Figure 3.
Quantitative analysis of Munc13-1 and Cav2.1 immunolabeling in AZs synapsing on Kv3.1b+ and mGluR1α+ INs
(A) The AZs are significantly (p = 7 × 10−9, MWU test) larger on mGluR1α+ INs (0.10 ± 0.048 μm2, n = 118) than on Kv3.1b+ cells (0.07 ± 0.035 μm2, n = 159).
(B) The normalized density of Munc13-1 immunolabeling is similar (p = 0.081, MWU test) in both synapse populations.
(C) Cumulative probability plots of the mean nearest neighbor distances (NNDs) of gold particles (data, solid lines) and those of randomly placed particles (dotted lines). The mean NNDs of the data are significantly (p < 0.001, Kruskal-Wallis [KW] test followed by Wilcoxon signed-rank [WSR] test with Holm-Bonferroni correction [HBC]) smaller than those of randomly distributed gold particles within both AZ populations. The experimental NNDs are slightly longer in AZs on Kv3.1b+ cells (26 ± 10 nm) compared to those (23 ± 4 nm) on mGluR1α+ cells (p = 0.02542, KW test followed by MWU test with HBC).
(D) Ripley analysis of individual synapses demonstrates that Munc13-1 gold particles are clustered (p < 0.05, maximum absolute deviation [MAD] test) in 66% and 81% of the AZs on Kv3.1b+ (n = 159) and mGluR1α+ (n = 118) dendrites, respectively.
(E) The number of Munc13-1 clusters is significantly (p = 0.000004, MWU test) larger in mGluR1α+ (7.9 ± 4.3, n = 96) than in Kv3.1b+ (5.4 ± 2.5, n = 105) dendrites contacting AZs.
(F) The Munc13-1 cluster density does not differ in the two AZ populations (p = 0.24, MWU test).
(G) The center-to-center NND between Munc13-1 clusters is similar in the two AZ populations (p = 0.43, MWU test).
(H) The density of gold particles labeling Cav2.1 is significantly (p = 0.0016, MWU test) larger in AZs contacting Kv3.1b+ dendrites.
(I) Cumulative probability plots of the mean NNDs of gold particles labeling Cav2.1 (data, solid lines) and those of randomly placed particles (dashed lines). The mean NNDs of the data are significantly (p < 0.001, WSR test with HBC) different from randomly distributed gold particles within the same AZ population, but they are comparable between the two AZ populations (p = 0.695, MWU test with HBC).
(J) In only a small fraction of the AZs (Kv3.1b+: 8%, n = 123; mGluR1α+: 16%, n = 69), gold particles for Cav2.1 are clustered (p < 0.05, MAD test), as revealed by Ripley’s analysis of individual AZs.
(K and L) Bivariate Ripley’s analysis of the Cav2.1 and Munc13-1 gold particles in double labeling experiments in AZs contacting Kv3.1b+ (K) and mGluR1α+ (L) dendrites reveals a repellent interaction between the two types of molecules. Normalized Hbiv(r) is plotted for individual AZs (thin lines), and the population average is shown with the thick line. The thick black line is the mean Hbiv(r) of 500 random distributions generated for each AZ. Gray area is the 99% confidence envelope (CE).
(M) The mean NNDs between Cav2.1 gold particles and the edges of Munc13-1 clusters are significantly shorter (p < 0.0001 for both synapse populations, MWU test with HBC) than those between the Munc13-1 clusters in both synapse populations (orange: Kv3.1b+, cyan: mGluR1α+).
Horizontal lines in the boxplots: 25th, 50th, and 75th percentiles, rectangle: mean, whiskers: SD.