the addition of terminal boutons lacking older red⁺ PSDs (arrow) at M4 versus the internal addition of new boutons lacking red⁺ PSDs at M6 (white outline and arrows). H, Quantification of the percent of GluR^Old PSDs to all PSDs at the terminal bouton versus the second bouton from the end. Older PSDs are enriched in the terminal bouton at M6/7 NMJs (terminal bouton GluR^Old positive PSDs as a percent of all PSDs, M6/7, 34.3% ± 3.4; n = 15 NMJs from 5 larvae; M4, 14.2% ± 2.7; n = 29 NMJs from 5 larvae; second bouton from end, M6/7, 29.7% ± 3.7; n = 14 NMJs from 5 larvae; M4, 38.6% ± 3.0; n = 26 NMJs from 5 larvae). Statistical significance determined with Student's t test, ns, not significant. Asterisks denote the following p value: ***p ≤ 0.001. Raw values and statistical details are provided in Data S1.

AZs lacking Cac can support spontaneous fusion. A, Representative third instar M4 NMJ from a larva expressing GluRIIB-GFP and Cac^RFP. Several PSDs lacking opposed Cac^RFP+ signal are individually numbered. Cac^RFP signal is shown in Panel 2, with spontaneous activity (release events per minute) presented as a heat map at AZs opposed to GluRIIB⁺ PSDs in Panel 3. The corresponding evoked P_r heatmap is shown on the right, highlighting lack of evoked release at Cac⁻ AZs. AZs lacking Cac can display low (Sites 1 and 5), moderate (Site 3), or high (Sites 2 and 4) rates of spontaneous release with no corresponding evoked output. B, Representative third instar larval M4 NMJ imaged 96 h after PC to identify GluR^Old PSDs (left panel). The corresponding heatmap for spontaneous activity (release events per minute) is shown on the right. C, Correlation between PSD age determined by red⁺ GluR^Old fluorescent intensity and spontaneous release rate (Pearson's r value = 0.31). D, Spontaneous release rate for AZs opposed to older PSDs containing any red⁺ GluR compared with younger PSDs with only green⁺/red⁻ GluR (red⁺ synapses, 0.841 ± 0.1 events per min; n = 80 AZs from 4 larvae; red⁻ synapses, 0.398 ± 0.04; n = 157 AZs from 4 larvae; p < 0.0001). E, Spontaneous release rate for AZs containing Cac^RFP compared with those without (Cac^RFP+, 1.11 ± 0.05 events per min; n = 366 AZs from 5 larvae; Cac^RFP−, 0.497 ± 0.07; n = 55 AZs from 5 larvae; p < 0.0001). Statistical significance determined with Student's t test; ns, not significant. Asterisks denote the following p values: ***p ≤ 0.001 and ****p ≤ 0.0001. Raw values and statistical details are provided in Data S1.

Time course for protein accumulation during AZ maturation. A, Representative second instar M4 synaptic boutons with an analysis of the distribution of fluorescent puncta for the indicated AZ proteins (left panel A) in relation to immunostained RBP (middle panel B) and endogenous CRISPR GFP-tagged or RFP-tagged Cac (right panel C). The merged image shows labeling for Cac and the AZ protein indicated in panel A, with representative AZs lacking Cac indicated by white arrows. The right panels show the percentage of individual puncta for each AZ protein lacking RBP or Cac (top values) versus RBP or Cac puncta lacking the indicated AZ protein (bottom values). N = 7 NMJs from two larvae for each correlation. B, Representative image of a second instar M4 synaptic bouton showing Liprin-α puncta compared with the distribution of GluRIIB. Most puncta overlap, although a few are positive for Liprin-α only (white arrows). C, Serial imaging of RBP and Cac appearance at second instar M4 AZs over three time points indicate RBP accumulates before Cac arrival. The appearance of new RBP or Cac accumulations within 6 h intervals was scored (n = 10 NMJs from 3 larvae), revealing 62% of newly formed puncta are only RBP⁺ and 38% are RBP⁺ and Cac⁺. No case of Cac at newly formed AZs lacking RBP was observed. Raw values and statistical details are provided in Data S1.

The abundance of early AZ scaffolds poorly predicts Cac accumulation and P_r. A, Representative images of third instar M4 synaptic boutons expressing endogenous CRISPR-tagged Cac^GFP (top row) and the indicated AZ scaffolding protein (bottom row). One of the brightest Cac⁺ puncta in each bouton is highlighted (white arrows) to compare abundance with the indicated scaffolding protein. B–E, Correlation between AZ abundance (fluorescent intensity) of Cac and Liprin-α (B, n = 1,422 AZs from 6 NMJs from 6 larvae), Syd1 (C, n = 1,579 AZs from 6 NMJs from 6 larvae), RBP (D, n = 996 AZs from 6 NMJs from 6 larvae), and Unc13A (E, n = 984 AZs from 6 NMJs from 6 larvae) at third instar M4 NMJs. F–J, Correlation between evoked P_r and AZ abundance (fluorescent intensity) of Liprin-α (F, n = 913 AZs from 6 NMJs from 6 larvae), Syd 1 (G, n = 525 AZs from 6 NMJs from 6 larvae), RBP (H, n = 407 AZs from 6 NMJs from 6 larvae), Unc13A (I, n = 462 AZs from 8 NMJs from 6 larvae), and Cac (J, n = 486 AZs from 7 NMJs from 6 larvae) at third instar M4 NMJs. Raw values and statistical details are provided in Data S1.

Presynaptic silencing results in AZ enlargement via a Rab3-independent mechanism. A, Representative images of Cac, BRP, and RBP immunolabeling within synaptic boutons of third instar M1 NMJs of controls or following expression of TeNT (middle panel) or Syt1^DN (right panel) with the MN1-Ib-Gal4 driver. B, Quantification of AZ Cac area in the three genotypes (Ctrl, n = 18 NMJs from 5 larvae; TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; Syt1^DN; n = 19 NMJs from 5 larvae; p < 0.0001). C–E, Quantification of AZ sum Cac fluorescence (C, Ctrl, n = 18 NMJs from 5 larvae; TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001), BRP (D, Ctrl, n = 18 NMJs from 5 larvae; TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001) and RBP (E, Ctrl, n = 18 NMJs from 5 larvae; TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001) across the three genotypes. F, Representative images of Cac, BRP, and RBP immunolabeling within synaptic boutons of third instar M1 NMJs of controls (left panel) or following expression of rab3 RNAi alone (second panel) or coexpressed with TeNT (third panel) or Syt1^DN (right panel) with the MN1-Ib-Gal4 driver. G, Quantification of AZ Cac area in the four genotypes (Ctrl, n = 19 NMJs from 5 larvae; rab3 RNAi, n = 19 NMJs from 5 larvae; p = 0.0002; rab3 RNAi + TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; rab3 RNAi + Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001). H–J, Quantification of AZ sum fluorescence for Cac (H, Ctrl, n = 19 NMJs from 5 larvae; rab3 RNAi, n = 19 NMJs from 5 larvae, p = 0.019; rab3 RNAi + TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; rab3 RNAi + Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001), BRP (I, Ctrl, n = 19 NMJs from 5 larvae; rab3 RNAi, n = 19 NMJs from 5 larvae; p = 0.0009; rab3 RNAi + TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; rab3 RNAi + Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001), and RBP (J, Ctrl, n = 19 NMJs from 5 larvae; rab3 RNAi, n = 19 NMJs from 5 larvae; p = 0.0009; rab3 RNAi + TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; rab3 RNAi + Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001) across the four genotypes. K, Representative images of Cac and HRP immunolabeling within synaptic boutons of M1 NMJs of controls or following expression of rab3 RNAi, Syt1^DN or TeNT alone, or rab3 RNAi coexpressed with TeNT or Syt1^DN, with the MN1-Ib-Gal4 driver. L, Quantification of Cac⁺ AZ number normalized to NMJ HRP area across the six genotypes (Ctrl, n = 22 NMJs from 5 larvae; rab3 RNAi, n = 11 NMJs from 4 larvae, p < 0.0001; TeNT, n = 21 NMJs from 5 larvae; p < 0.0001; Syt1^DN, n = 19 NMJs from 5 larvae; p < 0.0001; rab3 RNAi + TeNT, n = 15 NMJs from 4 larvae; p < 0.0001; rab3 RNAi + Syt1^DN, n = 24 NMJs from 5 larvae; p < 0.0001). Statistical significance determined with one-way ANOVA followed by Tukey's multiple-comparison test. Asterisks denote the following p values: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Raw values and statistical details are provided in Data S1.

Presynaptic knockdown of the Para sodium channel mimics effects of blocking SV fusion on AZ enlargement. A, Representative images of Cac immunolabeling within synaptic boutons of third instar M1 NMJs of controls (left panel) or following expression of para RNAi (second panel), rab3 RNAi (third panel), or para RNAi + rab3 RNAi (right panel) with the MN1-Ib-Gal4 driver. RNAi-mediated suppression of para also triggers AZ enlargement that is enhanced following Rab3 knockdown. Scale bar, 1 µm. B, Quantification of the M1 AZ Cac area in the four genotypes (Ctrl, n = 15 NMJs from 4 larvae; para RNAi, n = 14 NMJs from 4 larvae; p < 0.0001; rab3 RNAi, n = 15 NMJs from 4 larvae; p < 0.0001; para RNAi + rab3 RNAi, n = 14 NMJs from 4 larvae; p < 0.0001). C, Quantification of M1 AZ Cac sum fluorescence in the four genotypes (Ctrl, n = 15 NMJs from 4 larvae; para RNAi, n = 14 NMJs from 4 larvae; p < 0.0001; rab3 RNAi, n = 15 NMJs from 4 larvae; p = 0.0002; para RNAi + rab3 RNAi, n = 14 NMJs from 4 larvae; p < 0.0001). Statistical significance determined with Student's t test; asterisks denote the following p value: ***p ≤ 0.001. Raw values and statistical details are provided in Data S1.

Presynaptic silencing reduces seeding of both early and late AZ scaffolds. A, Representative images of Unc13B-mClover and Unc13A-mRuby localization within synaptic boutons of third instar M1 NMJs of controls (top panel) or following expression of rab3 RNAi (second panel), TeNT (third panel), or Syt1^DN (bottom panel) with the MN1-Ib-Gal4 driver. The merged image is shown on the right. Scale bar, 1 µm. B, Quantification of Unc13B⁺ AZ number (per 10 mm²) across the four genotypes (Ctrl, n = 19 NMJs from 5 larvae; rab3 RNAi, n = 28 NMJs from 6 larvae; p = 0.99; TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; Syt1^DN, n = 12 NMJs from 4 larvae; p < 0.0001). C, Quantification of the Unc13A⁺ AZ number (per 10 mm²) across the four genotypes (Ctrl, n = 19 NMJs from 5 larvae; rab3 RNAi, n = 28 NMJs from 6 larvae; p < 0.0001; TeNT, n = 19 NMJs from 5 larvae; p < 0.0001; Syt1^DN, n = 12 NMJs from 4 larvae; p < 0.0001). D, Quantification of the Unc13A⁺/Unc13B⁺ AZ ratio across the four genotypes (Ctrl, n = 19 NMJs from 5 larvae; rab3 RNAi, n = 28 NMJs from 6 larvae; p < 0.0001; TeNT, n = 19 NMJs from 5 larvae; p = 0.07; Syt1^DN, n = 12 NMJs from 4 larvae; p = 0.57). E, Representative images of third instar M4 boutons immunostained for BRP (cyan) after PC of GluRIIE^Maple at the first or second instar stage in rab3 mutants or the first instar stage in controls. BRP⁺ AZs were rarely observed at newly formed green⁺/red⁻ GluR^New PSDs (green arrowheads) compared with older red⁺ PSDs (red arrowheads) after PC at the first instar stage in rab3 mutants. When PC was performed in the second instar stage in rab3 mutants, only older red⁺ PSDs were opposed to AZs that were BRP⁺. AZs opposed to newly formed green⁺/red⁻ PSDs were BRP⁻. Controls showed a normal pattern of synapse maturation, with AZs opposed to older red⁺ PSDs containing BRP and 89% of newly formed green⁺/red⁻ PSDs opposed to AZs being BRP⁺. F, Quantification of BRP⁺ AZs opposed to newly formed green⁺/red⁻ PSDs after PC in the first instar stage in controls and rab3 mutants (Ctrl, 86.6 ± 1.3%; n = 18 NMJs from 3 larvae; rab3, 8.5 ± 2.0%; n = 11 NMJs from 3 larvae; p > 0.001) and after PC in the second instar stage in rab3 mutants (0.9 ± 0.3%; n = 14 NMJs from 3 larvae). G, Relative P_r frequency distribution across the AZ population for rab3 and control M4 NMJs reveals a right-shifted and more homogenous distribution in rab3 mutants. Statistical significance determined with one-way ANOVA followed by Tukey's multiple-comparison test. Asterisks denote the following p values: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; and ****p ≤ 0.0001. Raw values and statistical details are provided in Data S1.

Presynaptic silencing increases T-bar area, while rab3 mutants contain multiple T-bars per AZ. A, Representative EM images of synaptic boutons at third instar M1 NMJs of controls (top panel) or following expression of TeNT (second panel), rab3 RNAi (third panel), or rab3 RNAi + TeNT (bottom panel) with the MN1-Ib-Gal4 driver. Scale bar, 200 nm. B, Quantification of T-bar size (length of the top T-bar platform) across the four genotypes (Ctrl, n = 25 T-bars from 3 larvae; TeNT, n = 30 T-bars from 3 larvae; p < 0.0001; rab3 RNAi, n = 39 T-bars from 3 larvae; p = 0.88; rab3 RNAi + TeNT, n = 33 T-bars from 3 larvae; p = 0.0007). C, Quantification of T-bar number per AZ across the four genotypes (Ctrl, n = 25 AZs from 3 larvae; TeNT, n = 29 AZs from 3 larvae; p = 0.74; rab3 RNAi, n = 28 AZs from 3 larvae; p = 0.04; rab3 RNAi + TeNT, n = 21 AZs from 3 larvae; p < 0.0001). D, Quantification of synapse length (measurement of synaptic cleft electron-dense material length) across the four genotypes (Ctrl, n = 26 synapses from 3 larvae; TeNT, n = 37 synapses from 3 larvae; p = 0.90; rab3 RNAi, n = 45 synapses from 3 larvae; p = 0.09; rab3 RNAi + TeNT, n = 27 synapses from 3 larvae; p = 0.001). E, Representative confocal and MINFLUX images of synaptic boutons at third instar M1 NMJs of controls (top panel) or following expression of TeNT (second panel), rab3 RNAi (third panel), or rab3 RNAi + TeNT (bottom panel) with the MN1-Ib-Gal4 driver. The red dashed line denotes a representative BRP cluster diameter measurement. Scale bar, 500 nm. F, Representative SIM images of synaptic boutons at third instar M1 NMJs of controls (top panel) or following expression of TeNT (second panel), rab3 RNAi (third panel), or rab3 RNAi + TeNT (bottom panel) with the MN1-Ib-Gal4 driver. The blue dashed line denotes a representative BRP ring diameter measurement. Scale bar, 1 mm. G, Quantification of BRP cluster diameter measured with MINFLUX across the four genotypes (Ctrl, n = 49 AZs from 3 larvae; TeNT, n = 53 AZs from 3 larvae; p = 0.0001; rab3 RNAi, n = 65 AZs from 3 larvae; p < 0.0001; rab3 RNAi + TeNT, n = 28 AZs from 3 larvae; p < 0.0001). H, Quantification of BRP ring diameter measured with SIM across the four genotypes (Ctrl, n = 45 AZs from 3 larvae; TeNT, n = 59 AZs from 3 larvae; p < 0.0001; rab3 RNAi, n = 24 AZs from 3 larvae; p = 0.71; rab3 RNAi + TeNT, n = 95 AZs from 3 larvae; p < 0.0001). Statistical significance determined with one-way ANOVA followed by Tukey's multiple-comparison test. Asterisks denote the following p values: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Raw values and statistical details are provided in Data S1.

Changes in BRP turnover and role of GluRIIA in AZ enlargement following presynaptic silencing. A, Representative images of mEosEM-tagged BRP before (green BRP^New) and after (red BRP^Old) PC of M9 (control) and M1 (TeNT) NMJs in larvae expressing UAS-TeNT with the MN1-Ib-Gal4 driver. B, BRP^Old sum fluorescence per AZ puncta was compared before and 24 h after PC at M9 (control) and M1 (TeNT) NMJs (Ctrl 0 Hr, 121,279 ± 5,214; n = 28 NMJs from 7 larvae; Ctrl 24 Hr, 88,464 ± 4,428; n = 32 NMJs from 14 larvae; p < 0.0001; TeNT 0 Hr, 187,774 ± 7,831; n = 28 NMJs from 7 larvae; TeNT 24 Hr, 155,278 ± 6,958; n = 32 NMJs from 14 larvae; p = 0.0029). C, Decay in BRP^Old as a percentage of the signal detected immediately after PC (Ctrl, 72.94% ± 3.65; n = 32 NMJs from 14 larvae; TeNT, 82.69 ± 3.71; n = 32 NMJs from 14 larvae; p = 0.0034). D, Quantification of mean BRP^New fluorescence 24 h post-PC (normalized by pixel area of each AZ; Ctrl, 6,315 ± 613.4; n = 32 NMJs from 14 larvae; TeNT, 6,795 ± 533.8; n = 32 NMJs from 14 larvae; p = 0.0647). E, Quantification of sum BRP^New fluorescence 24 h post-PC (Ctrl, 84,669 ± 7,899; n = 32 NMJs from 14 larvae; TeNT, 110,841 ± 8,890; n = 32 NMJs from 14 larvae; p < 0.0001). F, Representative images showing BRP-NEMOm fluorescence in response to Ca²⁺ influx during 10 Hz stimulation in M9 (control) and M1 (TeNT) NMJs in larvae expressing UAS-TeNT with the MN1-Ib-Gal4 driver. Basal fluorescence of BRP-NEMOm without stimulation is undistinguishable from background (data not shown). G, Quantification of sum BRP-NEMOm fluorescence per AZ (Ctrl, 229,046 ± 15,421; n = 14 NMJs from 7 larvae; TeNT, 437,306 ± 27,187; n = 14 NMJs from 7 larvae; p < 0.0001). H, Quantification of mean BRP-NEMOm fluorescence per AZ (Ctrl, 9,784 ± 241.9; n = 14 NMJs from 7 larvae; TeNT, 11,362 ± 471.1; n = 14 NMJs from 7 larvae; p = 0.0044). Statistical significance determined with paired t test. I, Representative images of Cac (top panel) and GluRIIA (bottom panel) immunolabeling at synaptic boutons of third instar M9 and M1 NMJs of controls, GluRIIA^−/− null mutants or following expression of Syt1^DN or rab3 RNAi with the MN1-Ib-Gal4 driver in control GluRIIA^+/− heterozygotes or GluRIIA^−/− nulls. J, Quantification of the M1/M9 ratio for AZ Cac area across the six genotypes (Ctrl, n = 14 NMJs from 4 larvae; GluRIIA^+/− + Syt1^DN, n = 14 NMJs from 4 larvae; p < 0.0001; GluRIIA^−/− + Syt1^DN, n = 26 NMJs from 6 larvae; p = 0.84; GluRIIA^−/−, n = 14 NMJs from 4 larvae; p = 0.83; GluRIIA^+/− + rab3 RNAi, n = 13 NMJs from 4 larvae; p = 0.0002; GluRIIA^−/− + rab3 RNAi, n = 14 NMJs from 4 larvae; p < 0.0001). K, Quantification of AZ Cac area at control M1 and M9 NMJs and manipulated M1 NMJs across the five genotypes (Ctrl M9, n = 14 NMJs from 4 larvae; Ctrl M1, n = 14 NMJs from 4 larvae; GluRIIA^+/− + Syt1^DN, n = 14 NMJs from 6 larvae; p < 0.0001; GluRIIA^−/− + Syt1^DN, n = 26 NMJs from 4 larvae; p = 0.30; GluRIIA^−/−, n = 14 NMJs from 4 larvae; p = 0.98; GluRIIA^+/− + rab3 RNAi, n = 13 NMJs from 4 larvae; p < 0.0001; GluRIIA^−/− + rab3 RNAi, n = 14 NMJs from 4 larvae; p < 0.0001). L, Quantification of M1 and M9 Cac⁺ AZ number in controls or GluRIIA^−/− null mutants with or without expression of Syt1^DN using the MN1-Ib-Gal4 driver (Ctrl M1, n = 14 NMJs from 4 larvae; Ctrl M9, n = 15 NMJs from 4 larvae; p = 0.99; M1 in MN1-Ib-Gal4 > Syt1^DN, n = 20 NMJs from 5 larvae; p = 0.0003; M9 in MN1-Ib-Gal4 > Syt1^DN, n = 19 NMJs from 5 larvae; p = 0.7; M1 in GluRIIA^−/−, n = 13 NMJs from 4 larvae; p = 0.99; M9 in GluRIIA^−/−, n = 13 NMJs from 4 larvae; p = 0.99; M1 in GluRIIA^−/− + MN1-Ib-Gal4 > Syt1^DN, n = 17 NMJs from 4 larvae; p < 0.0001; M9 in GluRIIA^−/− + MN1-Ib-Gal4 > Syt1^DN, n = 16 NMJs from 4 larvae; p = 0.90). Statistical significance determined with one-way ANOVA followed by Tukey's multiple-comparison test. Asterisks denote the following p values: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, and ****p ≤ 0.0001. Raw values and statistical details are provided in Data S1.