Molecular Diversity of Glutamatergic and GABAergic Synapses from Multiplexed Fluorescence Imaging

Figures

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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.