Stereotaxic Exposure of the Central Nucleus of the Amygdala to Corticosterone Increases Colonic Permeability and Reduces Nerve-Mediated Active Ion Transport in Rats

Abstract

Background: Irritable bowel syndrome (IBS) is characterized by visceral pain and abnormal bowel habits that are worsened during stress. Evidence also suggests altered intestinal barrier function in IBS. Previously, we demonstrated that stereotaxic application of the stress hormone corticosterone (CORT) onto the central nucleus of the amygdala (CeA) induces colonic hyperalgesia and anxiety-like behavior in a rat model, however the effect on intestinal permeability and mucosal function remain to be evaluated. Methods: Male Fischer 344 rats underwent bilateral stereotaxic implantation of CORT or inert cholesterol (CHOL)-containing micropellets (30 μg) onto the dorsal margin of the CeA. Seven days later, colonic tissue was isolated to assess tissue permeability in modified Ussing chambers via transepithelial electrical resistance (TEER) and macromolecular flux of horseradish peroxidase (HRP). Secretory responses to electrical field stimulation (EFS) of submucosal enteric nerves as well as activation with forskolin were used to assess movements of ions across the isolated colonic tissues. In a separate cohort, colonic histology, and mast cell infiltration was assessed. Key Results: Compared to CHOL-implanted controls, we determined that exposing the CeA to elevated levels of CORT significantly increased macromolecular flux across the colonic epithelial layer without changing TEER. Nerve-mediated but not cAMP-mediated active transport was inhibited in response to elevated amygdala CORT. There were no histological changes or increases in mast cell infiltration within colonic tissue from CORT treated animals. Conclusion and Inferences: These observations support a novel role for the CeA as a modulator of nerve-mediated colonic epithelial function.

Keywords: amygdala; colon; corticosterone; permeability; rat; stress.

Figure 1

Elevated amygdala CORT significantly increases macromolecular flux across the colonic epithelium. (A) Experimental design: 32 male rats were randomly assigned to receive stereotaxic implantation of micropellets containing CORT or CHOL on the dorsal margin of the CeA. After a 7-day recovery, n = 10 rats (5 rats/treatment) were euthanized for tissue collection for evaluation of colonic histology, mast cell infiltration, and tight junction protein expression; n = 22 rats (11 rats/treatment) were euthanized and the colon was used for evaluation of mucosal permeability, macromolecular flux, neural mediated secretion, or receptor independent secretion. (B) Representative schematic of CORT or CHOL micropellet localization based upon the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 2014). (C) Amygdala CORT did not affect colonic TEER (Mann-Whitney U = 88.5, p = 0.339) compared to CHOL treated controls (n = 5–6/group, 14–16 viable Ussing Chambers samples/treatment). (D) CORT implantation onto the amygdala significantly increases the flux of HRP across the colonic epithelium (Mann–Whitney U = 16, **p = 0.0024) (CHOL n = 5, 11 preparations; CORT n = 4, 11 preparations). Values are mean ± SEM.

Figure 2

Active transport following electrical field stimulation is significantly inhibited in CORT implanted animals. Following EFS at frequencies of 32 and 64 Hz colonic tissue from CORT treated animals exhibited an attenuated Isc (Bonferroni post-hoc test, **p < 0.01) compared to CHOL treated controls. Values are expressed as mean ΔI_sc ± SEM following each stimulation pulse. Analyzed using 2-factor repeated measures ANOVA. (CHOL n = 4 rats, 8 chambers; CORT n = 5 rats, 14 chambers).

Figure 3

CORT and CHOL treated animals exhibit similar cellular responses to increased cAMP induced by forskolin. The cellular response to mucosal and serosal administration of forskolin (10 μM) to the baths is not significantly different between CORT and CHOL implanted animals (t = 0.005, df = 10, p = 0.996, n = 2 rats, 6 chambers/treatment). Values are expressed as mean ΔI_sc ± SEM.

Figure 4

Histological appearance and mast cell infiltration following CORT or CHOL amygdala implants onto the CeA. (A,B) Representative H&E stained colon section. A score of zero was given across to all specimens across both treatment groups, indicating no mucosal damage in CORT or CHOL amygdala implanted animals. (C–E) Representative toluidine blue stained colon section. Mast cell infiltration was not increased following exposure of the CeA to CORT compared to CHOL treated controls (Mann–Whitney U = 5, p = 0.429, n = 4/treatment). Values are mean ± SEM.