Fig. 3.

Associations between glutamate and GABA boutons. (A–C) Serial section images showing that GAD2-labeled puncta (yellow) were often found adjacent to VGLUT1–3–containing puncta (green, magenta, and light blue, respectively). Synapsin is depicted in blue. (D) Total density of objects where markers of two types of axon terminals and synapsin overlapped. The most common overlap was seen between VGLUT2 and GAD2 (*P < 0.001 vs. either VGLUT1 or VGLUT3). Lower densities of associations between different glutamatergic axons (e.g., VGLUT1/SYN/VGLUT2, VGLUT1/SYN/VGLUT3, and VGLUT2/SYN/VGLUT3) were also detected. Bars represent SEM (n = 3 rats). (E) Cross-correlation analysis of pixels between different types of boutons shows the highest degree of colocalization for pairs containing VGLUT2 and GAD2, consistent with their higher overall abundance. (F) Normalized cross-correlation analysis to evaluate the specificity of correlations, unbiased by abundance. All curves of VGLUT1–3/SYN/GAD2 associations drop sharply, suggesting a similar spatial proximity of all three VGLUT–SYN pairs with GAD2 puncta. Association curves between different types of glutamate axons drop, but to a higher plateau, indicating a greater fraction of stochastic associations.