Retinogeniculate synapses form multiple active zones during eye-specific competition.

(A) Experimental design: CTB-Alexa 488 was injected into the right eye of wild-type (WT) and β2KO mice. One day after the treatment, tissue was collected from the left dLGN at P2, P4, and P8. The red squares indicate the STORM imaging regions. (B) Representative multi-active zone (mAZ, left panels) and single-active zone (sAZ, right panels) synapses in WT (top panels) and β2KO mice (bottom panels) at each age. Arrowheads point to individual Bassoon clusters (active zones) within each synapse. (C) Representative images of mAZ retinogeniculate synapses in the mouse dLGN at P8. Electron microscopy images show darkly-stained dAPEX2(+) mitochondria within RGC boutons. Arrowheads point to electron-dense material at the postsynaptic density (PSD) apposed to individual active zones with clustered presynaptic synaptic vesicles. Scale bars are 1 µm in all images. (D) Representative CTB(+) dominant-eye (top panels) and CTB(-) non-dominant-eye (bottom panels) mAZ synapses in a WT P8 sample, showing synaptic (left panels), CTB (middle panels), and merged immunolabels (right panels). (E and F) Eye-specific mAZ synapse density (E) and mAZ synapse fraction (F) across development in WT (top panel) and β2KO mice (bottom panel). Black dots represent mean values from separate biological replicates and black lines connect measurements within each replicate (N=3 for each age and genotype). Error bars represent group means ± SEMs. Statistical significance between CTB(+) and CTB(-) synapse measurements was assessed using paired T-tests. *: p<0.05, **: p<0.01, ***: p<0.001.

Multi-active zone synapses undergo eye-specific vesicle pool maturation.

(A) Violin plots showing the distribution of VGluT2 cluster volume for mAZ synapses in WT and β2KO mice at each age. The width of each violin plot reflects the relative synapse proportions at each volume across the entire grouped data set (N=3 biological replicates). The maximum width of the violin plots was normalized across mAZ and sAZ groups. The black dots represent the median value of each biological replicate (N=3), and the black horizontal lines represent the median value of all synapses grouped across replicates. Black lines connect measurements of CTB(+) and CTB(-) populations from the same biological replicate. Statistical significance was determined using a mixed model ANOVA. Black asterisks indicate significant eye-specific differences at each developmental time point. (B) Violin plots similar to (A) show the distributions of VGluT2 cluster volume for sAZ synapses in WT and β2KO mice at each age. (C) Average number of AZs (individual bassoon clusters) per mAZ synapse in WT (top panel) and β2KO mice (bottom panel). Black dots represent mean values from separate biological replicates and black lines connect measurements within each replicate. (D and E) Total (D) and average/AZ (E) VGluT2 cluster volume as a function of AZ number for all synapses in WT P4 samples (top panel) and β2KO P4 samples (bottom panel). Black dots represent mean values from separate biological replicates and black lines connect measurements within each replicate. A table of the total eye-specific synapse numbers of each type (mAZ and sAZ) and AZ number for each biological replicate in both genotypes is included in the Supplemental Materials (Table S1). In (C-E), error bars represent means ± SEMs. Statistical significance was assessed using paired T-tests. In all panels, *: p<0.05, **: p<0.01, ***: p<0.001. N=3 biological replicates for each age and genotype.

Multi-active zone synapses are loci for synaptic clustering.

(A) A representative mAZ synapse (arrow) in a WT P8 dLGN with nearby sAZ synapses (arrowheads) clustered within 1.5 μm (dashed yellow ring). (B) Comparison of a normalized clustering index between CTB(+) dominant-eye mAZ and sAZ synapses at each time point in WT (left) and β2KO (right) mice. Black dots represent mean values from separate biological replicates and black lines connect measurements within each replicate (N=3 for each age and genotype). (C) Same as in (B) showing the normalized clustering index measurements for CTB(-) non-dominant-eye synapses. In B/C, paired T-tests were used to test the statistical significance between mAZ and sAZ clustering index measurements. Error bars represent means ± SEMs. *: p<0.05; **: p<0.01. (D) Cumulative distributions of VGluT2 volume for CTB(+) dominant-eye sAZ synapses near (<1.5 μm, black lines) or far from (>1.5 μm, red lines) like-eye mAZ synapses. Left panel: WT. Right panel: β2KO. (E) Same as in (D) showing the cumulative distributions of VGluT2 volume for CTB(-) non-dominant-eye sAZ synapses relative to like-eye mAZ synapses. The distributions in D/E show merged data across all developmental ages (P2/P4/P8; N=3 biological replicates at each time point). A nonparametric Kolmogorov-Smirnov test was used for statistical analysis. “n.s.” indicates no significant difference between near and far sAZ synapse distributions from the K-S test. Separate measurements of 5/95% confidence intervals show overlap of the two distributions in each case.

Synaptic clustering varies with distance to neighboring eye-specific multi-active zone synapses.

(A) VGluT2 volume of CTB(-) non-dominant-eye mAZ synapses relative to their distance to the nearest CTB(+) dominant-eye mAZ synapse in a WT P4 sample (left panel) and a β2KO P4 sample (right panel). Each black dot represents one mAZ synapse. The red solid line represents the result of linear regression fitting. (B) Distributions of distances between CTB(-) non-dominant-eye mAZ synapses and their nearest CTB(+) dominant-eye mAZ synapse separated by the number of AZs within each CTB(-) mAZ synapse in WT P4 samples (left panel) and β2KO P4 samples (right panel). The median value is indicated by the horizontal line within the box, while the box boundaries represent quartile values. The whiskers represent the maximum and minimum values. A mixed model ANOVA was used to perform statistical tests. “n.s.” indicates no significant differences. The red solid line represents the result of linear regression fitting. (C) Cumulative distributions of distances between CTB(+) dominant-eye mAZ synapses and their nearest CTB(+) dominant-eye mAZ synapse (cartoon) in WT P4 samples (left panel) and β2KO P4 samples (right panel). Red lines indicate clustered mAZ synapses with nearby (<1.5 μm) sAZ synapses and black lines indicate isolated mAZ synapses with no nearby sAZ synapses. (D) Same presentation as in (C), showing distances between CTB(-) mAZ synapses. (E) Same presentation as in (C), showing distances between CTB(-) non-dominant-eye mAZ synapses and their nearest CTB(+) dominant-eye mAZ synapse. (F) Same presentation as in (C), showing distances between CTB(+) dominant-eye mAZ synapses and the nearest CTB(-) non-dominant-eye mAZ synapse. For C-F, nonparametric Kolmogorov-Smirnov tests were used for statistical comparisons. “***” indicates p<0.001, while “n.s.” indicates no significant difference from the K-S test results. Separate measurements of 5/95% confidence intervals show overlap of the two distributions in each “n.s.” instance.

Eye-specific differences in sAZ synapse density in the first postnatal week, related to Figure 1.

(A) Representative sAZ (left panels) and mAZ (middle and right panels) synapses (maximum projection images, top panels) and their 3D renderings (bottom panels). Arrowheads point to individual active zones. (B) The density of eye-specific sAZ synapses across ages in WT (top panel) and β2KO mice (bottom panel). Error bars represent means ± SEMs (N=3 biological replicates for each age/genotype). Black dots represent mean values from separate biological replicates and black lines connect measurements within each replicate. Statistical significance between eye-specific synapse measurements was assessed using paired T-tests. *: P<0.05. **: p<0.01.

mAZ synapses undergo eye-specific vesicle pool maturation, related to Figure 2.

(A-B) VGluT2 cluster volume relative to AZ number for each synapse in WT (left panels) and β2KO mice (right panels) at P2 (A) and P8 (B). (C-D) Average VGluT2 volume per AZ (bassoon cluster) for all synapses in WT (left panels) and β2KO mice (right panels) at P2 (C) and P8 (D). For all panels, error bars indicate means ± SEMs (N=3 biological replicates for each age and genotype). Black dots represent mean values from separate biological replicates and black lines connect measurements within each replicate. Statistical significance between eye-specific synapse measurements was assessed using paired T-tests: *: p<0.05; **:p<0.01.

Eye-specific and distance-dependent synapse clustering, related to Figure 3.

(A) The ratio of CTB(-) sAZ synapses nearby like-eye mAZ synapses for increasing distance thresholds in WT P4 samples. The observed ratios are higher than a reshuffling of the data at distances of 1-4 μm across all time points. (B) Same presentation of results as in (A), but for CTB(+) sAZ synapses nearby CTB(+) mAZ synapses. (C) Percentage of CTB(-) non-dominant-eye sAZ synapses within 1.5 μm of an opposite-eye CTB(+) mAZ synapse in WT (left panel) and β2KO mice (right panel) at P4. (D) Same presentation as in (C), showing percentage of CTB(+) dominant-eye sAZ synapses near an opposite-eye mAZ synapse. For all panels, error bars represent means ± SEMs (N=3 biological replicates for each age and genotype). Black dots represent mean values from separate biological replicates and black lines connect measurements within each replicate. Statistical significance between original and shuffled measurements was assessed using paired T-tests: *: P<0.05; **: p<0.01; ***: p<0.001.

Multi-active zone synapses are loci for synaptic clustering.

(A) Comparison of the total percentage of dominant-eye (top panels) and non-dominant (bottom panels) sAZ synapses near like-eye mAZ synapses between the original and shuffled synapse positions across all time points. Left panels: WT; Right panels: β2KO. (B) Same analysis as in (A) but comparing the total percentage of sAZ synapses near like-eye sAZ synapses. Error bars represent means ± SEMs (N=3 biological replicates). Black dots represent mean values from separate biological replicates and black lines connect measurements between measured and shuffled values for each replicate. Statistical significance between original and shuffled measurements was assessed using paired T-tests: *: P<0.05; **: p<0.01; ***: p<0.001. (C) Comparison between the original data and 10 iterations of data shuffling from a WTP4 CTB(-) sample (left) and a WTP4 CTB(+) sample (right). Error bars represent 5/95% confidence intervals.

The ratio of clustered and isolated eye-specific mAZ synapses, related to Figure 4.

Clustered and isolated mAZ synapses are distinguished by the presence of sAZ synapses within a 1.5 μm radius shell. The figures show the ratio of clustered mAZ synapses for CTB(+) (A) and CTB(-) (B) synapses.

Eye-specific synaptic clustering varies based on distance to multi-active zone synapses, related to Figure 4.

(A) Cumulative histogram of the distances from CTB(+) mAZ synapses to their nearest CTB(+) (left panel) and CTB(-) (right panel) mAZ synapse in P4 WT data where sAZ synapse distributions were randomized. Black lines show distributions for isolated mAZ synapses with no nearby (<1.5 μm) sAZ synapses and red lines show distributions for clustered mAZ synapses with one or more sAZ synapses nearby. (B) Same presentation as in (A), showing distances from CTB(-) mAZ synapses to their nearest CTB(+) (left panel) and CTB(-) (right panel) mAZ synapse in P4 WT randomized data. (C and D) Same presentation as in A/B, showing WT P8 original data. (E and F) Same presentation as in C/D, showing β2KO P8 original data. Nonparametric Kolmogorov-Smirnov tests were used for statistical comparisons (N=3 biological replicates for each condition). ***: p<0.001. “n.s.” indicates no significant differences. Separate measurements of 5/95% confidence intervals show overlap of the two distributions in each “n.s.” instance.

VGluT2 volume near active zones is similar between multi-AZ and single-AZ synapses at P4.

(A) The violin plots show the distribution of VGluT2 volume within a 70nm shell surrounding each associated Bassoon cluster (AZ). The width of each violin plot reflects the relative synapse proportions at each volume across the entire grouped data set (N=3 biological replicates). The maximum width of the violin plots was normalized across mAZ and sAZ groups. The black dots represent the median value of each biological replicate (N=3), and the black horizontal lines represent the median value of all synapses grouped across replicates. Black lines connect measurements of sAZ and mAZ populations from the same biological replicate. Statistical significance was determined using a mixed model ANOVA. “n.s.” represents that no significant differences were found. Measurements of 5/95% confidence intervals show overlap of the two distributions in each “n.s.” instance. (B) VGluT2 signal volume near the AZ is similar for mAZ and sAZ synapses in β2KO mice. Data presentation and statistics are the same as those in (A).