Fig. 2.

Glutamatergic and GABAergic axons in the DR. (A–D) Immunolabeling for VGLUT1–3–containing (green, magenta, and light blue, respectively) or GAD2-containing (red) can be found in the same location in serial sections, and these puncta are often coincident with synapsin (blue). In these examples, axons are also adjacent to serotonin neuron dendrites (white), identified by the presence of TPOH labeling and the lack of synapsin labeling. (E) The total density of puncta containing synapsin in combination with either VGLUT1–3 or GAD2 in the neuropil. VGLUT2- and GAD2-containing axons are most abundant. Bars represent SEM (n = 3 rats; *P < 0.001 vs. either VGLUT1 or VGLUT3). (F) Cross-correlation analysis between VGLUT1–3 or GAD2 and synapsin (SYN). When imaging channels are aligned (x = 0-μm displacement), VGLUT2 and GAD2 show the highest absolute correlation with synapsin, and this is likely driven by their higher relative abundance (shown in E). When imaging channels are displaced with respect to each other, all of the correlations decrease. (G) The normalized cross-correlation analysis shows the relative decrease in correlations with image displacement; that is, all of the curves are divided by the initial correlation value at no shift. This analysis provides a measure of how specific the correlations are, unbiased by the abundance of antigens. All curves drop similarly, indicating that VGLUT1–3 and GAD2 each have a fairly equal likelihood of coexisting with synapsin, supporting the specificity of immunolabeling.