Quantification of the number and distribution of GABA release sites in MLI axons. Immunolabeling of VGAT in Alexa Fluor 488–loaded MLIs was performed to identify axonal release sites. (A) Confocal image stack Z projection of an Alexa Fluor 488–filled MLI. (B) Superimposition of Alexa Fluor (magenta) and VGAT immunolabeling (green). (C) Detail of the colocalization of VGAT and Alexa Fluor 488 labeling in a varicosity. XY, YZ, and XZ projections (middle, right, and bottom panels, respectively). (D) Intensity profiles of VGAT (green line) and Alexa Fluor 488 (magenta line) along the main branch of an MLI axon. The cartoon on top represents the MLI’s soma and axonal main branch; circles indicate detected GABA varicosities. The soma produced an intense labeling while the rest of the trace showed near baseline intensity interrupted by clearly defined peaks of VGAT and Alexa Fluor intensity, corresponding to varicosities. (Inset) Criteria used for detecting release sites. Threshold = baseline + 2 SD of the VGAT intensity profile (green trace in D); width, >500 nm. GABA release sites are heterogeneously distributed along the axon. (E) Number of release sites per axonal quarter. (F) Histogram of distances between release sites (mean ± SD: 6.1 ± 5.8 µm; median: 4.4 µm; n = 390 pairs of sites). Branching points are also heterogeneously distributed along the axon. (G) Number of branches per axonal quarter. (H) Histogram of collateral’s length (mean ± SD: 24.1 ± 18.8 µm; median: 19.4 µm; n = 89 collaterals). In E and G, the gray dots represent individual cells, and the black dots show mean ± SD; n = 7 reconstructed MLIs (*, P < 0.05; **, P < 0.01).