Article Figures & Data
Figures
- Figure 1.
Measuring synaptic content from excitatory and inhibitory synapses using CellProfiler. A, Multiplexed images of PRISM stained DIV21 hippocampal neurons are used as input for the CellProfiler pipeline. Raw images (top) are aligned using MAP2 signal. A white-top filter is applied to enhance puncta (middle). Puncta are thresholded and separated into individual puncta using peak intensity (bottom). B, Synapses are labeled excitatory (E) or inhibitory (I) using the presence or absence of excitatory-specific (vGlut1) or inhibitory-specific (vGAT) markers. Synapses with both markers are labeled as dual (D). Clusters that do not contain either marker are labeled as unknown (U). C, Representative image (top) and enlarged dendrite (bottom) from a MAP2-stained DIV21 hippocampal neuron with excitatory (red) and inhibitory (green) synapses labeled as colored circles. The size of the colored circle represents the relative synapsin1 area. Scale bar: 1 μm.
- Figure 2.
Synaptic cluster identification using UMAP. A, UMAP plots of individual synapses (n = 10,000) using CellProfiler output separates excitatory (red) and inhibitory (blue) synapses into two major clusters with multiple subclusters. B, Unique clusters identified by HDBSCAN. (–) indicates synaptic target below limit of detection. C, Representative synapses from each cluster. D, Heatmap indicates the average relative intensities for each synaptic target within each cluster. All values are normalized to untreated mean integrated intensity.
- Figure 3.
Comparison of synaptic intensity and protein relationships among all proteins within each cluster. Ridgeline plots of relative synaptic intensity for each cluster group (A–F) using HDBSCAN. Horizontal black line represents cluster mean intensity. All values are normalized to untreated mean integrated intensity. The first peak indicates synapses with integrated intensity of zero. Heatmap indicates the correlation coefficient between each protein. Correlation values between −0.4 and 0.4 obscured to highlight strong correlations. Dendrograms surrounding heatmap show hierarchical clustering of proteins within each cluster.
- Figure 4.
Excitatory and inhibitory synaptic density does not change in response to 48-h TTX treatment. A, Quantification of excitatory and inhibitory synaptic density from untreated (red) and TTX-treated (blue) cells. Bar height represents the mean number of synapses per 100-μm length of dendrite. B, Quantification of the excitatory:inhibitory synaptic ratio in untreated (red) and TTX-treated (blue) neurons. Bar height represents mean excitatory:inhibitory ratio. Error bars indicate 95% confidence intervals. Closed circles indicate results from individual replicates n = 6. C, Violin plots of relative synaptic intensity for synaptic targets at excitatory synapses (left) and inhibitory synapses (right) from untreated (red) and TTX-treated cells (blue). Black line indicates the mean intensity; p values are computed using Student’s t test on the mean Integrated Intensity with n = 6 (UT) and n = 5 (TTX) replicates. All values are normalized to untreated mean integrated intensity; **p < 0.01, *p < 0.05.
- Figure 5.
Characterizing changes within clusters in response to TTX treatment. A, UMAP plots of individual synapses (n = 10,000) separated by treatment group. B, Heatmap indicates the average relative intensities for each synaptic target within each cluster. Left side of the column is the untreated sample, the right side of the column is the TTX-treated sample. All values are normalized to untreated mean integrated intensity. C, Bar heights show synapse density, relative to untreated groups, within each HDBSCAN identified cluster following TTX treatment; p values are computed using Student’s t test with n = 6 (UT) and n = 5 (TTX) replicates; **p < 0.01, *p < 0.05. Error bars indicate 95% confidence intervals.
Tables
Antibody target Vendor Catalog
numberSpecies and
clonalityConjugation
strategyDocking
strand
sequenceWorking
concentration
(μg/ml)PSD-95 Cell Signaling Technology 3450 Rabbit monoclonal − − 0.1 Bassoon Enzo Life Sciences ADI-VAM-PS003 Mouse monoclonal SMCC p8 4 vGAT Synaptic Systems 131011 Mouse monoclonal SMCC p3 10 Gephyrin Synaptic Systems 147208 Rat chimeric − − 3 MAP2 Novus Biologicals NB300-213 Chicken polyclonal − − Phalloidin Bachem H-7634 − SMCC p2 50 Cortactin Millipore 05–180 Mouse monoclonal SMCC p4 20 Synapsin1 Santa Cruz sc-7379 Goat polyclonal SMCC p9 3 SHANK3 Santa Cruz sc-30193 Rabbit polyclonal SiteClick p6 7.4 Homer-1b/c Santa Cruz sc-20807 Rabbit polyclonal SMCC p10 4 NR2B NeuroMab 75–097 Mouse monoclonal − − 10 Anti-rabbit secondary Life Technologies A16126 Goat polyclonal SMCC p1 3 Anti-mouse secondary Life Technologies A16068 Goat polyclonal SMCC p12 3 Anti-rat secondary Invitrogen A18873 Goat polyclonal SMCC p7 8 MAP2 Abcam Ab5392 Chicken polyclonal − − 9.5 vGlut1 Synaptic Systems 135304 Guinea pig polyclonal − − 1:400 (dilution) Alexa Fluor 488 anti-
chicken secondaryThermo Fisher A11039 Goat polyclonal − − 4 Alexa Fluor 555 anti-
guinea pig secondaryThermo Fisher A21435 Goat polyclonal − − 4 All antibodies and the working concentration used in this study are listed with the company name catalog and conjugation style.
Sequence
nameDocking strand
sequence (5′ to 3′)ssLNA imaging probe
sequence (5′ to 3′)p1 TTATACATCTA T*AGAT*G*TATAA p2 TTATCTACATA TATGT*A*G*ATAA p3 TTTCTTCATTA TAAT*G*A*AGAAA p4 TTATGAATCTA TA*GAT*T*CATAA p5 Not used in this study Not used in this study p6 TTAATTGAGTA T*A*CTCAATTAA p7 TTAATTAGGAT A*T*CCT*AATTAA p8 TTATAATGGAT A*T*CC*ATTATAA p9 TTTAATAAGGT A*CC*T*TATTAAA p10 TTATAGAGAAG C*T*TC*TCTATAA p11 Not used in this study Not used in this study p12 TTATAGTGATT A*ATC*A*CTATAA LNA substitutions are indicated with * following the LNA nucleotide.
Metric Region of
interest
sourceTarget used for
measurementCompactness Synapsin1 Synapsin1 Eccentricity Synapsin1 Synapsin1 Euler number Synapsin1 Synapsin1 Extent Synapsin1 Synapsin1 Form factor Synapsin1 Synapsin1 Major axis length Synapsin1 Synapsin1 Maximum radius Synapsin1 Synapsin1 Mean radius Synapsin1 Synapsin1 Minor axis length Synapsin1 Synapsin1 Orientation Synapsin1 Synapsin1 Perimeter Synapsin1 Synapsin1 Solidity Synapsin1 Synapsin1 Puncta number Synapsin1 Homer-1b/c Puncta number Synapsin1 NR2B Puncta number Synapsin1 PSD-95 Puncta number Synapsin1 SHANK3 Puncta number Synapsin1 Actin Puncta number Synapsin1 Cortactin Puncta number Synapsin1 vGlut-1 Integrated intensity edge Synapsin1 DAPI Integrated intensity edge Synapsin1 Gephyrin Integrated intensity Synapsin1 NR2B Integrated intensity Synapsin1 PSD-95 Integrated intensity Synapsin1 SHANK3 Integrated intensity Synapsin1 Actin Integrated intensity Synapsin1 Synapsin1 Integrated intensity Synapsin1 vGlut-1 Lower quartile intensity Synapsin1 Synapsin1 MAD intensity Synapsin1 DAPI MAD intensity Synapsin1 Gephyrin MAD intensity Synapsin1 Homer-1b/c MAD intensity Synapsin1 MAP2 MAD intensity Synapsin1 PSD-95 MAD intensity Synapsin1 SHANK3 MAD intensity Synapsin1 Actin MAD intensity Synapsin1 Bassoon MAD intensity Synapsin1 Cortactin MAD intensity Synapsin1 Synapsin1 MAD intensity Synapsin1 vGAT Mass displacement Synapsin1 DAPI Mass displacement Synapsin1 Gephyrin Mass displacement Synapsin1 Homer-1b/c Mass displacement Synapsin1 MAP2 Mass displacement Synapsin1 NR2B Mass displacement Synapsin1 PSD-95 Mass displacement Synapsin1 SHANK3 Mass displacement Synapsin1 Actin Mass displacement Synapsin1 Bassoon Mass displacement Synapsin1 Cortactin Mass displacement Synapsin1 Synapsin1 Mass displacement Synapsin1 vGlut-1 Mass displacement Synapsin1 vGAT Min intensity edge Synapsin1 Homer-1b/c Min intensity edge Synapsin1 NR2B Min intensity edge Synapsin1 PSD-95 Min intensity edge Synapsin1 SHANK3 Min intensity edge Synapsin1 Actin Min intensity edge Synapsin1 Bassoon Min intensity edge Synapsin1 Cortactin Min intensity edge Synapsin1 vGlut-1 Min intensity Synapsin1 Gephyrin Min intensity Synapsin1 MAP2 Min intensity Synapsin1 Synapsin1 Min intensity Synapsin1 vGAT Std intensity Synapsin1 Gephyrin Std intensity Synapsin1 NR2B Std intensity Synapsin1 vGlut-1 Upper quartile intensity Synapsin1 DAPI Distance (centroid) Gephyrin Gephyrin Integrated intensity Gephyrin Gephyrin Mass displacement Gephyrin Gephyrin Std intensity Gephyrin Gephyrin Distance (centroid) Homer-1b/c Homer-1b/c Integrated intensity Homer-1b/c Homer-1b/c MAD intensity Homer-1b/c Homer-1b/c Mass displacement Homer-1b/c Homer-1b/c Min intensity edge Homer-1b/c Homer-1b/c Distance (minimum) NR2B NR2B Integrated intensity NR2B NR2B MAD intensity NR2B NR2B Mass dissplacement NR2B NR2B Distance (minimum) PSD-95 PSD-95 Integrated intensity PSD-95 PSD-95 MAD intensity PSD-95 PSD-95 Mass displacement PSD-95 PSD-95 Min intensity PSD-95 PSD-95 Distance (minimum) SHANK3 SHANK3 Integrated intensity SHANK3 SHANK3 MAD intensity SHANK3 SHANK3 Mass displacement Actin Actin Distance (centroid) Actin Actin Integrated intensity Actin Actin MAD intensity Actin Actin Mass displacement Actin Actin Distance (centroid) Bassoon Bassoon MAD intensity Bassoon Bassoon Mass displacement Bassoon Bassoon Distance (centroid) Cortactin Cortactin Integrated intensity Cortactin Cortactin MAD intensity Cortactin Cortactin Mass displacement Cortactin Cortactin Min intensity Cortactin Cortactin MAD intensity vGlut-1 vGlut-1 Mass displacement vGlut-1 vGlut-1 Min intensity edge vGlut-1 vGlut-1 Mass displacement vGAT vGAT Min intensity vGAT vGAT Std intensity edge vGAT vGAT The region of interest source is the image that was used to create the regions of interest (the puncta objects). The target used for measurement is the image source that the region of interest was used on. For example, synapsin1 puncta (regions of interest) are always created from synapsin1 images, but measurements using those regions can come from any image source, i.e., we can examine what the signal of vGlut-1, homer-1b/c, etc. is within the regions created by the synapsin1 objects.
Mean Effect (Cohen’s d) N Figure Target Group comparison Data
structureType of test Shuffles p value Difference 95% CI
lower95% CI
upperEffect 95% CI
lower95% CI
upperPower p < 0.05 UT TTX a Gephyrin distance to synapsin1 Gephyrin positive synapses (UMAP 6 vs UMAP1-5) Normal Permutation t test 5000 0.002 −0.44 −0.46 −0.40 −16.60 −20.65 −12.49 1.0 * 6 5 3A,F Gephyrin integrated intensity Gephyrin positive synapses (UMAP 6 vs UMAP1-5) Normal Permutation t test 5000 0.002 0.35 0.20 0.49 2.70 1.25 5.46 1.0 * 6 5 PSD-95 distance to synapsin1 PSD-95 positive synapses (UMAP6 vs UMAP1-5) Normal Permutation t test 5000 0.003 −0.56 −0.70 −0.47 −5.67 −7.51 −4.11 1.0 * 6 5 3A,F PSD-95 integrated intensity PSD-95 positive synapses (UMAP6 vs UMAP1-5) Normal Permutation t test 5000 0.007 −0.23 −0.31 −0.10 −2.56 −6.14 −0.69 1.0 * 6 5 3A,F Homer1-b/c integrated intensity Homer1-b/c positive synapses (UMAP6 vs UMAP1) Normal Permutation t test 5000 0.003 −0.56 −0.67 −0.43 −5.08 −7.32 −3.58 1.0 * 6 5 b 4A Excitatory synapse number Untreated vs TTX Normal Permutation t test 5000 0.626 2.99 −7.40 15.23 0.29 −1.10 1.59 0.1 6 6 Inhibitory synapse number Untreated vs TTX Normal Permutation t test 5000 0.592 3.62 −7.56 16.24 0.33 −1.03 1.69 0.1 6 6 4B Excitatory:inhibitory synapse ratio Untreated vs TTX Normal Permutation t test 5000 0.592 −0.86 −4.03 1.78 −0.32 −1.64 1.13 0.1 6 6 c 4C vGlut1 integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.006 0.17 0.10 0.24 2.82 1.17 5.03 1.0 * 6 5 vGAT integrated intensity Untreated vs TTX (inhibitory synapses) Normal Permutation t test 5000 0.001 0.60 0.44 0.75 4.31 2.84 6.50 1.0 * 6 5 Synapsin1 integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.110 0.10 −0.02 0.18 1.11 −0.52 4.00 0.4 6 5 Synapsin1 integrated intensity Untreated vs TTX (inhibitory synapses) Normal Permutation t test 5000 0.004 0.35 0.23 0.47 3.11 1.53 4.95 1.0 * 6 5 Bassoon integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.129 0.18 −0.04 0.38 1.02 −0.66 2.49 0.3 6 5 Bassoon integrated intensity Untreated vs TTX (inhibitory synapses) Normal Permutation t test 5000 0.360 0.11 −0.12 0.32 0.57 −0.98 2.07 0.1 6 5 SHANK3 integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.008 0.35 0.12 0.46 2.45 0.60 7.29 0.9 * 6 5 Homer1-b/c integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.037 0.15 0.02 0.26 1.54 −0.02 3.44 0.6 * 6 5 PSD-95 integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.017 0.15 0.06 0.24 1.93 0.71 3.31 0.8 * 6 5 Gephyrin integrated intensity Untreated vs TTX (inhibitory synapses) Normal Permutation t test 5000 0.610 0.07 −0.14 0.35 0.31 −1.24 1.72 0.1 6 5 NR2B integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.089 0.12 −0.02 0.22 1.18 −0.48 3.26 0.4 6 5 Cortactin integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.549 −0.09 −0.33 0.18 −0.39 −2.45 1.07 0.1 6 5 Cortactin integrated intensity Untreated vs TTX (inhibitory synapses) Normal Permutation t test 5000 0.589 −0.09 −0.41 0.24 −0.31 −1.87 1.14 0.1 6 5 Actin integrated intensity Untreated vs TTX (excitatory synapses) Normal Permutation t test 5000 0.475 −0.07 −0.26 0.04 −0.53 −1.80 1.06 0.1 6 5 Actin integrated intensity Untreated vs TTX (inhibitory synapses) Normal Permutation t test 5000 0.794 0.04 −0.22 0.38 0.17 −1.48 2.06 0.1 6 5 d 5C Synapsin1 UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.202 0.13 −0.05 0.30 0.84 −0.72 2.49 0.2 5 Synapsin1 UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.479 −0.03 −0.12 0.03 −0.47 −1.83 1.09 0.1 6 5 Synapsin1 UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.569 0.03 −0.10 0.09 0.36 −1.23 3.70 0.1 6 5 Synapsin1 UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.237 −0.06 −0.17 0.03 −0.82 −2.54 1.03 0.2 6 5 Synapsin1 UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.099 −0.09 −0.18 0.01 −1.09 −2.63 0.48 0.4 6 5 Synapsin1 UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.005 0.37 0.24 0.49 3.09 1.64 4.75 1.0 * 6 5 Gephyrin UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.464 0.02 −0.03 0.07 0.47 −0.92 1.96 0.1 6 5 Gephyrin UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.261 −0.03 −0.08 0.02 −0.68 −2.13 0.68 0.2 6 5 Gephyrin UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.345 −0.03 −0.08 0.02 −0.61 −1.96 0.76 0.1 6 5 Gephyrin UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.304 0.02 −0.02 0.06 0.69 −1.58 2.43 0.2 6 5 Gephyrin UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.647 0.02 −0.05 0.08 0.30 −1.82 2.69 0.1 6 5 Gephyrin UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.733 0.03 −0.12 0.25 0.20 −1.41 1.56 0.1 6 5 Homer1-b/c UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.221 0.09 −0.05 0.23 0.81 −0.89 2.60 0.2 6 5 Homer1-b/c UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.452 0.00 0.00 0.00 −0.81 −1.64 −0.40 0.2 6 5 Homer1-b/c UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.438 0.05 −0.07 0.16 0.49 −1.17 2.16 0.1 6 5 Homer1-b/c UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.660 0.01 −0.04 0.08 0.29 −1.70 2.07 0.1 6 5 Homer1-b/c UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.474 0.02 −0.05 0.08 0.41 −1.23 2.01 0.1 6 5 Homer1-b/c UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.031 0.19 0.07 0.29 1.80 0.48 4.20 0.8 * 6 5 NR2B UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.144 0.10 −0.03 0.22 0.97 −0.48 2.77 0.3 6 5 NR2B UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.827 −0.02 −0.14 0.11 −0.15 −1.71 1.68 0.1 6 5 NR2B UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.312 0.07 −0.04 0.17 0.63 −0.95 2.41 0.2 6 5 NR2B UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.661 0.05 −0.18 0.28 0.26 −1.50 2.35 0.1 6 5 NR2B UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.744 −0.03 −0.20 0.12 −0.20 −1.80 1.34 0.1 6 5 NR2B UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.533 0.14 −0.33 0.54 0.37 −1.08 1.97 0.1 6 5 PSD-95 UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.313 0.06 −0.04 0.17 0.65 −0.85 2.04 0.2 6 5 PSD-95 UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.053 0.06 0.01 0.11 1.43 −0.33 3.18 0.6 6 5 PSD-95 UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.778 0.02 −0.10 0.15 0.17 −1.28 1.56 0.1 6 5 PSD-95 UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.882 0.00 0.00 0.00 −0.12 −1.47 1.58 0.1 6 5 PSD-95 UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.184 0.00 0.00 0.01 0.86 0.40 1.50 0.2 6 5 PSD-95 UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.729 −0.01 −0.08 0.07 −0.21 −1.69 1.26 0.1 6 5 SHANK3 UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.018 0.23 0.11 0.35 2.07 0.81 4.11 0.9 * 6 5 SHANK3 UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.011 0.10 0.04 0.15 1.94 0.55 3.24 0.8 * 6 5 SHANK3 UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.019 0.20 0.08 0.30 1.85 0.55 4.35 0.8 * 6 5 SHANK3 UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.186 0.06 −0.03 0.14 0.85 −0.62 2.44 0.2 6 5 SHANK3 UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.285 0.06 −0.04 0.16 0.67 −0.80 2.17 0.2 6 5 SHANK3 UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.360 0.07 −0.06 0.22 0.58 −0.84 2.15 0.1 6 5 Actin UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.345 −0.10 −0.28 0.06 −0.60 −1.95 1.11 0.1 6 5 Actin UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.927 0.00 −0.05 0.06 −0.06 −1.54 1.59 0.1 6 5 Actin UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.407 −0.06 −0.18 0.06 −0.56 −2.01 1.13 0.1 6 5 Actin UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.721 −0.01 −0.07 0.05 −0.21 −1.70 1.26 0.1 6 5 Actin UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.923 −0.01 −0.18 0.07 −0.11 −1.72 2.14 0.1 6 5 Actin UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.876 0.01 −0.14 0.19 0.10 −1.31 1.65 0.1 6 5 Bassoon UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.250 0.08 −0.04 0.21 0.74 −0.90 1.94 0.2 6 5 Bassoon UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.593 −0.02 −0.11 0.06 −0.33 −1.97 1.56 0.1 6 5 Bassoon UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.064 0.09 0.02 0.22 1.16 −0.33 2.12 0.4 6 5 Bassoon UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.877 0.01 −0.09 0.13 0.09 −2.47 3.45 0.1 6 5 Bassoon UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.569 0.02 −0.05 0.08 0.33 −1.26 2.33 0.1 6 5 Bassoon UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.281 0.07 −0.06 0.19 0.64 −0.83 2.20 0.2 6 5 Cortactin UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.201 −0.09 −0.24 0.01 −0.84 −2.03 0.63 0.2 6 5 Cortactin UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.573 −0.02 −0.11 0.05 −0.34 −1.81 1.07 0.1 6 5 Cortactin UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.390 0.00 0.00 0.00 0.55 −0.98 2.37 0.1 6 5 Cortactin UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.867 0.00 0.00 0.00 0.11 −1.21 1.57 0.1 6 5 Cortactin UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.781 −0.02 −0.12 0.11 −0.16 −2.09 1.78 0.1 6 5 Cortactin UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.585 −0.06 −0.24 0.14 −0.33 −1.87 1.10 0.1 6 5 vGlut1 UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.044 0.19 0.01 0.30 1.49 −0.25 3.75 0.6 * 6 5 vGlut1 UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.252 0.03 −0.02 0.08 0.78 −0.83 2.69 0.2 6 5 vGlut1 UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.045 0.08 0.01 0.16 1.38 −0.39 3.00 0.5 * 6 5 vGlut1 UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.231 0.03 −0.02 0.07 0.77 −0.83 2.44 0.2 6 5 vGlut1 UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.722 0.01 −0.05 0.09 0.21 −2.28 3.33 0.1 6 5 vGlut1 UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.358 −0.01 −0.02 0.01 −0.57 −2.24 1.05 0.1 6 5 vGAT UMAP1 Untreated vs TTX Normal Permutation t test 5000 0.386 0.00 −0.01 0.00 −0.60 −1.69 1.01 0.1 6 5 vGAT UMAP2 Untreated vs TTX Normal Permutation t test 5000 0.883 0.00 0.00 0.00 0.08 −1.41 1.55 0.1 6 5 vGAT UMAP3 Untreated vs TTX Normal Permutation t test 5000 0.101 −0.01 −0.02 0.00 −1.03 −1.98 0.55 0.3 6 5 vGAT UMAP4 Untreated vs TTX Normal Permutation t test 5000 0.861 0.00 0.00 0.01 0.06 −1.31 1.57 0.1 6 5 vGAT UMAP5 Untreated vs TTX Normal Permutation t test 5000 0.101 −0.01 −0.01 0.00 −1.13 −2.16 −0.49 0.4 6 5 vGAT UMAP6 Untreated vs TTX Normal Permutation t test 5000 0.002 0.86 0.61 1.08 3.99 2.55 6.30 1.0 * 6 5 e 5C UMAP1 synapse number Untreated vs TTX Normal Permutation t test 5000 0.117 0.11 0.01 0.22 1.09 −0.56 2.56 0.4 6 5 UMAP2 synapse number Untreated vs TTX Normal Permutation t test 5000 0.042 −0.27 −0.47 −0.04 −1.52 −3.59 0.15 0.6 * 6 5 UMAP3 synapse number Untreated vs TTX Normal Permutation t test 5000 0.092 0.23 0.07 0.53 1.17 0.10 2.19 0.4 6 5 UMAP4 synapse number Untreated vs TTX Normal Permutation t test 5000 0.091 −0.14 −0.28 0.03 −1.13 −3.51 0.56 0.4 6 5 UMAP5 synapse number Untreated vs TTX Normal Permutation t test 5000 0.002 −0.42 −0.60 −0.30 −3.12 −4.32 −1.96 1.0 * 6 5 UMAP6 synapse number Untreated vs TTX Normal Permutation t test 5000 0.428 0.12 −0.11 0.42 0.51 −0.84 1.85 0.1 6 5 Two-tailed permutation t tests, mean confidence intervals, and effect size were calculated the from 5000 reshuffles of the untreated (n = 6) and TTX-treated (n = 5) samples. The p values represent the likelihood of observing the reported effect size, if the null hypothesis of zero difference is true. Power analysis represents the probability of observing effect size if the null hypothesis of zero difference is true.
Extended Data 1
CellProfiler multiplexed image analysis pipeline. The included image analysis pipeline automates the detection and quantification of synaptic puncta from multiplexed images. Download Extended Data 1, ZIP file.
Extended Data Figure 1-1
Overview of CellProfiler workflow. The CellProfiler pipeline takes maximum projection images as input then aligns the images and performs illumination correction by subtracting background signal. The pipeline next defines nuclei objects using DAPI, dendrite objects, using MAP2 and puncta objects using the various PRISM and IF staining. In the last stage of the pipeline, the puncta objects are organized into synapses. Puncta objects are considered synaptic if the objects are outside of the nuclei, are within eight pixels of the dendritic mask and are overlapping with synapsin1 objects. Data are exported to a csv file (green) and multiple images (purple) are created during each stage for quality control. Download Figure 1-1, TIF file.
Extended Data Figure 1-2
Synaptic properties measured by CellProfiler. The various intensity, shape, distance and object number measurements performed by CellProfiler. Each measurement is performed on each target from PRISM staining. Download Figure 1-2, PDF file.
Extended Data Figure 1-3
Classification of synapses using CellProfiler. Synapses are classified based on the presence of synapsin1, vGlut1, and vGAT. Synapses with synapsin1 and only vGlut1 are classified as excitatory. Synapses with synapsin1 and only vGAT are classified as inhibitory. The remaining synapses are unclassified and contain either synapsin1 alone, or synapsin1 and both vGlut1 and vGAT. Download Figure 1-3, TIF file.
Extended Data Figure 2-1
UMAP analysis of synapsin1 shape measurements. UMAP analysis of synapses colored for indicated synapsin1 shape metrics. Download Figure 2-1, TIF file.
Extended Data Figure 2-2
UMAP analysis of puncta number per synapse. UMAP analysis shows synapses colored for the number of puncta per synapse for each synaptic target. Download Figure 2-2, TIF file.
Extended Data Figure 2-3
UMAP analysis of relative integrated intensity. UMAP analysis of synapses colored for relative integrated intensity of each synaptic target. Scale bar shows log values. Download Figure 2-3, TIF file.
Extended Data Figure 2-4
UMAP analysis of relative integrated intensity of puncta edge. UMAP analysis of synapses colored for relative integrated intensity of the edge of each punctum for each synaptic target. Scale bar show log values. Download Figure 2-4, TIF file.
Extended Data Figure 2-5
UMAP analysis of lower quartile intensity. UMAP analysis of synapses colored for the lower quartile intensity (log values) of each punctum for each synaptic target. Download Figure 2-5, TIF file.
Extended Data Figure 2-6
UMAP analysis of median absolute deviation. UMAP analysis of synapses colored for median absolute deviation intensity (log values) of each punctum for each synaptic target. Download Figure 2-6, TIF file.
Extended Data Figure 2-7
UMAP analysis of puncta mass displacement. UMAP analysis of synapses colored for the mass displacement (log values) of each punctum for each synaptic target. Download Figure 2-7, TIF file.
Extended Data Figure 2-8
UMAP analysis of minimum pixel intensity of puncta edge. UMAP analysis of synapses colored for minimum pixel intensity on the edge of each punctum for each synaptic target. Download Figure 2-8, TIF file.
Extended Data Figure 2-9
UMAP analysis of puncta maximum intensity. UMAP analysis of synapses colored for maximum intensity (log values) of each punctum for each synaptic target. Download Figure 2-9, TIF file.
Extended Data Figure 2-10
UMAP analysis of puncta edge standard deviation. UMAP analysis of synapses colored for standard deviation of the edge (log values) of each punctum for each synaptic target. Download Figure 2-10, TIF file.
Extended Data Figure 2-11
UMAP analysis of puncta upper quartile intensity. UMAP analysis of synapses colored for the upper quartile intensity of each punctum for each synaptic target. Download Figure 2-11, TIF file.
Extended Data Figure 2-12
UMAP analysis of distance to synapsin1 puncta centroid. UMAP analysis of synapses colored for distance in pixels (log value) between puncta centroids (synapsin1 and each indicated target). Download Figure 2-12, TIF file.
Extended Data Figure 2-13
UMAP analysis of minimum distance to synapsin1 puncta. UMAP analysis of synapses colored for minimum distance in pixels (log values) between the indicated target and synapsin1. Download Figure 2-13, TIF file.
Extended Data Figure 2-14
Relative synaptic intensities of all targets for each synaptic subtype. Ridgeline plots of relative synaptic intensities for synaptic targets within cluster groups identified using HDBScan. Horizontal black line represents cluster mean intensity. All values are normalized to untreated mean integrated intensity. Percent of synapses with integrated intensity >0 for each cluster. Download Figure 2-14, TIF file.
Extended Data Figure 5-1
Synaptic cluster identification for individual replicates. UMAP plots of individual synapses using CellProfiler output separates excitatory and inhibitory synapses into approximately six unique clusters identified by HDBSCAN. Each cluster is present in each sample indicating unique cluster formation is not an artifact of individual differences in culture conditions across different samples. Download Figure 5-1, TIF file.
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