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

(A) Experimental design. CTB-Alexa 488 was injected into the right eye of wild-type and β2KO mice. One day after the treatment, tissue was collected from the left dLGN at P2, P4, and P8. Red squares indicate the STORM imaging regions that were analyzed. (B) Representative examples of individual sAZ and mAZ inputs, with corresponding active zone counts ranging from one to three. Upper panels show Z-projections of inputs and lower panels show the corresponding 3D volume. Arrowheads point to individual Bassoon clusters (active zones) paired with postsynaptic Homer1 labels within each input. All examples are from a WT P8 sample. (C) Electron micrographs of mAZ retinogeniculate inputs in a P8 ET33Cre::ROSA26LSL-Matrix-dAPEX2 mouse. Darkly-stained dAPEX2(+) mitochondria are present within ipsilaterally-projecting RGC terminals. Arrowheads point to electron-dense material at the postsynaptic density, apposed to individual active zones with clustered presynaptic synaptic vesicles.

Changes in eye-specific input density during synaptic competition.

(A) Representative Z-projection images of mAZ and sAZ inputs across ages and genotypes. Arrowheads point to individual Bassoon/Homer1 cluster pairs indicating release sites. (B) Representative CTB(+) dominant-eye (top panels) and CTB(-) non-dominant-eye (bottom panels) mAZ inputs in a WT P8 sample, showing synaptic (left panels), CTB (middle panels), and merged labels (right panels). Arrowheads point to individual Bassoon/Homer1 paired clusters. (C) Eye-specific mAZ (left) and sAZ (right) input density across development in WT (top panels) and β2KO mice (bottom panels). Black dots represent mean values from separate biological replicates and black lines connect eye-specific measurements within each replicate (N=3 for each age and genotype). Error bars represent group means ± SEMs. Statistical significance between eye-specific measurements was assessed using two-tailed paired T-tests at each age. *p<0.05, **p<0.01.

Dominant-eye inputs show larger vesicle pools that scale with active zone number.

(A and B) Violin plots showing the distribution of VGluT2 cluster volume for (A) mAZ and (B) sAZ inputs in WT (filled) and β2KO mice (striped) at each age. The width of each violin plot reflects the relative synapse proportions across the entire grouped data set at each age (N=3 biological replicates) and the maximum width was normalized across all 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 inputs grouped across replicates. Black lines connect measurements of CTB(+) and CTB(-) populations from the same biological replicate. Statistical significance was determined using a linear mixed model ANOVA with a post-hoc Bonferroni correction. Black asterisks indicate significant eye-specific differences at each age. *p<0.05, **p<0.01. ***p<0.001. (C) Eye-specific VGluT2 signal volume for all inputs separated by number of AZs in WT (left panel) and β2KO mice (right panel) at P4. (D) Average VGluT2 volume per AZ for all inputs separated by number of AZs in WT (left panel) and β2KO mice (right panel) at P4. In panels (C) and (D), error bars indicate group means ± SEMs (N=3 biological replicates for each age and genotype). Black dots represent mean values from separate biological replicates and black lines connect eye-specific measurements within each replicate. Statistical significance between eye-specific measurements was assessed using two-tailed paired T-tests: *p<0.05; **p<0.01.

Eye-specific synapse clustering before eye-opening.

(A) Representative mAZ (left panels) and sAZ (right panels) inputs in a WT P8 sample with nearby sAZ synapses (arrowheads) clustered within 1.5 μm (dashed yellow ring). Arrows point to the centered mAZ or sAZ inputs. (B) Ratio of clustered and isolated mAZ and sAZ inputs for CTB(+) (upper panels) and CTB(-) (lower panels) inputs in WT and β2KO mice at P4. (C) Comparison of the clustered input ratio between mAZ and sAZ inputs across different ages, genotypes, and eyes of origin. (D) Comparison of the average number of nearby sAZ synapses for clustered mAZ and sAZ inputs across different ages, genotypes, and eyes of origin. In panels B-D, 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. Two-tailed paired T-tests were used to test statistical significance between mAZ and sAZ inputs at each age. *p<0.05; **p<0.01.

Clustered mAZ inputs are closer than isolated inputs during competition.

(A-B) Distance between clustered and isolated mAZ inputs and the closest like-eye clustered mAZ input, shown for (A) CTB(+) and (B) CTB(-) projections at P4 in WT and β2KO mice. Boxes indicate the 25%-75% distribution of input measurements from N=3 biological replicates and whiskers extend to 1.5 times the interquartile range. Gray dots represent individual distance measurements for all mAZ inputs. Black and red dots represent mean values from separate biological replicates and black lines connect measurements within each replicate (N=3 for each age and genotype). Statistical significance was determined using a linear mixed model ANOVA with post-hoc Bonferroni correction. Black asterisks indicate significant differences. *p<0.05, ***p<0.001.

Fraction of mAZ inputs across development, related to Fig. 2.

Eye-specific mAZ input fraction 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 eye-specific measurements was assessed using two-tailed paired T-tests. **p<0.01, ***p<0.001

Quantification of docked vesicle pool volume and AZ number in mAZ and sAZ inputs, related to Fig. 2.

(A-C) Violin plots show the distribution of VGluT2 cluster volume within a 70 nm shell surround individual Bassoon clusters at (A) P2, (B) P4, and (C) P8. 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 all groups. Black horizontal lines represent the median value of all inputs grouped across replicates. The black dots represent the median value of each biological replicate and black lines between dots connect measurements from the same biological replicate. Statistical significance was determined using a linear mixed model ANOVA with post-hoc Bonferroni correction. See Table S3 for 5/95% confidence intervals. (D-E) Average AZ number per mAZ input in (D) WT and (E) β2KO mice. Black dots represent mean values from separate biological replicates (N = 3) and black lines connect measurements within each replicate. Error bars represent means ± SEMs. Statistical significance was assessed using two-tailed paired T-tests. *p<0.05.

Relationship between vesicle pool volume and active zone number, related to Fig. 3.

(A) Total VGluT2 volume per input and (B) average VGluT2 volume per AZ for all inputs separated by number of AZs in WT (filled bars) and β2KO mice (striped bars) at P2. (C) Total VGluT2 volume per input and (D) average VGluT2 volume per AZ for all inputs separated by number of AZs in WT (filled bars) and β2KO mice (striped bars) at P8. In all panels, error bars indicate group 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 measurements was assessed using two-tailed paired T-tests: *p<0.05; **p<0.01.

sAZ synapse clustering near like-eye mAZ inputs, related to Fig. 4.

(A) The ratio of CTB(+) dominant-eye and (B) CTB(-) non-dominant-eye sAZ synapses nearby like-eye mAZ inputs within increasing distance cutoffs in WT samples across ages. At distances of 1-2 μm across all ages, the observed ratios (filled bars) are higher than a reshuffling of the data (open bars) where the position of sAZ inputs was randomized within the neuropil. (C-D) Percentage of sAZ synapses within 1.5 μm of opposite-eye mAZ inputs across ages in WT and β2KO mice. Data are compared against a randomization of sAZ positions (open bars) as in panels A/B. For all panels, error bars represent group 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 was assessed using two-tailed paired T-tests: *p<0.05; **p<0.01; ***p<0.001.

Clustered and isolated mAZ inputs show similar spacing after competition, related to Fig. 5.

(A-B) Distance between clustered and isolated mAZ inputs and the closest like-eye clustered mAZ input, shown for (A) CTB(+) and (B) CTB(-) projections at P8 in WT and β2KO mice. Boxes indicate the 25%-75% distribution of input measurements from N=3 biological replicates and whiskers extend to 1.5 times the interquartile range. Gray dots represent individual distance measurements for all mAZ inputs. Black and red dots represent mean values from separate biological replicates and black lines connect measurements within each replicate (N=3 for each age and genotype). Statistical significance was determined using a linear mixed model ANOVA with post-hoc Bonferroni correction (see Table S3 for 5/95% confidence intervals).

sAZ synapse vesicle pool volume is independent of distance to mAZ inputs, related to Fig. 5.

(A) Cumulative distributions of VGluT2 volume for CTB(+) dominant-eye or (B) CTB(-) non-dominant-eye sAZ synapses near (<1.5 μm) or far from (>1.5 μm) like-eye mAZ inputs. The distributions show merged data across all ages (P2/P4/P8; N=3 biological replicates at each time point). A nonparametric Kolmogorov-Smirnov test was used for statistical analysis (see Table S3 for 5/95% confidence intervals).