Comparison between SC and FC, and optimal nuisance factor removal methods.

a, Schematic image of the comparison between SC matrices and FC matrices. (left) Three ROI types: Hemibrain anatomical 52 ROIs, SCKm 50 ROIs, and DistKm 50 ROIs. (center) SC matrices (neuron) and SC matrices (synapse) for each ROI type. (right) FC matrices for an average of 8 flies for each ROI type. b, Schematic image of calcium imaging data registration. c, Swarm plot of ROI volume for each ROI type. d, Results of nuisance factor removal method investigation. The vertical axis shows the correlation coefficient, and the horizontal axis shows the FC-SC correlation for each method. Results are plotted for the three ROI types. e, The vertical axis shows the averaged AUC of 100 thresholds, and the horizontal axis is the same as d. f, The vertical axis shows the FC-SC detection & correlation score, and the horizontal axis is the same as d.

Investigating spatial smoothing size, and comparison of FC-SC correlation between mammals and flies.

a, (top) Example z-slice of the reduced FDA template with DistKm ROIs from 20 to 1000 ROIs. (middle) SC matrices (neuron) of DistKm ROIs from 20 to 1000 ROIs. (bottom) FC matrices of DistKm ROIs from 20 to 1000 ROIs. b, From left to right, FC-SC correlation, FC-SC detection and FC-SC detection & correlation score, respectively. The solid line shows the FC vs. SC matrix (neuron), and the dashed line shows FC vs. SC matrix (synapse) at each ROI count. The vertical axis shows the correlation coefficient, averaged AUC and FC-SC detection & correlation score, respectively. The horizontal axis shows spatial smoothing size (voxels). c, Comparison between raw & smoothing (0 to 30 voxels) vs. Poly-tCompCor & smoothing (0 to 30 voxels) at each ROI count. Non-parametric Mann-Whitney U test was performed and Bonferroni correction was applied to correct for the familywise error rate (*** p<1.67e-4). d, Relation between FC-SC correlation and ROI count. (left vertical axis) Solid line shows FC-SC correlation (neuron) of SCKm and DistKm ROI type at each ROI count. (right vertical axis) The dashed line shows FC-SC detection (neuron) of SCKm and DistKm at each ROI count. e, Swarm plot of ROI volume of SCKm and DistKm at each ROI count.

Comparison between FlyEM and FlyWire connectome data.

a, Bar graph of post-synapse count in hemibrain region from FlyEM and FlyWire connectome data. The vertical axis shows post-synapse count, and the horizontal axis shows confidence thresholds for FlyEM and Cleft score thresholds for FlyWire. b, (top) Maximum Z-projection of synapse point cloud of FlyEM (confidence thresholds=0.8 [hereafter, cf0.8]) (bottom) Maximum Z-projection of synapse point cloud of FlyWire (Cleft score threshold=140 [hereafter, sc140]) c, Bar graph of post-synapse count in hemibrain primary ROIs. Blue bar shows FlyEM (cf0.8), red bar shows FlyWire (sc140). The vertical axis shows post-synapse count, and the horizontal axis shows hemibrain primary ROIs. d, Bar graphs of FC-SC correlation, FC-SC detection, and FC-SC detection & correlation score, respectively. The horizontal axis shows confidence thresholds for FlyEM and Cleft score thresholds for FlyWire. e, Bar graph of FC-SC correlation in hemibrain primary ROIs. Blue bars show FlyEM (cf0.8) vs FC (neuron), red bars show FlyWire (sc140) vs FC (neuron). The vertical axis shows FC-SC correlation, and the horizontal axis shows hemibrain primary ROIs. f, Bar graph of neurotransmitter rate of output neurons in hemibrain primary ROIs based on FlyWire (sc140) connectome data [45]. The horizontal axis shows hemibrain primary ROIs. (DA: dopamine, SER: serotonin, GABA, GLUT: glutamine, ACH: acetylcholine, OCT: octopamine) g, Scatter plots of neurotransmitter rate of output neurons vs. FC-SC correlation (neuron) in hemibrain primary ROIs (FlyWire sc140). Each of the six neurotransmitters was compared.

Quantification of pre- and post-synapse segregation, and the relationship between FC and synapses.

a, Unsegregated first (APL_L cell) and second (CT1_R cell) ranking in FlyWire (sc140) connectome data. A red dot shows pre-synapse, a blue dot shows post-synapse. b, The PPSSI histogram of all neurons (139,255) in FlyWire (sc140) connectome data. The vertical axis shows neuron count, and the horizontal axis shows PPSSI [0 1]. c, Segregated first (OA-AL2b2 cell) and second (OA-AL2b2 cell) ranking in FlyWire (sc140) connectome data. d, Schematic image of pre- and post-synapse clustering based on DBSCAN, and calculation of cPPSSI and PPSSI. e, Histogram of null SC matrices (blue bar) and extracted SC matrix with PPSSI (0-0.1) (red solid line). Black dotted line shows cumulative distribution function of the normal distribution, brown solid line shows cumulative distribution function of null & extracted SC matrices, and red dotted line shows Bonferroni-corrected p<0.05 threshold. Top shows SC matrix (synapse) total, middle shows FC-SC correlation (neuron), and bottom shows FC-SC correlation (synapse). f, Histogram of null SC matrices and extracted SC matrix with PPSSI (0.9-1). g, Example of a reciprocal-synapse in the EM image (left) and in 3D (right). h, Histogram of reciprocal-synaptic minimum distances in FlyWire (sc140) connectome data. The vertical axis shows reciprocal-synapse pair count, and the horizontal axis shows distance [μm]. i, Histogram of null SC matrices and extracted SC matrix for reciprocal-synapses (≤ 2μm).

General Linear Model analysis result and Wedge-AVLP GABA neuron.

a, Schematic image of GLM analysis. From the top, experimental image and walking trajectory, calcium response function, thresholded velocity, and GLM design matrix, respectively. b, 2nd level (group) GLM analysis result from 9 flies. Top shows Z=17 slice of the resized FDA template, and the bottom shows the Z=21 slice. The white arrow indicates the suppressed region during the fly walking task. c, Synapse point cloud of the left WAGN in FlyWire (sc140) connectome data. Top shows Z=17 slice of the resized FDA template, and bottom shows the Z=21 slice. d, Network graph of the left WAGN, ANs, WPNb tier 1 and tier 2/3 neurons from FlyWire codex (sc50, connection threshold>5). Blue is acetylcholine, ocher is GABA neuron. The small number on the edges shows synapse count. e, 3D image of the left WAGN, ANs, WPNb tier 1, tier 2/3 and left ER1 neurons, frontal view. Inside the black square shows the left WAGN, ANs and WPNb tier 2/3 from the back side. f, Histogram of closest distance from pre-synapse (to WPNb) to post-synapse (from ANs) in the left WAGN. Horizontal axis shows distance [μm]. Neurotransmitter indicates input ANs to post-synapses. g, Example 3D image of pre-synapse (to WPNb) to post-synapse (from ANs). h, All pre- and post-synapses of the left WAGN from the front. Color indicates cPPSSI in each synapse. i, Histogram of closest distance from pre-synapse to post-synapse in the left WAGN. The horizontal axis shows distance [μm]. Neurotransmitter indicates input neurons to post-synapses.

Schematic image of registration process and connectome data transformation in Drosophila melanogaster.

a, Schematic image of calcium image registration to the resized FDA template. Motion and slice timing correction were conducted using SPM12 [57], and the averaged NIfTI image was registered by ANTs [58] to the resized FDA template. Lastly, 4D NIfTI images were transformed using the transformation information. b, Schematic image of synapse points

Pre- and Post-synapses of pyramidal cell in human temporal cortex layer 3-4.

a, (left) Input to (post-synapses) a pyramidal cell in the human temporal cortex, from EM connectome data [39]. (center insert) A slice of human temporal cortex and target pyramidal cell. (right) Output from (pre-synapses) a pyramidal cell in the human temporal cortex, from EM connectome data. Blue dots show post-synaptic sites, Red dots show pre-synaptic sites.

Investigating spatial smoothing size, and high-pass filtering.

a, (top) Example z-slice of reduced FDA template with SCKm ROIs from 20 to 1000 ROIs. (middle) SC matrices (neuron) of SCKm ROIs from 20 to 1000 ROIs. (bottom) FC matrices of SCKm ROIs from 20 to 1000 ROIs. b, From left to right, FC-SC correlation, FC-SC detection, and FC-SC detection & correlation score, respectively. Solid lines shows FC vs. SC matrix (neuron), and dashed lines shows FC vs. SC matrix (synapse) for each ROI count. The vertical axis shows the correlation coefficient, averaged AUC, and FC-SC detection & correlation score, respectively. The horizontal axis shows spatial smoothing size (voxels). c, Scatter plot of FC-SC correlation in the marmoset left cortex [10] (145 injection×9862 voxels, r=0.379). d, Result of high-pass filtering and nuisance factor removal combination of DistKm ROIs. From left to right, FC-SC correlation, FC-SC detection, and FC-SC detection & correlation score, respectively. The high-pass filter degraded the FC-SC relationship.

Comparison between FlyEM and FlyWire connectome data.

a, (left) A scatter plot of regional post-synapse count between FlyEM (cf0.8) and FlyWire (sc140) in the hemibrain primary 63 ROIs. The Pearson correlation is not so similar, r=0.778, but the Spearman correlation is much more similar, r=0.9. (right) A scatter plot of log10 of the regional post-synapse count between FlyEM (cf0.8) and FlyWire (sc140) in the hemibrain primary ROIs. Pearson correlation shows r=0.919, a higher similarity. b, From left to right, FC-SC correlation, FC-SC detection, and FC-SC detection & correlation score, respectively. Solid lines shows FC vs. SC matrix (neuron), and dashed lines shows FC vs. SC matrix (synapse) of FlyEM (cf0.5-0.9) and FlyWire (sc50-140) corresponding Fig.3d. The vertical axis shows the correlation coefficient, averaged AUC, and FC-SC detection & correlation score, respectively. The horizontal axis shows spatial smoothing & nuisance factor removal combinations. c, From left to right, SC matrix (neuron) (FlyEM cf0.8), SC matrix (neuron) (FlyWire sc140) and FC matrix (Poly-tCompCor, no smoothing) in the hemibrain primary 63 ROIs. d, (right) SC matrix (neuron) result of FlyEM (cf0.8) minus FlyWire (sc140) in hemibrain primary ROIs. (left) SC matrix (synapse) result of FlyEM (cf0.8) minus FlyWire (sc140). FlyEM shows a higher number of synaptic connections.

In-neuron neurotransmitters and comparison between FlyEM and FlyWire connectome data.

a, Bar graph of neurotransmitter rate of input neurons in hemibrain primary 63 ROIs based on FlyWire (sc140) connectome data [44]. The horizontal axis shows hemibrain primary ROIs. (DA: dopamine, SER: serotonin, GABA, GLUT: glutamine, ACH: acetylcholine, OCT: octopamine) b, Scatter plots of neurotransmitter rate of input neurons vs. FC-SC correlation (neuron) in hemibrain primary ROIs (FlyWire sc140). Each of the six neurotransmitters was compared. c, Comparison between FlyEM and FlyWire APL right neuron (synapse point cloud mask). Red voxels are FlyEM, green voxels are FlyWire, and yellow voxels are both. Sørensen-Dice index (0.716) shows similarity, so neuropil shape is similar between two. d, (left) A scatter plot of post-synapse count between FlyEM and FlyWire APL right neuron. Pearson correlation shows r=0.104, so not similar at all. (center) Same as left, but synapse point cloud was spatially smoothed by gaussian kernel (2 voxels). Similarity was increased (r=0.379). (right) Same as center, but smoothed by gaussian kernel (8 voxels). Similarity was increased (r=0.66). These results support that spatial smoothing can absorb inter-individual variability.

Quantification of pre- and post-synapse segregation and relationship between FC and synapses (FlyEM).

a, Unsegregated first (APL_L cell) and second (DPM_R cell) ranked cells in the FlyEM (cf0.8) connectome data. Red dots shows pre-synapses, blue dot shows post-synapses. b, The PPSSI histogram of hemibrain neurons (99,644) in FlyEM (cf0.8) connectome data. The vertical axis shows neuron count, and the horizontal axis shows PPSSI [0 1]. c, Segregated first (AOTU019_R cell) and second (DNa02_R cell) ranked cells in the FlyEM (cf0.8) connectome data. d, Histogram of null SC matrices (blue bar) and extracted SC matrix with PPSSI (0-0.1) (red solid line). The black dotted line shows the cumulative distribution function of the normal distribution, the brown solid line shows the cumulative distribution function of null & extracted SC matrices, and the red dotted line shows the Bonferroni-corrected p<0.05 threshold. Top shows the SC matrix (synapse) total, middle shows FC-SC correlation (neuron), and bottom shows FC-SC correlation (synapse). e, Histogram of null SC matrices and extracted SC matrix with PPSSI (0.9-1). f, Example of a reciprocal-synapse in the EM image (left) and 3D image (right). g, Histogram of reciprocal-synaptic minimum distances in the FlyEM (cf0.8) connectome data. The vertical axis shows reciprocal-synapse pair count, and the horizontal axis shows distance [μm]. h, Histogram of null SC matrices and extracted SC matrix with reciprocal-synapses (≤2μm). i, The segregation index histogram of all neurons (139,255) in FlyWire (sc140) connectome data. The horizontal axis shows segregation index [0 1]. j, The segregation index histogram of hemibrain neurons (99,644) in the FlyEM (cf0.8) connectome data. The horizontal axis shows the segregation index [0 1].

Reciprocal synapses of CT1 and APL neurons, and synapse point cloud for several conditions.

a, Histogram of reciprocal-synaptic minimum distances of the CT1-R neuron in the FlyWire (sc140) connectome data. The vertical axis shows reciprocal-synapse pair count, and the horizontal axis shows distance [μm]. b, Skeleton view of the CT1-R neuron. Red dots are pre-synapses and blue dots are post-synapses of reciprocal synapses (≤ 2μm). c, Histogram of reciprocal-synaptic minimum distances of the APL-R neuron in the FlyWire (sc140) connectome data. d, Skeleton view of the APL-R neuron, the same way as b. e, (top) Maximum Z-projection of pre & post synapse point cloud within PPSSI (0-0.1) in the FlyEM data (cf0.8). (bottom) Maximum Z-projection of pre & post synapse point cloud within PPSSI (0-0.1) in the FlyWire data (sc140). f, The same as in e, but with the condition PPSSI in 0.9-1. g, The same as in e, but with the condition of only reciprocal synapses (≤ 2μm).

Unsegregated and segregated cell ranking examples (FlyWire sc140).

a, Unsegregated cell ranking from 3rd to 11th in the FlyWirerd(sc140) connectome data. Red dots shows pre-synapses, blue dots shows post-synapses. b, Segregated cell ranking from 3 to 11th.

Unsegregated and segregated cell ranking (FlyEM cf0.8).

a, Unsegregated cell ranking from 3 to 11th in the FlyErdM (cf0.8) connectome data. Red dots shows pre-synapse, blue dots show post-synapses. b, Segregated cell ranking from 3 to 11th.

Mechanosensory pathway from Wedge to Ellipsoid Body.

a, 3D image of WL-L right, WED016 left, WPNb tier 1, and ER1 left neurons, frontal view. Black square insert shows WL-L right, WED016 left, WPNb tier 1 and ER1 left neurons from the left lateral view. b, Network graph of WL-L right, WED016 left, WPNb tier 1 and ER1 left neurons from the FlyWire codex (sc50, connection threshold≥5). Blue is for acetylcholine, ocher for GABA neurons. The small number on the edges shows synapse count.