Fig. 3

Expression of CGRP after chronic NTG administration. A Schematic illustration of the rostro-caudal anatomical location of CeA relative to the position of bregma. B Representative images of CGRP expression in the CeA after chronic NTG administration. Red dashed square indicates the high magnification of the CeA (left). The CGRP fiber intensity in the CeA after chronic NTG induction was significantly higher than that in the control group (right, n = 4 per group; CeA_R; p = 0.003; CeA_L; p = 0.0003). Scale bar: 1 mm (left), 50 μm (right). C The representative pERK positive neurons double-labeled with CGRP fibers. Scale bar: 100 μm. D Representative low and high magnification images of co-labeled CGRP receptor type 1 (CGRPR)- and PKC-δ- positive neurons in the CeA (left) and quantified results (right, n = 4 per group; CGRPR/PKC-δ; p = 0.593; PKC-δ/CGRPR; p = 0.687). Scale bar: 50 μm. E Representative low magnification images of co-labeled CGRP- and pERK- positive neurons in the PBN. Scale bar: 500 μm. F Representative low and high magnification images of co-labeled CGRP- and pERK- positive neurons in the PBN (left). The CGRP positive neurons in the PBN was significantly higher after chronic NTG administration than controls (right, n = 4 per group; CGRP/pERK; p < 0.0001; pERK/CGRP; p = 0.002). Scale bar: 100 μm. G Representative images of bilateral retrograde CTB594 labeling and co-labeled CGRP fibers in the CeA. Scale bar: 100 μm. H Representative images of CTB594 co-labeled CGRP positive neurons in the PBN, confirming that the CGRP positive neurons project from the PBN to CeA (left). The CGRP positive neurons in the PBN was significantly higher after chronic NTG administration than controls (right, n = 4 per group; CTB594/CGRP; p = 0.002; CGRP/CTB594; p = 0.0006). Scale bar: 100 μm. All data shown are mean ± SEM and analyzed by Mann-Whitney U test. Significance levels set at p < 0.05 *, p < 0.01 **, and p < 0.001 ***