A unique set of complex synapses shows eye-specific differences during retinogeniculate segregation.

(A) Experimental design: CTB-Alexa 488 was injected into the right eye of wild-type (WT) and β2KO mice. Tissue was collected from the left dLGN at P2, P4, and P8. The red squares indicate the STORM imaging regions. (B) Representative complex (left panels) and simple synapses (right panels) in WT (top panels) and β2KO mice (bottom panels) at each age. (C) Representative CTB(+) dominant-eye (top panels) and CTB(-) non-dominant-eye (bottom panels) complex synapses in a WT P8 sample, showing synaptic (left panels), CTB (middle panels), and merged immunolabels (right panels). (D) Eye-specific complex synapse density across development in WT (top panel) and β2KO mice (bottom panel). (E) Eye-specific complex synapse fraction across development in WT (top panel) and β2KO mice (bottom panel). In (D) and (E), error bars represent means ± SEMs. Statistical significance between CTB(+) and CTB(-) synapse measurements was assessed using one-way ANOVA. *: p<0.05, **: p<0.01. N=3 biological replicates for each age/genotype.

Complex synapses undergo eye-specific vesicle pool maturation.

(A) Violin plots showing the distribution of VGluT2 cluster volume for complex synapses in WT and β2KO mice at each age. 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. Black lines connect measurements of CTB(+) and CTB(-) populations from the same biological replicate. Statistical significance was determined using a mixed model ANOVA with a post hoc Bonferroni test. Black asterisks indicate eye-specific differences and colored asterisks indicate differences across time points. (B) Violin plots similar to (A) show the distribution of VGluT2 cluster volume for simple synapses in WT and β2KO mice at each age. (C) Average number of active zones (AZs = individual bassoon clusters) per complex synapse in WT (top panel) and β2KO mice (bottom panel). (D) VGluT2 cluster volume as a function of AZ number for all synapses in WT P4 samples (top panel) and β2KO P4 samples (bottom panel). (E) Average VGluT2 cluster volume per AZ for all synapses in WT P4 samples (top panel) and β2KO P4 samples (bottom panel). In (C-E), error bars represent means ± SEMs from N=3 biological replicates. Statistical significance was assessed using one-way ANOVA with a post hoc Tukey’s test. Black asterisks indicate eye- specific differences and colored asterisks indicate differences between simple (1 AZ) and complex (>1 AZ) synapses. “n.s.” indicates no significance between simple and complex synapses. In all panels, *: p<0.05, **: p<0.01, ***: p<0.001.

Complex synapses are loci for synaptic clustering.

(A) A representative complex synapse in a WT P8 dLGN (arrow) with nearby simple synapses (arrowheads) clustered within 1.5 μm (dashed yellow ring). (B) The fraction of CTB(+) dominant-eye simple synapses near like-eye complex synapses (cartoon) across development in WT (middle panel) and β2KO mice (right panel). Colored lines show the measured distributions and black lines show results of a randomized simple synapse distribution within the sample imaging volume. (C) Same as in (B) showing results for CTB(-) non-dominant-eye simple synapses near like-eye complex synapses. In B/C, a one-way ANOVA was used to test the statistical significance between original and randomized data. Error bars represent means ± SEMs. *: p<0.05; **: p<0.01; ***: P<0.001. (D) Cumulative distribution of simple synapse VGluT2 volume for CTB(+) dominant-eye simple synapses near (<1.5 μm, black lines) or far from (>1.5 μm, red lines) like-eye complex synapses. (E) Same as in (D) showing the cumulative distribution of simple synapse VGluT2 volume for CTB(-) non-dominant-eye simple synapse relative to like-eye complex synapses. The distributions in (D/E) show merged data across all developmental ages. A nonparametric Kolmogorov-Smirnov test was used for statistical analysis. “n.s.” indicates no significant difference between near and far simple synapse distributions.

Complex synapses mediate distance-dependent synaptic stabilization and punishment underlying eye-specific competition.

(A) VGluT2 volume of CTB(-) non-dominant-eye complex synapses relative to their distance to the nearest CTB(+) dominant-eye complex synapse in a WT P4 sample (left panel) and a β2KO P4 sample (right panel). Each black dot represents one synapse. (B) Distributions of distances between CTB(-) non-dominant-eye complex synapses and their nearest CTB(+) dominant-eye complex synapse separated by the number of AZs within each CTB(-) complex 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 significance differences. (C) Cumulative distributions of distances between CTB(+) dominant-eye complex synapses and their nearest CTB(+) dominant-eye complex synapse (cartoon) in WT P4 samples (left panel) and β2KO P4 samples (right panel). Red lines indicate clustered complex synapses with nearby (<1.5 μm) simple synapses and black lines indicate isolated complex synapses with no nearby simple synapses. (D) Same presentation as in (C), showing distances between CTB(-) complex synapses. (E) Same presentation as in (C), showing distances between CTB(-) non-dominant-eye complex synapses and their nearest CTB(+) dominant- eye complex synapse (cartoon). (F) Same presentation as in (C), showing distances between CTB(+) dominant-eye complex synapses and the nearest CTB(-) non-dominant-eye complex synapse. For C- F, nonparametric Kolmogorov-Smirnov tests were used for statistical comparisons. “***” indicates p<0.001, while “n.s.” indicates no significant differences.

Eye-specific differences in simple synapse density in the first postnatal week, related to

Fig 1. (A) Volumetric STORM imaging enables the differentiation of complex (arrows) versus simple (arrowheads) synapses (top panels), which cannot be distinguished in diffraction-limited conventional images (bottom panels). (B) The density of eye-specific simple synapses across ages in WT (top panel) and β2 KO mice (bottom panel). Error bars represent means ± SEMs (N=3 biological replicates for each age/genotype). Statistical tests were performed using a one-way ANOVA. *: P<0.05. **: p<0.01.

Complex synapses undergo eye-specific vesicle pool maturation, related to

Fig 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). Error bars indicate means ± SEMs (N=3 biological replicates for each age and genotype). A one-way ANOVA was used to assess statistical significance between eye-of-origin (black asterisks) and eye-specific synapses with different AZ numbers (colored asterisks). A post hoc Tukey’s test was conducted for pairwise comparisons between simple (1 AZ) and complex (>1 AZ) synapses. (C-D) VGluT2 volume per AZ (bassoon cluster) for all synapses in WT (left panels) and β2KO mice (right panels) at P2 (C) and P8 (D). Figure presentation and statistical tests were the same as shown in (A) and (B). In all panels: *: p<0.05; **:p<0.01; ***:p<0.001.

Complex synapses are loci for synaptic clustering, related to

Fig 3. (A) Percentage of CTB(-) non-dominant-eye simple synapses near an opposite-eye complex synapse in WT (top panel) and β2KO mice (bottom panel). (B) Same presentation as in (A), showing percentage of CTB(+) dominant-eye simple synapses near an opposite-eye complex synapse. (C) To further validate our selection of a 1.5 um search radius, we performed additional control measurements with varying local search radii. For complex synapses of both eyes-of-origin, the detection of non-random clustering increased when the search radius was expanded from 1 μm to 2 μm and then decreased as the radius was further expanded to sample the average simple synapse density (3-4 μm) (example with CTB(-) non-dominant-eye synapses). The figure shows the percentage of CTB(-) non-dominant-eye simple synapses near like-type CTB(-) complex synapses across development as a function of increasing distance cutoffs from the surface of complex synapses. Distributions are shown for cutoff distances of 1.0 μm (top left panel), 2.0 μm (top right panel), 3.0 μm (bottom left panel), and 4.0 μm (bottom right panel). For all panels, grey and purple lines represent the original data, and black lines represent the results from a randomized simple synapse distribution. Error bars represent means ± SEMs (N=3 biological replicates for each age and genotype). Statistical tests between original and randomized data were performed using one-way ANOVA. *: P<0.05; **: p<0.01; ***: p<0.001. “n.s.” indicates no significant differences.

Complex synapses mediate distance-dependent synaptic stabilization and punishment underlying eye-specific competition, related to

Fig. 4. (A) Cumulative histogram of the distances from CTB(+) complex synapses to their nearest CTB(+) (left panel) and CTB(-) (right panel) complex synapse in P4 WT data where simple synapse distributions were randomized. Black lines show distributions for isolated complex synapses with no nearby (<1.5 μm) simple synapses and red lines show distributions for clustered complex synapses with one or more simple synapses nearby. (B) Same presentation as in (A), showing distances from CTB(-) complex synapses to their nearest CTB(+) (left panel) and CTB(-) (right panel) complex 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.