Figure 2. Virtually all NTS→lPBN fiber terminals in close apposition to lPBN CGRP neurons coexpress VGLUT2. A, Image of caudal NTS indicating BDA tracer injection placement (blue), with VGLUT2 (green) also imaged to reveal tissue structure and landmarks. ap, Area postrema; cc, central canal; dmnx, dorsal motor nucleus of the vagus; ts, solitary tract. B, Location of lPBN region analyzed. C, Epifluorescence micrograph showing triple labeling of BDA+ axons (blue), VGLUT2 (green), and CGRP+ neurons (red) within the lPBN; 20× objective. bc, Brachium conjunctivum. #Region of the lPBN analyzed for triple labeling. D, Higher-magnification confocal image showing triple labeling of BDA+ axons and terminals (blue), VGLUT2 (green), and CGRP (red) within the lPBN; 100× oil objective and 3× digital zoom. E, Confocal grayscale image: colocalized pixels (white) are those in which both BDA and VGLUT2 signals are above their respective thresholds. Because all BDA-positive profiles within the field also are VGLUT2 positive, they appear gray/white. F, Table of Pearson's and Mander's correlation coefficients for colocalization of BDA and VGLUT2. Rtotal, Pearson's correlation coefficient for all pixels above background (no thresholds applied); Rcoloc, Pearson's correlation coefficient for all pixels in which both BDA and VGLUT2 channels are above their respective threshold; tM-BDA, tM-VGLUT2, Mander's split channel colocalization coefficient using threshold.