Elyanne M. Ratcliffe

Physiological Modulation of Intestinal Motility by Enteric Dopaminergic Neurons and the D2 Receptor: Analysis of Dopamine Receptor Expression, Location, Development, and Function in Wild-Type and Knock-Out Mice

Dopaminergic neurons are present in both plexuses of the murine bowel and are upregulated after extrinsic denervation but play unknown roles in enteric nervous system (ENS) physiology. Transcripts encoding dopamine (DA) receptors D1–D5 were analyzed by reverse transcription-PCR in stomach ≈ duodenum ≈ ileum ≈ proximal ≫ distal colon. Dissected muscle and myenteric plexus contained transcripts encoding D1–D3 and D5, whereas mucosa contained D1 and D3–D5. D1–D5 expression began in fetal gut [embryonic day 10 (E10)], before the appearance of neurons (E12), and was sustained without developmental regulation through postnatal day 1. In situ hybridization revealed that subsets of submucosal and myenteric neurons contained mRNA encoding D2 or D3. Immunoblots confirmed that D1, D2, and D5 receptor proteins were present from stomach through distal colon. Subsets of submucosal and myenteric neurons were also D1, D2, or D3 immunoreactive. When double labeled by in situ hybridization, these neurons contained mRNA encoding the respective receptors. Total gastrointestinal transit time (TGTT) and colonic transit time (CTT) were measured in mice lacking D2, D3, or D2 plus D3. Both TGTT and CTT were decreased significantly (motility increased) in D2 and D2 plus D3, but not D3, knock-out animals. Mice lacking D2 and D2 plus D3 but not D3 were smaller than wild-type littermates, yet ate significantly more and had greater stool frequency, water content, and mass. Because motility is abnormal when D2 is absent, the net inhibitory DA effect on motility is physiologically significant. The early expression of DA receptors is also consistent with the possibility that DA affects ENS development.

Intestinal microbiota influence the early postnatal development of the enteric nervous system

Normal gastrointestinal function depends on an intact and coordinated enteric nervous system (ENS). While the ENS is formed during fetal life, plasticity persists in the postnatal period during which the gastrointestinal tract is colonized by bacteria. We tested the hypothesis that colonization of the bowel by intestinal microbiota influences the postnatal development of the ENS.

Of the bugs that shape us: maternal obesity, the gut microbiome, and long-term disease risk

Chronic disease risk is inextricably linked to our early-life environment, where maternal, fetal, and childhood factors predict disease risk later in life. Currently, maternal obesity is a key predictor of childhood obesity and metabolic complications in adulthood. Although the mechanisms are unclear, new and emerging evidence points to our microbiome, where the bacterial composition of the gut modulates the weight gain and altered metabolism that drives obesity. Over the course of pregnancy, maternal bacterial load increases, and gut bacterial diversity changes and is influenced by pre-pregnancy- and pregnancy-related obesity. Alterations in the bacterial composition of the mother have been shown to affect the development and function of the gastrointestinal tract of her offspring. How these microbial shifts influence the maternal–fetal–infant relationship is a topic of hot debate. This paper will review the evidence linking nutrition, maternal obesity, the maternal gut microbiome, and fetal gut development, bringing together clinical observations in humans and experimental data from targeted animal models.

Development of the Vagal Innervation of the GUT: Steering the Wandering Nerve

Background  The vagus nerve is the major neural connection between the gastrointestinal tract and the central nervous system. During fetal development, axons from the cell bodies of the nodose ganglia and the dorsal motor nucleus grow into the gut to find their enteric targets, providing the vagal sensory and motor innervations respectively. Vagal sensory and motor axons innervate selective targets, suggesting a role for guidance cues in the establishment of the normal pattern of enteric vagal innervation.

Laminin terminates the Netrin/DCC mediated attraction of vagal sensory axons

Vagal sensory axons navigate to specific sites in the bowel during fetal life. Netrin/deleted in colorectal cancer (DCC) were found to mediate the attraction of vagal sensory axons to the fetal mouse gut. We tested the hypothesis that laminin‐111 can reverse the chemoattractive effects of netrin and act as a stop signal for vagal sensory axons. Laminin‐111‐expressing cells were located in the E12 and E16 mouse bowel by in situ hybridization. At E12, these cells extended centrifugally from the endoderm; by E16, laminin‐111 expressing cells were found in the mucosa and outer gut mesenchyme. A similar pattern was seen in preparations of E13 and E15 mouse gut labeled with antibodies to laminin. Application of DiI to nodose ganglia identified vagal sensory axons growing into the fetal bowel. These terminals were found to avoid concentrations of laminin or to terminate at laminin‐delimited boundaries. Soluble laminin inhibited the preferential growth of nodose neurites toward netrin‐secreting cells (p < 0.01). This effect was mimicked by a peptide, YIGSR, a sequence within the β1 chain of laminin‐111 (p < 0.004) and antagonized by a peptide, IKVAV, a sequence within the α1 chain of laminin‐111. Antibodies to β1‐integrins were also able to reverse the inhibitive effects of laminin and restore the attraction of nodose neurites towards netrin‐1‐secreting cells. Soluble laminin inhibited the preferential growth of nodose neurites toward a cocultured explant of foregut. These findings suggest that laminin terminates the attraction of vagal sensory axons towards sources of netrin in the developing bowel.

CHOLINE ACETYLTRANSFERASE (CHAT) IMMUNOREACTIVITY IN PARAFFIN SECTIONS OF NORMAL AND DISEASED INTESTINES

There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin-streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6-24 hr of fixation in neutral buffered formalin and overnight incubation in 1 μg/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprungs disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders.

Enteric neurons synthesize netrins and are essential for the development of the vagal sensory innervation of the fetal gut

During fetal life, vagal sensory fibers establish a reproducible distribution in the gut that includes an association with myenteric ganglia. Previous work has shown that netrin is expressed in the bowel wall and, by acting on its receptor, deleted in colorectal cancer (DCC), mediates the guidance of vagal sensory axons to the developing gut. Because the highest concentration of netrins in fetal bowel is in the endoderm, we tested the hypothesis that the ingrowth of vagal afferents to the gut would be independent of the presence of enteric neurons, although enteric neurons might influence the internal distribution of these fibers. Surprisingly, experiments indicated that the vagal sensory innervation is intrinsic neuron‐dependent. To examine the vagal innervation in the absence of enteric ganglia, fetal Ret −/− mice were labeled by applying DiI bilaterally to nodose ganglia. In Ret −/− mice, DiI‐labeled vagal sensory axons descended in paraesophageal trunks as far as the proximal stomach, which contains neurons, but did not enter the aganglionic bowel. To determine whether neurons produce netrins, enteric neural‐crest‐derived cells (ENCDCs) were immunoselected from E15 rat gut. Transcripts encoding netrin‐1 and ‐3 were not detected in the ENCDCs, but appeared after they had given rise to neurons. When these neurons were cocultured with cells expressing c‐Myc‐tagged netrin‐1, the neurons displayed netrin‐1, but not c‐Myc, immunoreactivity. Enteric neurons thus synthesize netrins. The extent to which neuronal netrin accounts for the dependence of the vagal sensory innervation on intrinsic neurons, remains to be determined.

Molecular development of the extrinsic sensory innervation of the gastrointestinal tract

The extrinsic sensory innervation of the gastrointestinal tract is the conduit through which the gut and the central nervous system communicate. The hindbrain receives information directly from the bowel via the vagus nerve, while information from spinal afferents arrives in the central nervous system through the dorsal root ganglia. This review focuses on the molecular development of these vagal and spinal innervations, with an emphasis on mechanisms that involve axon guidance. During development, axons from both the nodose ganglia and dorsal root ganglia grow into the gut, innervate their appropriate enteric targets and avoid inappropriate cells in the gut wall. These developmental outcomes suggest that both attractive and repellent molecules are important in establishing the normal pattern of the extrinsic sensory innervation. In the fetal mouse gut, the guidance of vagal sensory axons is mediated by axon guidance molecules, such as netrin and the netrin receptor, deleted in colorectal cancer (DCC), as well as extracellular matrix molecules, such as laminin-111. Dorsal root ganglion neurons are known to express, and their axons to respond to, axon guidance molecules. The question of whether or not these molecules are involved in guiding spinal afferents to the bowel, however, has not yet been examined. It is anticipated that a better understanding of how vagal and spinal afferents innervate the fetal gut and reach specific enteric locations will provide deeper insights into the underlying mechanisms of normal and abnormal sensation from the gastrointestinal tract.

Slit/Robo-mediated chemorepulsion of vagal sensory axons in the fetal gut.

Background: The vagus nerve descends from the brain to the gut during fetal life to reach specific targets in the bowel wall. Vagal sensory axons have been shown to respond to the axon guidance molecule netrin and to its receptor, deleted in colorectal cancer (DCC). As there are regions of the gut wall into which vagal axons do and do not extend, it is likely that a combination of attractive and repellent cues are involved in how vagal axons reach specific targets. We tested the hypothesis that Slit/Robo chemorepulsion can contribute to the restriction of vagal sensory axons to specific targets in the gut wall. Results: Transcripts encoding Robo1 and Robo2 were expressed in the nodose ganglia throughout development and mRNA encoding the Robo ligands Slit1, Slit2, and Slit3 were all found in the fetal and adult bowel. Slit2 protein was located in the outer gut mesenchyme in regions that partially overlap with the secretion of netrin‐1. Neurites extending from explanted nodose ganglia were repelled by Slit2. Conclusions: These observations suggest that vagal sensory axons are responsive to Slit proteins and are thus repelled by Slits secreted in the gut wall and prevented from reaching inappropriate targets. Developmental Dynamics 242:9–15, 2013.

Endoscopic visualization of a gastric polypoid mass: a rare pediatric presentation of an inflammatory myofibroblastic tumor

An 11-year-old girl was referred to the Pediatric Gastronterology Clinic for assessment of a potential GI etiology f a 5-year history of iron deficiency anemia. On funcional inquiry, she reported fatigue, poor weight gain, and ccasional abdominal discomfort. She denied any other igns or symptoms of GI disease. On physical examination, she was noticeably pale with eight and height at the fifth percentile for her age. Her eneral physical examination was unremarkable. There as no lymphadenopathy and no palpable abdominal ass. Upper endoscopy visualized an unexpected polypoid ass with an ulcerated surface that emanated from the tomach wall (Fig. 1). A CT of the chest and abdomen efined a 4.7 3.8 3.5-cm mass originating from the esser curvature of the stomach, extending into the lumen, nd a 2.2 1.1-cm mass between the gastric angle and the iver (Fig. 2). Both masses contained coarse calcifications. djacent bulky lymphadenopathy was noted in the gasrohepatic and gastrosplenic ligament. After consultations ith pediatric surgery and pediatric oncology, the patient nderwent a distal gastrectomy and gastroduodenostomy. he main differential diagnosis included inflammatory yofibroblastic tumor (IMT), leiomyoma, leiomyosaroma, GI stromal tumor, and neural tumor. Gross pathologic inspection found a large submucosal ass adherent to the anterior wall of the stomach. On icroscopy, the tumor was composed of spindle cells, eavily infiltrated by lymphoplasmacytic cells, and inluded sclerotic areas with calcifications (Fig. 3). The spin-