Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract

Abstract

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.

Fig. 1

DiI-labeled vagal anterior gastric branches adjacent to the lower esophageal sphincter at P0 (A,B) and axons in the forestomach at E16.5 with axon bundles overexposed to permit visualization of individual fibers (C,D). A. A fluorescence photomicrograph illustrating the reduced visibility of labeled fibers 6 wk after application of dried DiI oil (Experiment 1). B. A confocal image illustrating the more rapid DiI diffusion, more complete labeling, and absence of DiI leakage 2.5 wk after DiI crystal application and use of EDTA in the fixative. C. Confocal image of DiI-labeled tissue mounted in 70% glycerol, 5% n-propyl gallate. D. A confocal image of DiI-labeled axons from a comparable stomach region to that shown in C, but imaged from a stomach mounted in PBS. Scale bars = 200 μm (A,B), and 25 μm (C,D).

Fig. 2

Confocal images illustrating the development of vagal axon bundles in the myenteric plexus of the forestomach. A. At E13.5 a plexus-like pattern started to emerge and axon bundles were small in diameter. B. At E14.5 axon bundle diameters and distances between axon bundles increased, the latter due to stomach growth. C. By E16.5 axon bundle diameters and interbundle distances had again increased; a putative IGLE precursor is present in the center of this image. Scale bars = 25 μm (A,B,C).

Fig. 3

A. DiI-labeled fibers in the myenteric plexus and putative IMA precursors in the underlying circular muscle. B. The same field as in A with imaging restricted to the circular muscle layer. DiI-labeled putative IMA precursors were present with two of their telodendria connected by a cross-bridge fiber (arrowhead). C. DiI-labeled fibers in the myenteric plexus and a putative IGLE precursor immediately below the plexus. D. The same field as in C with imaging restricted to the tissue plane immediately below the myenteric plexus, which contained the putative IGLE precursor. E. DiI-labeled fiber bundles and numerous putative efferent terminals in the myenteric plexus. F. The same field as in E with only the submucosa and mucosa imaged, which contained a network of fiber bundles, single axons and nerve terminals that originated in the myenteric plexus. All images were from P0 mice. Scale bars = 10 μm (A–D) or 100 μm (E,F).

Fig. 4

A. A small number of fibers encircled a single myenteric neuron soma (not stained) to form immature putative efferent terminals in a P0 stomach wall. Several growth cone-like structures were present (e.g., arrowheads), two contributing to the efferent terminals. B. Immature putative efferent terminals appeared to partially encircle a myenteric neuron soma (at center of image) that was lightly stained with one dendrite extending upward and another downward in a P0 stomach wall. This neuronal staining was probably due to transfer of DiI from the labeled terminals to their target cell, which occurred rarely. C. A small number of immature putative efferent terminals appeared to be forming where an axon bundle branched in a P0 duodenum. Two myenteric neurons were completely encircled by a small number of fibers, and 2–3 more appeared to be partially encircled. D. An image of forestomach bundles and numerous putative efferent terminal precursors at P0. These appeared to be fairly mature with several fibers surrounding each myenteric neuron and bundled tightly so that individual DiI-labeled fibers could not typically be distinguished. E. An image of a field of axon bundles and numerous efferent terminals covering a large proportion of the forestomach at P0, illustrating the completeness of DiI labeling. Scale bars =10 μm (A,B,C), 20 μm (D), and 100 μm (E).

Fig. 5

Postnatal maturation of putative IMA (A, C, and E) and IGLE (B, D, and F) precursors. A. Initial formation of a putative IMA precursor at P0. The individual axon shown here exited the myenteric plexus in the lower left and continued diagonally to the upper right within the smooth muscle layer giving rise to short neurites – some with small growth cone-like structures - that might represent the initial formation of an IMA or its telodendria (arrow, neurite enlarged in inset). The other axons present are in the myenteric plexus. B. Cluster of growth cone-like terminals that may represent an initial stage of IGLE formation at P0. C. A putative IMA precursor at P0 at a later stage of maturation compared with that shown in A. Two axons arose from the myenteric plexus (at left out of field of view), entered the longitudinal muscle layer, distributed additional axons and short rectilinear fibers forming IMA telodendria (e.g., arrows) that paralleled the muscle fibers, and an interconnecting crossbridge fiber (arrowhead). D. Putative IGLE precursor at P0 that may be more mature than the one illustrated in panel B. It exhibited more terminals and its morphology combined structural qualities of growth cones and terminal puncta. E. Putative IMA precursor at P0 in the circular muscle layer at a later stage of maturation as compared with that shown in C, in which the telodendria had lengthened and were connected by cross-bridge fibers (arrowhead). F. A putative IGLE precursor at P8 at a stage of maturation beyond that shown in B and D. There were more terminal structures that were more densely packed, and were arranged in several groups with some lying below the plane of the myenteric plexus, and the others lying above it, suggesting some developing IGLEs “surround” a myenteric ganglion. Scale bars = 10 μm (A–F) and 25 μm (inset, A).

Fig. 6

A. Vagal myenteric network of the esophagus labeled at P0. B. Individual labeled fibers with small growth-cone like structures in the esophageal myenteric plexus at P0. C. DiI-labeled myenteric fiber bundles, axons and putative efferent terminals in the duodenum at P0. D. Labeled fiber bundle, single axons and large growth-cone like structure in the duodenal myenteric plexus at P0. Scale bars = 20 μm (A – D).