Figure 3

Identification of the central terminals of VGluT2-expressing primary afferents. High resolution confocal microscope imaging was performed to identify VGluT2-IR terminals labeled with Neurobiotin and therefore of primary afferent origin. Images are a projection of 3 optical z-sections captured 0.25 μm apart from the mid superficial dorsal horn at L3 spanning lamina I and II. A: Neurobiotin (green), after application to all 6 lumbar dorsal roots. B: VGluT2-IR (magenta). C: merged image of A and B, with terminals containing both Neurobiotin and VGluT2-IR appearing whiter. VGluT2-IR terminals labeled with Neurobiotin are abundant in upper lamina I (arrows, and see enlargements of boxed area in C; D-F). VGluT2-IR terminals without Neurobiotin also are present in lamina I. A second band of dense VGluT2-IR terminals is visible in outer lamina II, but none are labeled with Neurobiotin in this example. VGluT2-IR varicosities are overall less dense in inner lamina II. Few VGluT2-IR varicosities in inner lamina II contain Neurobiotin: they are mostly large varicosities only weakly labeled with VGluT2-IR (arrowheads; and see enlargements of boxed area in C; G-I). Scale bar in C represents 10 μm and also applies to A and B. Scale bar in I represents 5 μm and also applies to D-H. LT; Lissauer's Tract, I; lamina I, IIo; outer lamina II, Iii; inner lamina II.