Volume 124, Issue 1, Pages 129-139 (January 2003) Localization of orexin-1 receptors to vagal afferent neurons in the rat and humans  Galina Burdyga, Simon Lal, David Spiller, Wen Jiang, David Thompson, Stephen Attwood, Shakeel Saeed, David Grundy, Andrea Varro, Rod Dimaline, Graham J. Dockray  Gastroenterology  Volume 124, Issue 1, Pages 129-139 (January 2003) DOI: 10.1053/gast.2003.50020 Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 1 RT-PCR showing orexin receptor expression in rat and human nodose ganglia and vagal nerve trunk. (A) Rat nodose ganglion, (B) rat vagal nerve trunk, (C) human nodose ganglion, and (D) human vagal nerve trunk. Primers specific for OX-R1 and the vanilloid receptor (VR1) revealed bands of the predicted size in rat and primers specific for OX-1 and OX-R2 yielded products in human samples. Primers specific for the orexin precursor did not reveal a band in either rat or human material. Predicted sizes shown by arrows to the left. There was no product in reactions without template (H2O). Gastroenterology 2003 124, 129-139DOI: (10.1053/gast.2003.50020) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 2 Localization of OX-R1 in rat and human nodose ganglia. (A) In rats nodose ganglion immunoreactive OX-R1 neurons (arrowheads) are found throughout the ganglion. (B) In humans nodose ganglion large immunoreactive nerve cell bodies (arrowhead) are found dispersed through the ganglion. Bars: A, 100 μm; B, 150 μm. Gastroenterology 2003 124, 129-139DOI: (10.1053/gast.2003.50020) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 3 Colocalization of OX-R1 in rat nodose ganglia with Ob-R, CCKA, CART, or CGRP. Panels on left (A, D, G, and J) show individual neurons expressing OX-R1. Middle panels show the corresponding neurons exhibiting Ob-R (B), CCKA (E), CART (H), or CGRP (K) immunoreactivities. Panels on right (C, F, I, and L) show overlay of the relevant left and center panels. Note in panels J–L there is a relatively uncommon neuron expressing both OX-R1 and CGRP. Bars: 10 μm. Gastroenterology 2003 124, 129-139DOI: (10.1053/gast.2003.50020) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 4 Relative abundance of neurons coexpressing OX-R1, Ob-R, CCKA, CART, and CGRP in rat and human nodose ganglia. Data were obtained as the total number of cells in well-oriented sections passing through the caudal and middle regions of the rat (left) and human (right) ganglia, stained with the combinations of antibody indicated. They are expressed as the percentage of the immunoreactive neurons assigned to the 2 regions and to 1 of 3 classes of neuron: OX-R1 alone, OX-R1, and either (A, E) Ob-R, (B, F) CCKA, (C, G) CART, or (D, H) CGRP or to Ob-R, CCKA, CART, or CGRP alone. Note approximately similar numbers of immunoreactive cells in the 2 regions of the ganglia in both species; most OX-R1 immunoreactive cells also express Ob-R, CCKA, and CART immunoreactivity. In man, but not rat, most OX-R1 immunoreactive neurons also express CGRP. Results are based on counts of 200–300 neurons in rat and 350–450 neurons in human samples, in each case, and are means ± standard error for n = 3–6. Gastroenterology 2003 124, 129-139DOI: (10.1053/gast.2003.50020) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 5 Colocalization of OX-R1 in human nodose ganglia with Ob-R, CCKA, CART, or CGRP. Panels on left (A, D, G, and J) show individual neurons expressing OX-R1. Middle panels show the corresponding neurons exhibiting (B) Ob-R, (E) CCKA, (H) CART, or (K) CGRP immunoreactivity. Panels on right (C, F, I, and L) show overlay of the relevant left and center panels. Bars: 25 μm. Gastroenterology 2003 124, 129-139DOI: (10.1053/gast.2003.50020) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 6 Colocalization of OX-R1 in human nodose ganglia with GFAP and VR1. Staining of glial cells with (A) GFAP identifies dense networks surrounding a (B) large OX-R1 immunoreactive neuron (open arrowhead), and shows (C) OX-R1 immunoreactivity in GFAP-positive cells (closed arrows). (D) Staining of glial cells with GFAP confirms the presence of (E) dense networks surrounding large neurons identified by neurone-specific enolase and (F) no overlap in the 2 markers. (G) Staining with VR1 identified chains of small neurons (VR1 green, cell nuclei stained red with propidium idodide). (H) At higher power 3 VR1-positive cells are shown, one of which exhibits (I) OX-R1 immunoreactivity; (J) overlay of H and I. Bars: A–G, 25 μm; H–J, 10 μm. Gastroenterology 2003 124, 129-139DOI: (10.1053/gast.2003.50020) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 7 Inhibition of CCK-stimulated jejunal afferent nerve discharge by orexin-A. (A) Representative mesenteric afferent nerve response to CCK8 (100 pmol IV, administered at ↓). Nerve discharge is displayed in the upper panel; the uppermost trace represents activity of a single nerve unit from the time point on the whole-nerve activity trace denoted by the dotted lines. The activity of this single unit is quantified in the rate histogram in the lower panel. (B) Representative trace of mesenteric afferent nerve discharge in response to CCK8 after prior administration of orexin-A (10 nmol, IV). Single unit discharge is again quantified in the rate histogram in the lower panel. (C) Summary of data showing the effect of CCK alone (open bars) and the effect of orexin-A on responses to CCK (shaded bars) on whole-nerve discharge with regard to the duration of response, peak afferent nerve discharge, and mean afferent discharge. Note that this is normalized data showing the relative magnitude of the response to CCK after orexin compared with vehicle treatment. Results are means ± SE, n = 5–8. (*P < 0.05 vs. control, Mann-Whitney rank sum test.) Gastroenterology 2003 124, 129-139DOI: (10.1053/gast.2003.50020) Copyright © 2003 American Gastroenterological Association Terms and Conditions