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Research ArticleResearch Article: Methods/New Tools, Novel Tools and Methods

Molecular Diversity of Glutamatergic and GABAergic Synapses from Multiplexed Fluorescence Imaging

Eric Danielson, Karen Perez de Arce, Beth Cimini, Eike-Christian Wamhoff, Shantanu Singh, Jeffrey R. Cottrell, Anne E. Carpenter and Mark Bathe
eNeuro 21 December 2020, 8 (1) ENEURO.0286-20.2020; DOI: https://doi.org/10.1523/ENEURO.0286-20.2020
Eric Danielson
1Department of Biological Engineering, MIT, Cambridge 02142, MA
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Karen Perez de Arce
2Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge 02142, MA
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Beth Cimini
3Imaging Platform, Broad Institute of MIT and Harvard, Cambridge 02142, MA
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Eike-Christian Wamhoff
1Department of Biological Engineering, MIT, Cambridge 02142, MA
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Shantanu Singh
3Imaging Platform, Broad Institute of MIT and Harvard, Cambridge 02142, MA
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Jeffrey R. Cottrell
2Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge 02142, MA
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Anne E. Carpenter
3Imaging Platform, Broad Institute of MIT and Harvard, Cambridge 02142, MA
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Mark Bathe
1Department of Biological Engineering, MIT, Cambridge 02142, MA
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  • Figure 1.
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    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.

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    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.

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    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.

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    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.

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    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

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    Table 1

    Antibody information

    Antibody targetVendorCatalog
    number
    Species and
    clonality
    Conjugation
    strategy
    Docking
    strand
    sequence
    Working
    concentration
    (μg/ml)
    PSD-95Cell Signaling Technology3450Rabbit monoclonal−−0.1
    BassoonEnzo Life SciencesADI-VAM-PS003Mouse monoclonalSMCCp84
    vGATSynaptic Systems131011Mouse monoclonalSMCCp310
    GephyrinSynaptic Systems147208Rat chimeric−−3
    MAP2Novus BiologicalsNB300-213Chicken polyclonal−−
    PhalloidinBachemH-7634−SMCCp250
    CortactinMillipore05–180Mouse monoclonalSMCCp420
    Synapsin1Santa Cruzsc-7379Goat polyclonalSMCCp93
    SHANK3Santa Cruzsc-30193Rabbit polyclonalSiteClickp67.4
    Homer-1b/cSanta Cruzsc-20807Rabbit polyclonalSMCCp104
    NR2BNeuroMab75–097Mouse monoclonal−−10
    Anti-rabbit secondaryLife TechnologiesA16126Goat polyclonalSMCCp13
    Anti-mouse secondaryLife TechnologiesA16068Goat polyclonalSMCCp123
    Anti-rat secondaryInvitrogenA18873Goat polyclonalSMCCp78
    MAP2AbcamAb5392Chicken polyclonal−−9.5
    vGlut1Synaptic Systems135304Guinea pig polyclonal−−1:400 (dilution)
    Alexa Fluor 488 anti-
    chicken secondary
    Thermo FisherA11039Goat polyclonal−−4
    Alexa Fluor 555 anti-
    guinea pig secondary
    Thermo FisherA21435Goat polyclonal−−4
    • All antibodies and the working concentration used in this study are listed with the company name catalog and conjugation style.

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    Table 2

    Docking strand and imaging probe sequences used in PRISM

    Sequence
    name
    Docking strand
    sequence (5′ to 3′)
    ssLNA imaging probe
    sequence (5′ to 3′)
    p1TTATACATCTAT*AGAT*G*TATAA
    p2TTATCTACATATATGT*A*G*ATAA
    p3TTTCTTCATTATAAT*G*A*AGAAA
    p4TTATGAATCTATA*GAT*T*CATAA
    p5Not used in this studyNot used in this study
    p6TTAATTGAGTAT*A*CTCAATTAA
    p7TTAATTAGGATA*T*CCT*AATTAA
    p8TTATAATGGATA*T*CC*ATTATAA
    p9TTTAATAAGGTA*CC*T*TATTAAA
    p10TTATAGAGAAGC*T*TC*TCTATAA
    p11Not used in this studyNot used in this study
    p12TTATAGTGATTA*ATC*A*CTATAA
    • LNA substitutions are indicated with * following the LNA nucleotide.

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    Table 3

    Specific features from CellProfiler used as input for UMAP

    MetricRegion of
    interest
    source
    Target used for
    measurement
    CompactnessSynapsin1Synapsin1
    EccentricitySynapsin1Synapsin1
    Euler numberSynapsin1Synapsin1
    ExtentSynapsin1Synapsin1
    Form factorSynapsin1Synapsin1
    Major axis lengthSynapsin1Synapsin1
    Maximum radiusSynapsin1Synapsin1
    Mean radiusSynapsin1Synapsin1
    Minor axis lengthSynapsin1Synapsin1
    OrientationSynapsin1Synapsin1
    PerimeterSynapsin1Synapsin1
    SoliditySynapsin1Synapsin1
    Puncta numberSynapsin1Homer-1b/c
    Puncta numberSynapsin1NR2B
    Puncta numberSynapsin1PSD-95
    Puncta numberSynapsin1SHANK3
    Puncta numberSynapsin1Actin
    Puncta numberSynapsin1Cortactin
    Puncta numberSynapsin1vGlut-1
    Integrated intensity edgeSynapsin1DAPI
    Integrated intensity edgeSynapsin1Gephyrin
    Integrated intensitySynapsin1NR2B
    Integrated intensitySynapsin1PSD-95
    Integrated intensitySynapsin1SHANK3
    Integrated intensitySynapsin1Actin
    Integrated intensitySynapsin1Synapsin1
    Integrated intensitySynapsin1vGlut-1
    Lower quartile intensitySynapsin1Synapsin1
    MAD intensitySynapsin1DAPI
    MAD intensitySynapsin1Gephyrin
    MAD intensitySynapsin1Homer-1b/c
    MAD intensitySynapsin1MAP2
    MAD intensitySynapsin1PSD-95
    MAD intensitySynapsin1SHANK3
    MAD intensitySynapsin1Actin
    MAD intensitySynapsin1Bassoon
    MAD intensitySynapsin1Cortactin
    MAD intensitySynapsin1Synapsin1
    MAD intensitySynapsin1vGAT
    Mass displacementSynapsin1DAPI
    Mass displacementSynapsin1Gephyrin
    Mass displacementSynapsin1Homer-1b/c
    Mass displacementSynapsin1MAP2
    Mass displacementSynapsin1NR2B
    Mass displacementSynapsin1PSD-95
    Mass displacementSynapsin1SHANK3
    Mass displacementSynapsin1Actin
    Mass displacementSynapsin1Bassoon
    Mass displacementSynapsin1Cortactin
    Mass displacementSynapsin1Synapsin1
    Mass displacementSynapsin1vGlut-1
    Mass displacementSynapsin1vGAT
    Min intensity edgeSynapsin1Homer-1b/c
    Min intensity edgeSynapsin1NR2B
    Min intensity edgeSynapsin1PSD-95
    Min intensity edgeSynapsin1SHANK3
    Min intensity edgeSynapsin1Actin
    Min intensity edgeSynapsin1Bassoon
    Min intensity edgeSynapsin1Cortactin
    Min intensity edgeSynapsin1vGlut-1
    Min intensitySynapsin1Gephyrin
    Min intensitySynapsin1MAP2
    Min intensitySynapsin1Synapsin1
    Min intensitySynapsin1vGAT
    Std intensitySynapsin1Gephyrin
    Std intensitySynapsin1NR2B
    Std intensitySynapsin1vGlut-1
    Upper quartile intensitySynapsin1DAPI
    Distance (centroid)GephyrinGephyrin
    Integrated intensityGephyrinGephyrin
    Mass displacementGephyrinGephyrin
    Std intensityGephyrinGephyrin
    Distance (centroid)Homer-1b/cHomer-1b/c
    Integrated intensityHomer-1b/cHomer-1b/c
    MAD intensityHomer-1b/cHomer-1b/c
    Mass displacementHomer-1b/cHomer-1b/c
    Min intensity edgeHomer-1b/cHomer-1b/c
    Distance (minimum)NR2BNR2B
    Integrated intensityNR2BNR2B
    MAD intensityNR2BNR2B
    Mass dissplacementNR2BNR2B
    Distance (minimum)PSD-95PSD-95
    Integrated intensityPSD-95PSD-95
    MAD intensityPSD-95PSD-95
    Mass displacementPSD-95PSD-95
    Min intensityPSD-95PSD-95
    Distance (minimum)SHANK3SHANK3
    Integrated intensitySHANK3SHANK3
    MAD intensitySHANK3SHANK3
    Mass displacementActinActin
    Distance (centroid)ActinActin
    Integrated intensityActinActin
    MAD intensityActinActin
    Mass displacementActinActin
    Distance (centroid)BassoonBassoon
    MAD intensityBassoonBassoon
    Mass displacementBassoonBassoon
    Distance (centroid)CortactinCortactin
    Integrated intensityCortactinCortactin
    MAD intensityCortactinCortactin
    Mass displacementCortactinCortactin
    Min intensityCortactinCortactin
    MAD intensityvGlut-1vGlut-1
    Mass displacementvGlut-1vGlut-1
    Min intensity edgevGlut-1vGlut-1
    Mass displacementvGATvGAT
    Min intensityvGATvGAT
    Std intensity edgevGATvGAT
    • 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.

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    Table 4

    Statistics table

    MeanEffect (Cohen’s d)N
    FigureTargetGroup comparisonData
    structure
    Type of testShufflesp valueDifference95% CI
    lower
    95% CI
    upper
    Effect95% CI
    lower
    95% CI
    upper
    Powerp < 0.05UTTTX
    aGephyrin distance to synapsin1Gephyrin positive synapses (UMAP 6 vs UMAP1-5)NormalPermutation t test50000.002−0.44−0.46−0.40−16.60−20.65−12.491.0*65
    3A,FGephyrin integrated intensityGephyrin positive synapses (UMAP 6 vs UMAP1-5)NormalPermutation t test50000.0020.350.200.492.701.255.461.0*65
    PSD-95 distance to synapsin1PSD-95 positive synapses (UMAP6 vs UMAP1-5)NormalPermutation t test50000.003−0.56−0.70−0.47−5.67−7.51−4.111.0*65
    3A,FPSD-95 integrated intensityPSD-95 positive synapses (UMAP6 vs UMAP1-5)NormalPermutation t test50000.007−0.23−0.31−0.10−2.56−6.14−0.691.0*65
    3A,FHomer1-b/c integrated intensityHomer1-b/c positive synapses (UMAP6 vs UMAP1)NormalPermutation t test50000.003−0.56−0.67−0.43−5.08−7.32−3.581.0*65
    b4AExcitatory synapse numberUntreated vs TTXNormalPermutation t test50000.6262.99−7.4015.230.29−1.101.590.166
    Inhibitory synapse numberUntreated vs TTXNormalPermutation t test50000.5923.62−7.5616.240.33−1.031.690.166
    4BExcitatory:inhibitory synapse ratioUntreated vs TTXNormalPermutation t test50000.592−0.86−4.031.78−0.32−1.641.130.166
    c4CvGlut1 integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.0060.170.100.242.821.175.031.0*65
    vGAT integrated intensityUntreated vs TTX (inhibitory synapses)NormalPermutation t test50000.0010.600.440.754.312.846.501.0*65
    Synapsin1 integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.1100.10−0.020.181.11−0.524.000.465
    Synapsin1 integrated intensityUntreated vs TTX (inhibitory synapses)NormalPermutation t test50000.0040.350.230.473.111.534.951.0*65
    Bassoon integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.1290.18−0.040.381.02−0.662.490.365
    Bassoon integrated intensityUntreated vs TTX (inhibitory synapses)NormalPermutation t test50000.3600.11−0.120.320.57−0.982.070.165
    SHANK3 integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.0080.350.120.462.450.607.290.9*65
    Homer1-b/c integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.0370.150.020.261.54−0.023.440.6*65
    PSD-95 integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.0170.150.060.241.930.713.310.8*65
    Gephyrin integrated intensityUntreated vs TTX (inhibitory synapses)NormalPermutation t test50000.6100.07−0.140.350.31−1.241.720.165
    NR2B integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.0890.12−0.020.221.18−0.483.260.465
    Cortactin integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.549−0.09−0.330.18−0.39−2.451.070.165
    Cortactin integrated intensityUntreated vs TTX (inhibitory synapses)NormalPermutation t test50000.589−0.09−0.410.24−0.31−1.871.140.165
    Actin integrated intensityUntreated vs TTX (excitatory synapses)NormalPermutation t test50000.475−0.07−0.260.04−0.53−1.801.060.165
    Actin integrated intensityUntreated vs TTX (inhibitory synapses)NormalPermutation t test50000.7940.04−0.220.380.17−1.482.060.165
    d5CSynapsin1 UMAP1Untreated vs TTXNormalPermutation t test50000.2020.13−0.050.300.84−0.722.490.25
    Synapsin1 UMAP2Untreated vs TTXNormalPermutation t test50000.479−0.03−0.120.03−0.47−1.831.090.165
    Synapsin1 UMAP3Untreated vs TTXNormalPermutation t test50000.5690.03−0.100.090.36−1.233.700.165
    Synapsin1 UMAP4Untreated vs TTXNormalPermutation t test50000.237−0.06−0.170.03−0.82−2.541.030.265
    Synapsin1 UMAP5Untreated vs TTXNormalPermutation t test50000.099−0.09−0.180.01−1.09−2.630.480.465
    Synapsin1 UMAP6Untreated vs TTXNormalPermutation t test50000.0050.370.240.493.091.644.751.0*65
    Gephyrin UMAP1Untreated vs TTXNormalPermutation t test50000.4640.02−0.030.070.47−0.921.960.165
    Gephyrin UMAP2Untreated vs TTXNormalPermutation t test50000.261−0.03−0.080.02−0.68−2.130.680.265
    Gephyrin UMAP3Untreated vs TTXNormalPermutation t test50000.345−0.03−0.080.02−0.61−1.960.760.165
    Gephyrin UMAP4Untreated vs TTXNormalPermutation t test50000.3040.02−0.020.060.69−1.582.430.265
    Gephyrin UMAP5Untreated vs TTXNormalPermutation t test50000.6470.02−0.050.080.30−1.822.690.165
    Gephyrin UMAP6Untreated vs TTXNormalPermutation t test50000.7330.03−0.120.250.20−1.411.560.165
    Homer1-b/c UMAP1Untreated vs TTXNormalPermutation t test50000.2210.09−0.050.230.81−0.892.600.265
    Homer1-b/c UMAP1Untreated vs TTXNormalPermutation t test50000.4520.000.000.00−0.81−1.64−0.400.265
    Homer1-b/c UMAP1Untreated vs TTXNormalPermutation t test50000.4380.05−0.070.160.49−1.172.160.165
    Homer1-b/c UMAP1Untreated vs TTXNormalPermutation t test50000.6600.01−0.040.080.29−1.702.070.165
    Homer1-b/c UMAP1Untreated vs TTXNormalPermutation t test50000.4740.02−0.050.080.41−1.232.010.165
    Homer1-b/c UMAP1Untreated vs TTXNormalPermutation t test50000.0310.190.070.291.800.484.200.8*65
    NR2B UMAP1Untreated vs TTXNormalPermutation t test50000.1440.10−0.030.220.97−0.482.770.365
    NR2B UMAP2Untreated vs TTXNormalPermutation t test50000.827−0.02−0.140.11−0.15−1.711.680.165
    NR2B UMAP3Untreated vs TTXNormalPermutation t test50000.3120.07−0.040.170.63−0.952.410.265
    NR2B UMAP4Untreated vs TTXNormalPermutation t test50000.6610.05−0.180.280.26−1.502.350.165
    NR2B UMAP5Untreated vs TTXNormalPermutation t test50000.744−0.03−0.200.12−0.20−1.801.340.165
    NR2B UMAP6Untreated vs TTXNormalPermutation t test50000.5330.14−0.330.540.37−1.081.970.165
    PSD-95 UMAP1Untreated vs TTXNormalPermutation t test50000.3130.06−0.040.170.65−0.852.040.265
    PSD-95 UMAP2Untreated vs TTXNormalPermutation t test50000.0530.060.010.111.43−0.333.180.665
    PSD-95 UMAP3Untreated vs TTXNormalPermutation t test50000.7780.02−0.100.150.17−1.281.560.165
    PSD-95 UMAP4Untreated vs TTXNormalPermutation t test50000.8820.000.000.00−0.12−1.471.580.165
    PSD-95 UMAP5Untreated vs TTXNormalPermutation t test50000.1840.000.000.010.860.401.500.265
    PSD-95 UMAP6Untreated vs TTXNormalPermutation t test50000.729−0.01−0.080.07−0.21−1.691.260.165
    SHANK3 UMAP1Untreated vs TTXNormalPermutation t test50000.0180.230.110.352.070.814.110.9*65
    SHANK3 UMAP2Untreated vs TTXNormalPermutation t test50000.0110.100.040.151.940.553.240.8*65
    SHANK3 UMAP3Untreated vs TTXNormalPermutation t test50000.0190.200.080.301.850.554.350.8*65
    SHANK3 UMAP4Untreated vs TTXNormalPermutation t test50000.1860.06−0.030.140.85−0.622.440.265
    SHANK3 UMAP5Untreated vs TTXNormalPermutation t test50000.2850.06−0.040.160.67−0.802.170.265
    SHANK3 UMAP6Untreated vs TTXNormalPermutation t test50000.3600.07−0.060.220.58−0.842.150.165
    Actin UMAP1Untreated vs TTXNormalPermutation t test50000.345−0.10−0.280.06−0.60−1.951.110.165
    Actin UMAP2Untreated vs TTXNormalPermutation t test50000.9270.00−0.050.06−0.06−1.541.590.165
    Actin UMAP3Untreated vs TTXNormalPermutation t test50000.407−0.06−0.180.06−0.56−2.011.130.165
    Actin UMAP4Untreated vs TTXNormalPermutation t test50000.721−0.01−0.070.05−0.21−1.701.260.165
    Actin UMAP5Untreated vs TTXNormalPermutation t test50000.923−0.01−0.180.07−0.11−1.722.140.165
    Actin UMAP6Untreated vs TTXNormalPermutation t test50000.8760.01−0.140.190.10−1.311.650.165
    Bassoon UMAP1Untreated vs TTXNormalPermutation t test50000.2500.08−0.040.210.74−0.901.940.265
    Bassoon UMAP2Untreated vs TTXNormalPermutation t test50000.593−0.02−0.110.06−0.33−1.971.560.165
    Bassoon UMAP3Untreated vs TTXNormalPermutation t test50000.0640.090.020.221.16−0.332.120.465
    Bassoon UMAP4Untreated vs TTXNormalPermutation t test50000.8770.01−0.090.130.09−2.473.450.165
    Bassoon UMAP5Untreated vs TTXNormalPermutation t test50000.5690.02−0.050.080.33−1.262.330.165
    Bassoon UMAP6Untreated vs TTXNormalPermutation t test50000.2810.07−0.060.190.64−0.832.200.265
    Cortactin UMAP1Untreated vs TTXNormalPermutation t test50000.201−0.09−0.240.01−0.84−2.030.630.265
    Cortactin UMAP2Untreated vs TTXNormalPermutation t test50000.573−0.02−0.110.05−0.34−1.811.070.165
    Cortactin UMAP3Untreated vs TTXNormalPermutation t test50000.3900.000.000.000.55−0.982.370.165
    Cortactin UMAP4Untreated vs TTXNormalPermutation t test50000.8670.000.000.000.11−1.211.570.165
    Cortactin UMAP5Untreated vs TTXNormalPermutation t test50000.781−0.02−0.120.11−0.16−2.091.780.165
    Cortactin UMAP6Untreated vs TTXNormalPermutation t test50000.585−0.06−0.240.14−0.33−1.871.100.165
    vGlut1 UMAP1Untreated vs TTXNormalPermutation t test50000.0440.190.010.301.49−0.253.750.6*65
    vGlut1 UMAP2Untreated vs TTXNormalPermutation t test50000.2520.03−0.020.080.78−0.832.690.265
    vGlut1 UMAP3Untreated vs TTXNormalPermutation t test50000.0450.080.010.161.38−0.393.000.5*65
    vGlut1 UMAP4Untreated vs TTXNormalPermutation t test50000.2310.03−0.020.070.77−0.832.440.265
    vGlut1 UMAP5Untreated vs TTXNormalPermutation t test50000.7220.01−0.050.090.21−2.283.330.165
    vGlut1 UMAP6Untreated vs TTXNormalPermutation t test50000.358−0.01−0.020.01−0.57−2.241.050.165
    vGAT UMAP1Untreated vs TTXNormalPermutation t test50000.3860.00−0.010.00−0.60−1.691.010.165
    vGAT UMAP2Untreated vs TTXNormalPermutation t test50000.8830.000.000.000.08−1.411.550.165
    vGAT UMAP3Untreated vs TTXNormalPermutation t test50000.101−0.01−0.020.00−1.03−1.980.550.365
    vGAT UMAP4Untreated vs TTXNormalPermutation t test50000.8610.000.000.010.06−1.311.570.165
    vGAT UMAP5Untreated vs TTXNormalPermutation t test50000.101−0.01−0.010.00−1.13−2.16−0.490.465
    vGAT UMAP6Untreated vs TTXNormalPermutation t test50000.0020.860.611.083.992.556.301.0*65
    e5CUMAP1 synapse numberUntreated vs TTXNormalPermutation t test50000.1170.110.010.221.09−0.562.560.465
    UMAP2 synapse numberUntreated vs TTXNormalPermutation t test50000.042−0.27−0.47−0.04−1.52−3.590.150.6*65
    UMAP3 synapse numberUntreated vs TTXNormalPermutation t test50000.0920.230.070.531.170.102.190.465
    UMAP4 synapse numberUntreated vs TTXNormalPermutation t test50000.091−0.14−0.280.03−1.13−3.510.560.465
    UMAP5 synapse numberUntreated vs TTXNormalPermutation t test50000.002−0.42−0.60−0.30−3.12−4.32−1.961.0*65
    UMAP6 synapse numberUntreated vs TTXNormalPermutation t test50000.4280.12−0.110.420.51−0.841.850.165
    • 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

  • Figures
  • Tables
  • 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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Molecular Diversity of Glutamatergic and GABAergic Synapses from Multiplexed Fluorescence Imaging
Eric Danielson, Karen Perez de Arce, Beth Cimini, Eike-Christian Wamhoff, Shantanu Singh, Jeffrey R. Cottrell, Anne E. Carpenter, Mark Bathe
eNeuro 21 December 2020, 8 (1) ENEURO.0286-20.2020; DOI: 10.1523/ENEURO.0286-20.2020

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Molecular Diversity of Glutamatergic and GABAergic Synapses from Multiplexed Fluorescence Imaging
Eric Danielson, Karen Perez de Arce, Beth Cimini, Eike-Christian Wamhoff, Shantanu Singh, Jeffrey R. Cottrell, Anne E. Carpenter, Mark Bathe
eNeuro 21 December 2020, 8 (1) ENEURO.0286-20.2020; DOI: 10.1523/ENEURO.0286-20.2020
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