Receptor-selective agonists induce emesis and Fos expression in the brain and enteric nervous system of the least shrew (Cryptotis parva)

Abstract

Research on the mechanisms of emesis has implicated multiple neurotransmitters via both central (dorsal vagal complex) and peripheral (enteric neurons and enterochromaffin cells) anatomical substrates. Taking advantage of advances in receptor-specific agonists, and utilizing Fos expression as a functional activity marker, this study demonstrates a strong, but incomplete, overlap in anatomical substrates for a variety of emetogens. We used cisplatin and specific agonists to 5-HT(3) serotonergic, D(2)/D(3) dopaminergic, and NK(1) tachykininergic receptors to induce vomiting in the least shrew (Cryptotis parva), and quantified the resulting Fos expression. The least shrew is a small mammal whose responses to emetic challenges are very similar to its human counterparts. In all cases, the enteric nervous system, nucleus of the solitary tract, and dorsal motor nucleus of the vagus demonstrated significantly increased Fos immunoreactivity (Fos-IR). However, Fos-IR induction was notably absent from the area postrema following the dopaminergic and NK(1) receptor-specific agents. Two brain nuclei not usually discussed regarding emesis, the dorsal raphe nucleus and paraventricular thalamic nucleus, also demonstrated increased emesis-related Fos-IR. Taken together, these data suggest the dorsal vagal complex is part of a common pathway for a variety of distinct emetogens, but there are central emetic substrates, both medullary and diencephalic, that can be accessed without directly stimulating the area postrema.

Figure 1. Examples of Fos immunoreactivity in the brainstem related to emetogen administration

In all Fos-IR panels, immunolabeling was performed on free-floating tissue sections using avidin-biotin-peroxidase amplification and nickel-enhanced DAB visualization. A) Coronal section through the DVC stained with cresyl violet. Note the different cytoarchitectonic details such as cell size and packing, which differentiate each subnucleus within the DVC. B) Sagittal section demonstrating Fos-IR in the DVC of a vehicle-injected shrew. Relatively few Fos-IR nuclei are found. C) The cisplatin-injected shrew DVC had numerous Fos-IR nuclei. Weakly labeled nuclei were not quantified. D) In a quinpirole-injected shrew, Fos-IR+ nuclei (the dark, filled ovals) were found through the NTS, but only infrequently in the AP. Abbreviations: 12 – hypoglossal nucleus; AP – area postrema; cc – central canal; DMNX – dorsal motor nucleus of the vagus; DVC – dorsal vagal complex; NTS – nucleus of the solitary tract. Scale bars: A, 200 μm; B-C, 100 m; D, 100 μm.

Figure 2. Examples of Fos and other immunohistochemical labeling related to emetogen administration

A-B) Examples of Fos-IR in the ENS following cisplatin administration. The thin layers of the nerve plexi prevented quantification of ENS labeling specific to submucosal versus myenteric labeling, so results are expressed for Fos-IR in both nerve plexi. C) Fos-IR in the PVT following cisplatin injection. The image is a sagittal section, with the ventricle to the right of the image. D) Sagittal section demonstrating Fos-IR in the DRN following cisplatin injection. In all of the above panels, immunolabeling was performed on free-floating tissue sections using avidin-biotin-peroxidase amplification and nickel-enhanced DAB visualization. E) Fos-IR in the DRN colocalized with 5-HT immunoreactivity. Grey 5-HT+ somata (arrow) sometimes had bright white, Fos-IR+ nuclei (arrowheads). In the upper left corner, a white nucleus with no 5-HT colocalization (a non-serotonergic raphe neuron) can be seen. F) Immunofluorescent labeling for SP fibers in the DRN overlaid onto a brightfield image of nickel-DAB-visualized Fos-IR from the same cisplatin-injected shrew demonstrates frequent fibers (arrow) coursing between, and making putative contacts with, Fos-IR neurons (arrowhead). SP immunolabeling indicated a dense fiber plexus in the DRN. For E and F, immunostaining was performed as above, but substituted fluorochrome-conjugated tyramide for nickel-enhanced DAB. Images were taken as fluorescent color images and converted to greyscale. G) SP-IR in the DVC of the least shrew visualized using nickel-enhanced DAB. The sagittal section demonstrates extremely dense fibers and putative terminals throughout the NTS, and to a lesser extent, in the AP. Abbreviations: 5-HT – serotonin; AP – area postrema; C – intestinal crypts; DRN – dorsal raphe nucleus; DVC – dorsal vagal complex; ENS – enteric nervous system; NTS – nucleus of the solitary tract; PVT – paraventricular thalamic nucleus; SP – substance P. Scale bars: A-D, 150 μm; E, 25 μm; F, 10 μm; G, 150 μm.