Expression of VGluT2 in Neurobiotin-labeled primary afferent terminals. High resolution confocal microscope imaging was performed to identify VGluT2-IR terminals labeled with Neurobiotin and therefore of primary afferent origin. Images represent a projection of 3 optical z-sections taken 0.25 μm apart. A: Neurobiotin (green), B: VGluT2-IR (magenta), C: CGRP-IR (red) and D: merged image. Varicosities containing both Neurobiotin and VGluT2-IR were common in upper lamina I, and many lacked CGRP-IR (arrows). Conversely, many of the varicosities containing both Neurobiotin and CGRP-IR lacked VGluT2-IR (arrowheads). Occasionally both VGluT2-IR and CGRP-IR occurred within the same Neurobiotin-labeled varicosity (short arrows with asterisks). The arrow with a single asterisk shows an example of a Neurobiotin-labeled varicosity with strong VGluT2-IR and weak CGRP-IR. The arrow with the double asterisks shows an example of a Neurobiotin-labeled varicosity with weak VGluT2-IR and strong CGRP-IR. Varicosities labeled with both Neurobiotin and VGluT2-IR were scarce below lamina I. Panels E-H and I-L are enlargements of regions indicated by white boxes in D. Scale bar represents 10 μm in D (also applies to panels A-C) and 5 μm in L (also applies to panels E-K). LT; Lissauer's Tract, I; lamina I, IIo; outer lamina II, Iii; inner lamina II.