A Genetic Approach to Pinning Down the Wanderer: Mapping Vagal Afferent Innervation of the Mouse Intestinal Mucosa
Vagal afferents that supply the small intestine mucosa play a key role in signaling satiation and regulating digestion. Little is known about the morphology or distribution of this innervation. A major obstacle has been the ability to selectively label a large proportion of vagal mucosal afferents independent of other extrinsic and intrinsic gastrointestinal tract innervation. A recent breakthrough revealed that Nav1.8Cre- Tomato control (CON) mice express Tomato protein in ~80% nodose and dorsal root ganglion (DRG) cells and in mucosal afferents throughout the intestines (Gautron, 519:3085, 2011). Subdiaphragmatic vagotomy in CON mice (VAGX) resulted in a complete loss of this mucosal innervation, suggesting the tdTomato innervation was all of vagal origin. However, Gautron and others relied on visualization of native Tomato fluorescence and qualitative analysis. To quantitatively explore the distribution, more thoroughly evaluate the vagal origin, and examine the morphology of tdTomato- containing nerve terminals throughout the entire small intestine, innervation at 13 sites along the small intestine of CON and VAGX mice were compared. Two different sham approaches, varying in degrees of invasiveness, were also utilized to further explore and evaluate the vagal origin and distribution. After perfusion fixation, 1 cm length blocks of small intestine tissue from a random subset of 5 out of the 13 sampling sites from each mouse were harvested, frozen, cross-sectioned, and stained. The tomato protein was stained to amplify its native fluorescence signal for more sensitive visualization. CON, VAGX, and sham tissue was harvested, processed, and mucosal innervation quantified in parallel. Several features of nerve terminals in villi or adjacent to crypts were quantified blind. VAGX mice had a significant reduction in the mean number of axons crossing a gridline at the base of a villus, of terminal branch crossings at a mid-height villus gridline, and of crypts in close apposition with nerve fibers compared to CON and sham mice. There was not a significant difference between CON and sham mice in any of these measures. Both CON and sham experimental conditions exhibited a large proximal to distal decrease in all measures, while the surviving label in the VAGX experimental condition was stable along the entire small intestine length. These findings suggest labeled afferents in the small intestinal mucosa are mainly vagal in origin, especially in the proximal afferents which are crucial for regulating food intake. Thus, this mouse model will be valuable for studying the morphology, plasticity, and function of vagal mucosal afferents at all levels of the small intestine.
Fox, Purdue University.
Off-Campus Purdue Users:
To access this dissertation, please log in to our