Spatial characterization of Brp::rGFP clusters at single-AZ level.

(A) Schematic of the split-GFP tagging strategy. The GFP11 fragment is inserted by CRISPR/Cas9 just prior to the stop codon of brp, while GFP1-10 is expressed in specific cell types via GAL4-UAS system.

(B) Brp::rGFP signals in DPM. The entire MB lobe structure is shown. The black box in the left panel indicates the zoom-in area (right panel). GFP1-10 and CD4::tdTomato (magenta) were co-expressed using VT64246-GAL4. Scale bar, 20 μm (left); 5 μm (right).

(C) Image processing pipeline. Raw images were processed with image deconvolution to improve the signal quality. 3D maxima (green pixels) are detected for each Brp::rGFP cluster. Pixels around the maxima were clustered to created 3D ROIs, indicated by yellow circles. The “AZ density” is the number of Brp::rGFP clusters within r (3 × mean NND). Scale bar, 1 μm.

(D) Heatmaps of F-scores showing the detection performance across in different cell types. An intensity threshold (threshold) is applied to reduce the background noise when detecting 3D maxima. The noise tolerance is the parameter that adjusts the sensitivity of 3D maxima detection. The color represents the F-score value.

(E) Number of Brp::rGFP clusters detected in DPM and APL. The dashed lines indicate the number of electron dense projections identified in the hemibrain connectome for DPM (17530) and APL (10789) on the MB lobes and peduncle (cite the resource here). For both DPM and APL, n = 5. Lower panels show the representative 3D reconstructions of Brp::rGFP clusters in DPM and APL. Scale bars, 20 μm. Data were presented as mean ± s.e.m.

Compartmentalized AZ structures of KCs.

(A) Histograms of the signal intensity of individual Brp::rGFP clusters from three KC subtypes. Values are normalized by the mean intensity in each data set (each sample). Each line represents the histogram of one independent sample. γ KCs (MB009B-GAL4), n = 6; α/β KCs (MB370B-GAL4), n = 5; α’/β’ KCs (MB008B-GAL4), n = 5.

(B) 3D reconstructions of Brp::rGFP clusters in three KC subtypes, colored by the Brp::rGFP intensity. The approximate locations of compartments are as indicated. Min. and Max. were set to represent the lowest and highest 5% of Brp::rGFP intensity value in the dataset respectively. Scale bars, 20 μm.

(C) Signal intensity of Brp:rGFP clusters in each compartment. Brp::rGFP puncta were quantified compartmentally. Medians in different compartments are showed as the ratio against the average of five compartments in the corresponding KC subtype. Each line represents one independent sample.

The variability of Brp::rGFP concentrations depends on cell type.

(A) Brp::rGFP clusters with distinct intensities but similar size in DPM. The left panel shows a cropped image of Brp::rGFP in DPM. White boxes indicate areas zoomed-in in panels 1 and 2. Transparent white lines in panels 1 and 2 show the lines on which the intensity profiles were plotted. Intensity profiles were plotted for AZ1 (magenta) and AZ2 (blue) respectively.

(B-D) Scatter plots showing the correlation between the Brp::rGFP intensity and cluster volume in DPM, APL and α/β KCs. Data of three representative samples were shown. Pearson correlation coefficient R were calculated for each sample.

(E) Correlations between the intensity and volume of Brp::rGFP clusters in different cell types. DPM (n = 6), APL (n = 5), γ KCs (n = 6, MB009B-GAL4), α’/β’ KCs (n = 5, MB370B-GAL4), α/β KCs (n = 5, MB008B-GAL4). DPM vs. α’/β’ KCs: P = 0.0253; DPM vs. α/β KCs: P = 0.0009; APL vs. α/β KCs: P = 0.0035; γ KCs vs. α/β KCs: P = 0.0418. Values marked with different lowercase letters represent significant difference (P < 0.05); Data were presented as box plots showing center (median), whiskers (Min. to Max.).

Stereotyped AZ distribution of PPL1-α3 DAN.

(A) Brp::rGFP in PPL1 DANs. UAS-GFP1-10 was expressed using MB504B-GAL4. Dashed line marks the rough boundary between α3 and α2α’2 compartments. Scale bar, 20 μm.

(B) Brp::rGFP intensity profiles of α3 and α2α’2 compartments. Left panels show the max-projection images of α3 and α2α’2 optical coronal sections. Transparent gray stripes indicate areas where intensity profiles are plotted in the right panels. Scale bars, 10 μm.

(C) 3D reconstruction colored by the AZ density in PPL-1 DANs. Color scale: Min. = 0, Max. = 40. Dashed line indicates the rough boundary between α3 and α2α’2 compartments. Scale bar, 20 μm.

(D) Stereotyped AZ distribution pattern in PPL1-α3 across individuals. 3D reconstructions show the AZ density across different brain samples. Scale bar, 20 μm.

Cell-type-specific stereotypy of AZ spatial distributions.

(A) 3D reconstructions of Brp::rGFP clusters in DPMs, colored by AZ density. Color scale: Min. = 0, Max. = 35. Black arrows indicate consistently high AZ density regions across brain samples. Scale bars, 20 μm.

(B) 3D reconstructions of Brp::rGFP clusters in APLs, colored by AZ density. Color scale: Min. = 0, Max. = 30. Black dashed square indicates the area zoomed-in. APL reconstructions are arranged from left to right according to the overall AZ density in α3.

Local intensity analysis revealed sub-compartmental AZ structures.

(A) Correlation of Brp::rGFP intensities between nearest neighbor AZs in different cell types. Brp::rGFP intensities were log transformed. DPM vs. γ KCs: P = 0.0066; DPM vs. α’/β’ KCs: P = 0.0027; DPM vs. α/β KCs: P = 0.0066; Scale bar: 1 μm.

(B) 3D reconstructions of Brp::rGFP clusters in a γ KC sample, colored by local intensity and AZ density. Black arrows indicate areas with both high local intensities and high AZ densities. Min. and Max. were set to represent the lowest and highest 5% in local intensity value respectively. Color scale of AZ density: Min. = 0 and Max = 35.

(C) 3D reconstructions of Brp::rGFP clusters in a DPM neuron, colored by local intensity and AZ density. Min. and Max. are set to represent the lowest and highest 5% in local intensity value respectively. Color scale of AZ density: Min. = 0, Max. = 35.

(D) Correlations between AZ density and local intensity in different cell types. DPM (n = 6), APL (n = 5), γ KCs (n = 6, MB009B-GAL4), α’/β’ KCs (n = 5, MB370B-GAL4), α/β KCs (n = 5, MB008B-GAL4). DPM vs. γ KCs: P = 0.0171; DPM vs. α’/β’ KCs: P = 0.0171; APL vs. γ KCs: P = 0.0014; APL vs. α’/β’ KCs: P = 0.0014; APL vs. α/β KCs: P = 0.0171.

Associative conditioning re-organizes sub-compartmental active zone clusters.

(A) Brp::rGFP (green) and CD4::tdTtomato cytoplasm membrane marker (magenta) in KCs, visualized by using R13F02-GAL4.

(B) The experimental design of the aversive single odor conditioning. Files in the pair group receive concurrent presentation of 2% 4-MCH and 90 V electric shock. Flies in the unpair group first receive the electric shock first and then 4-MCH 1 min later. Flies were dissected at different time points after the conditioning.

(C) Correlation coefficient (AZ density vs. local intensity of individual Brp::rGFP puncta) of each compartment at 90 min after conditioning. Pair (n = 11) vs. Unpair (n = 12), γ2 (P = 0.0261), β2 (P = 0.0060), α1 (P = 0.0026), α’3 (P = 0.0026).

(D) Heatmap showing the difference of correlation coefficient (AZ density vs. local intensity of Brp::rGFP puncta) between the pair and unpair group at different time points after conditioning. The color indicates the difference and the asterisks in the compartments indicate the significant difference. For 3 min, Pair (n = 12), Unpair (n = 11); For 20 min, Pair (n = 12), Unpair (n = 11); For 90 min, Pair (n = 11) vs. Unpair (n = 12), P = 0.0247; For 270 min, Pair (n = 11), Unpair (n = 11), ; For 1 D, Pair (n = 12), Unpair (n = 9); For all results involved statistical comparison, only significant results are shown. Scale bars, 20 μm. Mann-Whitney test with original False Discovery Rate (FDR) method of Benjamini and Hochberg correction. *P<0.05. Data were presented as box plots showing centre (median), whiskers (Min. to Max.).