Mice deficient in brain-derived neurotrophic factor have altered development of gastric vagal sensory innervation

Abstract

Vagal sensory neurons are dependent on neurotrophins for survival during development. Here, the contribution of brain-derived neurotrophic factor (BDNF) to survival and other aspects of gastric vagal afferent development was investigated. Post-mortem anterograde tracing with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate (DiI) was used to label selectively vagal projections to the stomach on postnatal days (P) 0, 3, 4, and 6 in wild types and heterozygous or homozygous BDNF mutants. Sampling sites distributed throughout the ventral stomach wall were scanned with a confocal microscope, and vagal axon bundles, single axons, putative mechanoreceptor precursors (intraganglionic laminar endings, IGLEs; intramuscular arrays, IMAs), and efferent terminals were quantified. Also, myenteric neurons, which are innervated by IGLEs, were stained with cuprolinic blue and counted. Quantitative comparisons across wild-type stomach compartments demonstrated that the adult distribution of IMAs was not present at P0 but began to form by P3-6. Among all the quantified elements, at P0, only IGLE density was significantly different in homozygous mutants compared with wild types, exhibiting a 50% reduction. Also, antrum innervation appeared disorganized, and some putative IMA precursors had truncated telodendria. At P3-6, the effect on IGLEs had recovered, the disorganization of antrum innervation had partially recovered, and some IMA telodendria were still truncated. The present results suggest that gastric IGLEs are among the vagal sensory neurons dependent on BDNF for survival or axon guidance. Alternatively, BDNF deficiency may delay gastric IGLE development. Also, BDNF may contribute to IMA differentiation and patterning of antral vagal innervation.

Fig. 1

(A) A schematic diagram that illustrates the DiI (1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) application site. A single DiI crystal was applied to the anterior vagal trunk at the bifurcation of the hepatic and gastric branches as they course along the abdominal esophagus. This location was determined to be optimal for comprehensive labeling of vagal innervation of the stomach wall (Murphy and Fox, 2007). (B) A schematic diagram adapted from Swithers et al. (2002) that illustrates where the estimated boundaries of three major stomach compartments were located in early postnatal mice and how the sampling sites were distributed among them. The antrum included columns 1–3 (light shading), the corpus columns 4–6 (dark shading), and the forestomach columns 7–10 (medium shading). (C) A schematic diagram that illustrates the sampling and counting method modified from Wang and Powley (2000) and Fox et al. (2000). Based on length and width of each ventral stomach wall sampled, eighty sampling points were established at the intersections of a sampling grid that consisted of 8 equidistant rows and 10 equidistant columns. The microscope objective was systematically moved from one intersection to the next, and the underlying tissue imaged. Then a counting grid was laid over each successive optical section of the z-series collected at each sampling site and used to quantify vagal fibers, bundles and terminals. Sampling points with less than half of the imaged area containing stomach tissue were not included in the quantification and are represented by gray circles in the sampling grid portion of the schematic.

Fig. 2

Putative Intraganglionic laminar ending (IGLE; A,B) and intramuscular array (IMA; C,D,E) precursors and mucosal/submucosal processes and terminals (F) labeled with DiI in the stomach wall of wild-type P0 mice. (A) A confocal image illustrating putative IGLE precursors (in outlined area) in the forestomach. At this age IGLEs are small and often consist of only a few terminal processes that vary from small puncta to relatively large growth cone-like structures. (B) Higher magnification image of the area outlined in A that consists of optical sections restricted to a tissue plane immediately below a myenteric ganglion that contains the putative IGLE leaves (arrows). (C) A confocal image taken from the forestomach that includes optical sections through the longitudinal muscle, myenteric plexus and circular muscle layers that contain IMAs and myenteric vagal axons and fiber bundles. (D) A confocal image of the same region as in C, but optical sections were restricted to the circular muscle layer containing putative IMA precursors (arrows). (E) A confocal image of the same region as in C and D, but optical sections were restricted to the longitudinal muscle layer containing putative IMAs (arrows). (F) Vagal fibers (arrows) and free endings (arrowheads) with imaging restricted to the mucosal and submucosal layers of the forestomach. Scale bars = 25 μm (A, C–F) or 10μm (B).

Fig. 3

The organization of vagal fibers and myenteric axon bundles is different in each of the three major stomach compartments at P0. (A–C) Confocal images of DiI labeled vagal bundles (arrows), fibers (smaller open arrows), and efferent terminals in myenteric ganglia (arrowheads) within the antrum (A), forestomach (B), and corpus (C). In the antrum fiber bundles were large in diameter and along with efferent terminals were tightly packed, whereas in the forestomach axon bundles were smaller in diameter and along with fibers were more loosely organized. Organization of fibers, axon bundles and efferents and in the corpus was similar to the forestomach, but additionally large diameter bundles extended from the cardia. Scale bars = 25μm.

Fig. 4

Quantitative comparisons of DiI-labeled vagal elements among wild types and heterozygous or homozygous bdnf mutants at P0. Elements were quantified over the entire ventral stomach wall and included axon bundle number (A), individual fiber density (B), putative IGLE precursor number (C), putative IMA precursor density (D), and efferent terminal number (E). IMA density in D plots circular and longitudinal IMAs separately and combined (total); the histogram bar shading represent the genotypes in a similar manner as in the other panels. The only significant difference was a 50% reduction of putative IGLEs in bdnf −/− mice compared with wild types. * Significantly different from wild type, p<0.05.

Fig. 5

At P0, bdnf KO was associated with altered vagal innervation of the antrum (A,B) and changes in the morphology of developing putative IMAs in the forestomach (C,D). (A,B) In bdnf KO mice vagal innervation of the antrum appeared to become disorganized as compared with wild types and heterozygous mutants. In particular, spacing between myenteric ganglia was reduced and there was an increase of growth cone-like structures. Confocal images illustrating the normal organization of vagal fibers, bundles and ganglia in the antrum of a wild-type mouse (A), and the apparent disorganization of these elements in a bdnf KO mouse (B). (C) Confocal image that includes optical sections restricted to the circular muscle layer, demonstrating normal IMA structure (arrows) observed in the forestomach of a wild-type mouse. Normal IMA telodendria are long rectilinear processes. (D) Similarly restricted confocal image as in C, illustrating the shorter, larger diameter IMA telodendria (arrows) present in some regions of the forestomach of a bdnf KO mouse. Scale bars = 25μm.

Fig. 6

Putative IGLE (A,B) and IMA (C,D,E) precursors and mucosal/submucosal processes and terminals (F) labeled with DiI in the stomach wall of wild-type P3-6 mice. (A) A confocal image illustrating putative IGLE precursors (in outlined area) in the forestomach. At this age IGLEs were slightly larger than at P0 and often began to take on a more laminar appearance, but otherwise had not changed significantly in structure. (B) Higher magnification image of the area outlined in A that consists of optical sections restricted to the tissue plane that contained the putative IGLE leaves, which was immediately below a myenteric ganglion. (C) A confocal image taken from the forestomach that includes optical sections through the longitudinal muscle, myenteric plexus and circular muscle layers and thus contains putative IMAs and vagal axons and fiber bundles. At this age IMA telodendria appeared to have increased in number and length as compared with P0. (D) A confocal image of the same region as in C, but optical sections were restricted to the circular muscle layer containing putative IMA precursors (arrows). (E) A confocal image of the same region as in C and D, but optical sections were restricted to the longitudinal muscle layer containing putative IMAs (arrows). (F) Vagal fibers (arrows) with imaging restricted to the mucosal and submucosal layers of the forestomach. Scale bars = 25μm (A, C–F) or 10μm (B).

Fig. 7

The organization of vagal fibers and myenteric axon bundles was different in each of the three major stomach compartments at P3-6. (A–C) Confocal images of DiI labeled vagal bundles (arrow), fibers (open arrow), and efferent terminals in myenteric ganglia (arrowheads) within the antrum (A), forestomach (B), and corpus (C). Although the vagal innervation pattern in each stomach compartment had matured slightly by P3-6 as compared with P0, the differences described between compartments at P0 were largely maintained at P3-6. Scale bars = 25μm.

Fig. 8

Quantitative comparisons of DiI-labeled vagal elements among the antrum, corpus and forestomach of wild-type mice at P3-6. The quantified elements included axon bundle number (A), individual fiber density (B), putative IGLE precursor number (C), putative IMA precursor density (D), and efferent terminal number (E). The forestomach contained more fiber bundles than the antrum (*, p<0.05). Moreover, the approximate doubling of IMA density in the forestomach as compared with the corpus and antrum was significant for both of these compartments (#, p<0.05).

Fig. 9

At P3-6, bdnf KO was associated with altered vagal innervation of the antrum (A,B) and changes in the morphology of developing putative IMAs in the forestomach (C). In bdnf KO mice vagal innervation of the antrum still appeared to be disorganized and denser compared with wild types and heterozygous mutants, but to a lesser degree that observed at P0. (A,B) Confocal images illustrating these differences between the antrum of a wild-type mouse (A), and a bdnf KO mouse (B). Also, in P3-6 bdnf KO mice, some IMA telodendria were stunted, but normal diameter. (C) Confocal image that includes optical sections restricted to the longitudinal muscle layer, illustrating the shorter IMA telodendria (arrows) observed in the forestomach of a bdnf KO mouse. To compare these IMAs with those of a wild type see Fig. 6E, which is a similarly restricted confocal image from a wild-type at this age. Scale bars = 25μm.

Fig. 10

Analysis of gastric myenteric neurons. (A) Brightfield photomicrograph of a myenteric ganglion in the stomach muscle wall of a wild-type P0 mouse stained with cuprolinic blue (three neuron cell bodies are indicated by arrows). The differentiation of the stain from background was not as prominent as in the adult GI muscle wall, but was sufficient to identify neurons according to the criteria. Scale bar = 25μm. (B,C) Quantification of myenteric neurons stained with cuprolinic blue at P0 among wild types and heterozygous or homozygous bdnf mutants, grouped by whole stomach (B) or by stomach compartment (C; antrum, corpus and forestomach). The histogram bar fills represent the genotypes in a similar manner as in B. When grouped by whole stomach neither heterozygous nor homozygous mutants were different from wild types (B). However, the decreased counts in heterozygotes compared with wild types were significantly different from the increased counts in homozygotes. This difference was mainly due to the significant difference between these groups in the antrum (C). # Significant difference between heterozygous and homozygous bdnf mutants at p<0.05 level.