Evaluating the Role of the Endocannabinoid System in Axon Guidance: A Literature Review.

Introduction: The endocannabinoid system (ECS) mediates the actions of cannabis and has been implicated in playing critical roles in key developmental events, including axon guidance. Although several recent studies have demonstrated ECS involvement in neurodevelopment, an emphasis on its putative role in axon guidance has not been reviewed comprehensively. Objective: The purpose of this literature review is to evaluate the interrelationships between the ECS and axon guidance. Methodology: This literature review analyzes existing literature demonstrating the normal role of endocannabinoid (eCB) signaling in axon guidance, with evidence from diverse animal models. Studies were obtained from a search strategy involving terms related to the ECS and axon guidance, and cross-checking cited literature to ensure a complete evaluation. Discussion: Cannabinoid receptors, as well as eCB synthesis and degradation machinery, appear necessary for normal axon guidance during neurodevelopment. Genetic and/or pharmacological disruption of eCB signaling results in axon growth and guidance errors, implying high sensitivity to exogenous cannabinoids. Conclusion: Overall, this review highlights the intricate connections between the ECS and axon guidance in normal neurodevelopment. The mechanistic evidence discussed suggests that alterations of the ECS through genetic and pharmacological interference disrupt its normal functioning and by extension its normal role in regulating neural circuitry formation. A comprehensive understanding of this topic will be valuable in potentially uncovering the mechanisms responsible for the neurodevelopmental defects associated with pre-natal cannabis use.

Comparing the suitability of virtual versus in-person care: Perceptions from pediatricians.

OBJECTIVES: The COVID-19 pandemic compelled a portion of healthcare to be delivered virtually. As the pandemic waned, health systems strived to find a balance between re-incorporating in-person care while maintaining virtual care. To find when virtual or in-person encounters are more appropriate, we surveyed pediatricians' perceptions when comparing the suitability of virtual care to in-person care. METHODS: We surveyed a Canadian tertiary-level pediatric hospital where pediatricians assessed whether specific clinical encounters or tasks were more or less effective virtually than when performed in person. Pediatricians also rated the importance of clinical and patient factors when deciding if a patient needs to be seen in person. RESULTS: Of 160 pediatrics faculty members, 56 (35%) responded to the survey. When assessing different types of clinical encounters, triage, multidisciplinary meetings, discharge, and follow ups were more likely to favor virtual encounters. However, first consultations and family meetings were more likely to favor in-person encounters. Regarding clinical tasks, pediatricians were more likely to endorse explaining test results, offering treatment recommendations, and obtaining patient histories virtually. On the contrary, there was a preference for physical examinations, assessing patients visually, and assessing developmental milestones to be performed in person. When deciding if a patient should be seen in person versus virtual, pediatricians rated the patient's condition and communication barriers as the most important factors favoring an in-person appointment. DISCUSSION: These results offer an initial framework for pediatricians when choosing which encounter type may be most appropriate for their patients between virtual or in-person appointments.

Effects of in utero exposure to fluoxetine on the gastrointestinal tract of rat offspring.

Perinatal exposure to selective serotonin reuptake inhibitors (SSRIs) has been shown to disrupt the development of serotonergic signaling pathways in the brain and enteric nervous system. Serotonin (5-hydroxytryptamine; 5-HT) signaling is critical for gastrointestinal homeostasis; changes in 5-HT expression and regulation have been associated with gastrointestinal diseases of motility and inflammation. We tested the hypothesis that perinatal exposure to the SSRI fluoxetine can influence the development of the gastrointestinal tract in exposed offspring. Female nulliparous Wistar rats were given fluoxetine (10 mg/kg) or vehicle control from 2 wk before mating until weaning; small and large intestines of female and male offspring were collected at postnatal days 1, 21 (P1, P21, respectively), and 6 mo of age. In histological preparations, the proportion of serotonergic neurons significantly increased in the colons of both female and male fluoxetine-exposed compared with control offspring at P21, a time point that signifies maximal exposure to fluoxetine. At 6 mo of age, male but not female fluoxetine-exposed offspring had a significant increase in circulating 5-HT, with a significant decrease in transcripts encoding the 5-HT2A receptor and monoamine oxidase as compared with control offspring. Measurement of spatiotemporal mapping of contractile activity of the small and large intestine at 6 mo of age revealed no changes in motility in the small bowel of fluoxetine-exposed offspring but revealed a significant increase in the frequency of colonic contractions in the female fluoxetine-exposed compared with control animals. Susceptibility to inflammation was examined at 6 mo using the dextran sulfate sodium model of acute colitis. In utero exposure to fluoxetine was not found to exacerbate colitis severity. These findings suggest that fluoxetine exposure during fetal and early postnatal development can lead to changes in serotonergic neurons at the peak of exposure with sex-specific changes in 5-HT signaling and colonic motility in adulthood.NEW & NOTEWORTHY There is increasing recognition of the relevance of in utero and early postnatal exposures in the developmental programming of the gastrointestinal tract. Perinatal exposure to selective serotonin reuptake inhibitors and antidepressant medications is of particular relevance as they are commonly prescribed during pregnancy, and serotonergic pathways play key roles during gastrointestinal development and in postnatal homeostasis. Here, we provide a comprehensive evaluation of clinically relevant outcomes of gastrointestinal motility and susceptibility to colitis in fluoxetine-exposed offspring and highlight changes in colonic serotonergic neurons at the peak of perinatal fluoxetine exposure with sex-dependent changes in serotonin signaling and colonic motility in adulthood.

Who's talking to whom: microbiome-enteric nervous system interactions in early life.

The enteric nervous system (ENS) is the intrinsic nervous system of the gastrointestinal tract (GI) and regulates important GI functions, including motility, nutrient uptake, and immune response. The development of the ENS begins during early organogenesis and continues to develop once feeding begins, with ongoing plasticity into adulthood. There has been increasing recognition that the intestinal microbiota and ENS interact during critical periods, with implications for normal development and potential disease pathogenesis. In this review, we focus on insights from mouse and zebrafish model systems to compare and contrast how each model can serve in elucidating the bidirectional communication between the ENS and the microbiome. At the end of this review, we further outline implications for human disease and highlight research innovations that can lead the field forward.

Impact of antegrade enema initiation on healthcare utilization in pediatric patients: A population-based cohort study.

BACKGROUND: When constipation is refractory to first-line interventions, antegrade enema use may be considered. We aimed to assess the impact of this intervention on healthcare utilization. METHODS: We conducted a population-based, quasi-experimental study with pre-post comparison of the intervention group and a non-equivalent control group using linked clinical and health administrative data from Ontario, Canada. Subjects included children (0-18 years) who underwent antegrade enema initiation from 2007 to 2020 and matched controls (4:1) from the general population. To assess the change in healthcare utilization following antegrade enema initiation, we used negative binomial generalized estimating equations with covariates selected a priori. KEY RESULTS: One hundred thirty-eight subjects met eligibility criteria (appendicostomy = 55 (39.9%); cecostomy tube = 83 (60.1%)) and were matched to 550 controls. There was no significant difference in the change in the rate of hospitalizations (rate ratio (RR) 1.05, 95% confidence interval (CI) 0.35-1.75), outpatient visits (RR 1.05, 95% CI 0.91-1.18), or same-day surgical procedures (RR 1.51, 95% CI 0.60-2.43) across cases in 2 years following antegrade enema initiation compared with controls. Cases had an increased rate of emergency department (ED) visits, which was not observed in controls (RR 1.52, 95% CI 1.11-1.79), driven in part by device-related complications. CONCLUSIONS AND INFERENCES: Understanding healthcare utilization patterns following antegrade enema initiation allows for effective health system planning and aids medical decision-making. The observed increase in ED visits for device-related complications speaks to the need to improve preventive management to help mitigate emergency care after initiation of antegrade enemas.

Therapeutic Effects of Medicinal Cannabinoids on the Gastrointestinal System in Pediatric Patients: A Systematic Review.

Changes in cannabis legalization have generated interest in medicinal cannabinoids for therapeutic uses, including those that target the gastrointestinal (GI) tract. These effects are mediated through interactions with the endocannabinoid system. Given the increasing societal awareness of the therapeutic potential of cannabinoids, it is important to ensure pediatric representation in clinical studies investigating cannabinoid use. This systematic review aims to assess the efficacy of medicinal cannabinoids in treating GI symptoms in pediatric patients. A literature search of Medline, Embase, CINAHL, Web of Science, and the Cochrane Library was performed from inception until June 23, 2020. Study design, patient characteristics, type, dose and duration of medicinal cannabinoid therapy, and GI outcomes were extracted. From 7303 records identified, 5 studies met all inclusion criteria. Included studies focused on chemotherapy-induced nausea, inflammatory bowel disease, and GI symptoms associated with severe complex motor disorders. Results varied based on the symptom being treated, the type of cannabinoid, and the patient population. Medicinal cannabinoids may have a potential role in treating specific GI symptoms in specific patient populations. The limited number and heterogenicity of included studies highlight the demand for future research to distinguish effects among different cannabinoid types and patient populations and to examine drug interactions. As interest increases, higher quality studies are needed to understand the efficacy of cannabinoids as a pediatric GI treatment and whether these benefits outweigh the associated risks (Registration Number: PROSPERO CRD42020202486).

Kiwifruit and Kiwifruit Extracts for Treatment of Constipation: A Systematic Review and Meta-Analysis.

Introduction: This systematic review aimed to summarize evidence to determine the effectiveness of kiwifruit or kiwifruit extracts in the treatment of constipation. Methods: Electronic databases were searched from inception to May 2022 without any age or language limitations. Eligible studies enrolled participants with constipation who were randomized to receive kiwifruit or kiwifruit extracts vs. any nonkiwifruit control. Standardized mean difference (SMD) and mean difference (MD) with confidence intervals (CI) were determined for the following outcomes: frequency of spontaneous bowel movements (SBM), abdominal pain and straining, as well as stool type as determined by the Bristol Stool Scale (BSS). The Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach was used to rate the certainty of evidence. Our review was registered on PROSPERO (CRD42021239397). Results: Seven RCTs, including 399 participants (82% female; mean age: 42 years (SD 14.6)), were included. Compared with placebo (n = 95), kiwifruit extracts might increase the weekly frequency of SBM (MD: 1.36; 95% CI: -0.44, 3.16) with low certainty of evidence; moreover, it had an uncertain effect on BSS (SMD: 1.54; 95% CI: -1.33, 4.41) with very low certainty of evidence. Additionally, compared with placebo (n = 119), kiwifruit or its extracts reduced abdominal pain (SMD: -1.44, 95% CI -2.83, -1.66) with moderate certainty of the evidence and improved frequency of straining (SMD: -0.29; 95% CI: -1.03, 0.47). Compared with psyllium, kiwifruit may increase the weekly frequency of SBM (MD: 1.01; 95% CI: -0.02, 2.04) with moderate certainty evidence, and may increase the value on the BSS (indicating softer stools) (MD: 0.63; 95% CI: 0.01, 1.25)with low certainty of evidence. Compared to placebo, kiwifruit-encapsulated extracts may result in an increase in minor adverse events (relative risk: 4.58; 95% CI: 0.79, 26.4). Conclusions: Among individuals with constipation, there is an overall low certainty of evidence indicating that kiwifruit may increase SBM when compared to placebo or psyllium. Although overall results are promising, establishing the role of kiwifruit in constipation requires large, methodologically rigorous trials. Protocol Registration: PROSPERO registration number CRD42021239397.

Development of Entrustable Professional Activities and Standards in Training in Pediatric Neurogastroenterology and Motility: North American Society for Pediatric Gastroenterology, Hepatology and Nutrition and American Neurogastroenterology and Motility Society Position Paper.

ABSTRACT: Neurogastroenterology and motility (NGM) disorders are common in childhood and are often very debilitating. Although pediatric gastroenterology fellows are expected to obtain training in the diagnosis and management of patients with these disorders, there is an ongoing concern for unmet needs and lack of exposure and standardized curriculum. In the context of tailoring training components, outcome and expressed needs of pediatric gastroenterology fellows and programs, members of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) and American Neurogastroenterology and Motility Society (ANMS) developed guidelines for NGM training in North America in line with specific expectations and goals of training as delineated through already established entrustable professional activities (EPAs). Members of the joint task force applied their expertise to identify the components of knowledge, skills, and management, which are expected of NGM consultants. The clinical knowledge, skills and management elements of the NGM curriculum are divided into domains based on anatomic regions including esophagus, stomach, small bowel, colon and anorectum. In addition, dedicated sections on pediatric functional gastrointestinal (GI) disorders, research and collaborative approach, role of behavioral health and surgical approaches to NGM disorders and transition from pediatric to adult neurogastroenterology are included in this document. Members of the NASPGHAN-ANMS task force anticipate that this document will serve as a resource to break existing barriers to pursuing a career in NGM and provide a framework towards uniform training expectations at 3 hierarchical tiers corresponding to EPA levels.

Influence of bacterial components on the developmental programming of enteric neurons.

BACKGROUND: Intestinal bacteria have been increasingly shown to be involved in early postnatal development. Previous work has shown that intestinal bacteria are necessary for the structural development and intrinsic function of the enteric nervous system in early postnatal life. Furthermore, colonization with a limited number of bacteria appears to be sufficient for the formation of a normal enteric nervous system. We tested the hypothesis that common bacterial components could influence the programming of developing enteric neurons. METHODS: The developmental programming of enteric neurons was studied by isolating enteric neural crest-derived cells from the fetal gut of C57Bl/6 mice at embryonic day 15.5. After the establishment of the cell line, cultured enteric neuronal precursors were exposed to increasing concentrations of a panel of bacterial components including lipopolysaccharide, flagellin, and components of peptidoglycan. KEY RESULT: Exposure to bacterial components consistently affected proportions of enteric neuronal precursors that developed into nitrergic neurons. Furthermore, flagellin and D-gamma-Glu-mDAP were found to promote the development of serotonergic neurons. Proportions of dopaminergic neurons remained unchanged. Proliferation of neuronal precursor cells was significantly increased upon exposure to lipopolysaccharide and flagellin, while no significant changes were observed in the proportion of apoptotic neuronal precursors compared to baseline with exposure to any bacterial component. CONCLUSIONS AND INTERFACES: These findings suggest that bacterial components may influence the development of enteric neurons.

Starving the Gut: A Deficit of Butyrate in the Intestinal Ecosystem of Children With Intestinal Failure.

BACKGROUND: Intestinal epithelial integrity is influenced by short-chain fatty acids (SCFAs) and is of critical importance for children with intestinal failure (IF) given the known devastating infectious and gastrointestinal complications. The composition of the microbiome in IF represents an important variable in the physiology and prognosis of this disease. AIM: We sought to compare the intestinal microbiome and SCFA concentration of children who require parenteral nutrition (PN) with that of children with short-bowel syndrome (SBS) who have discontinued PN and with age-matched controls, using high-throughput sequencing to investigate host-microbe interactions. METHODS: Fifty-three samples were submitted over 6-15 months. Six children with SBS + IF submitted 34 samples, and 6 children with SBS with discontinued PN submitted 15 samples; these were compared with samples from 5 control children. Fecal samples were analyzed by 16S ribosomal RNA partial gene sequencing using the MiSeq Illumina sequencer. SCFAs were measured in stool samples by mass spectrometry. RESULTS: Butyrate quantity was near absent in children with IF compared with that in controls (median 0.37 nmol/mg vs 10.92 nmol/mg; P < .0001). Similarly, commensal anaerobes known to produce SCFA, including Ruminococcaceae and Lachnospiraceae, were reduced in those with SBS. SBS + IF enteric samples demonstrated a 168-fold increase in the relative abundance of the Escherichia genus seemingly attributable to the species Escherichia coli. CONCLUSION: The reduced relative abundance of butyrate-producing Clostridia as well as decreased intestinal butyrate concentration in children with IF support further investigation in therapeutic options that target butyrate-producing bacterial communities or butyrate supplementation.

Functional Constipation and the Gut Microbiome in Children: Preclinical and Clinical Evidence.

Functional constipation is a common condition in childhood with significant impact on patients' quality of life and on health care resources. Functional constipation is characterized by decreased bowel movements and/or hard stools, which cause significant distress for children and their caregivers. While the term "functional" may imply the absence of organic causes with a focus on behavioral aspects, 40% of children continue to have symptoms beyond conventional management with one in four children continuing to experience constipation into adulthood. The refractory and chronic nature of constipation highlights the importance of considering a range of pathophysiological mechanisms, including the potential role of the gut microbiome. In this review, we provide an overview of preclinical and clinical studies that focus on the potential mechanisms through which the gut microbiome might contribute to the clinical presentation of functional constipation in pediatrics.

Characterization of Simultaneous Pressure Waves as Biomarkers for Colonic Motility Assessed by High-Resolution Colonic Manometry.

Simultaneous pressure waves (SPWs) in manometry recordings of the human colon have been associated with gas expulsion. Our hypothesis was that the SPW might be a critical component of most colonic motor functions, and hence might act as a biomarker for healthy colon motility. To that end, we performed high-resolution colonic manometry (HRCM), for the first time using an 84-sensor (1 cm spaced) water-perfused catheter, in 17 healthy volunteers. Intraluminal pressure patterns were recorded during baseline, proximal and rectal balloon distention, after a meal and following proximal and rectal luminal bisacodyl administration. Quantification was performed using software, based on Image J, developed during this study. Gas expulsion was always associated with SPWs, furthermore, SPWs were associated with water or balloon expulsion. SPWs were prominently emerging at the termination of proximal high amplitude propagating pressure waves (HAPWs); we termed this motor pattern HAPW-SPWs; hence, SPWs were often not a pan-colonic event. SPWs and HAPW-SPWs were observed at baseline with SPW amplitudes of 12.0 ± 8.5 mmHg and 20.2 ± 7.2 mmHg respectively. The SPW occurrence and amplitude significantly increased in response to meal, balloon distention and luminal bisacodyl, associated with 50.3% anal sphincter relaxation at baseline, which significantly increased to 59.0% after a meal, and 69.1% after bisacodyl. Often, full relaxation was achieved. The SPWs associated with gas expulsion had a significantly higher amplitude compared to SPWs without gas expulsion. SPWs could be seen to consist of clusters of high frequency pressure waves, likely associated with a cluster of fast propagating, circular muscle contractions. SPWs were occasionally observed in a highly rhythmic pattern at 1.8 ± 1.2 cycles/min. Unlike HAPWs, the SPWs did not obliterate haustral boundaries thereby explaining how gas can be expelled while solid content can remain restrained by the haustral boundaries. In conclusion, the SPW may become a biomarker for normal gas transit, the gastrocolonic reflex and extrinsic neural reflexes. The SPW assessment reveals coordination of activities in the colon, rectum and anal sphincters. SPWs may become of diagnostic value in patients with colonic dysmotility.

Intrapartum antibiotics for GBS prophylaxis alter colonization patterns in the early infant gut microbiome of low risk infants.

Early life microbial colonization and succession is critically important to healthy development with impacts on metabolic and immunologic processes throughout life. A longitudinal prospective cohort was recruited from midwifery practices to include infants born at full term gestation to women with uncomplicated pregnancies. Here we compare bacterial community succession in infants born vaginally, with no exposure to antibiotics (n = 53), with infants who were exposed to intrapartum antibiotic prophylaxis (IAP) for Group B Streptococcus (GBS; n = 14), and infants born by C-section (n = 7). Molecular profiles  of the 16 S rRNA genes indicate that there is a delay in the expansion of Bifidobacterium, which was the dominate infant gut colonizer, over the first 12 weeks and a persistence of Escherichia when IAP for GBS exposure is present during vaginal labour. Longer duration of IAP exposure increased the magnitude of the effect on Bifidobacterium populations, suggesting a longer delay in microbial community maturation. As with prior studies, we found altered gut colonisation following C-section that included a notable lack of Bacteroidetes. This study found that exposure of infants to IAP for GBS during vaginal birth affected aspects of gut microbial ecology that, although dramatic at early time points, disappeared by 12 weeks of age in most infants.

A comparison of intestinal microbiota in a population of low-risk infants exposed and not exposed to intrapartum antibiotics: The Baby & Microbiota of the Intestine cohort study protocol.

BACKGROUND: The intestinal microbiota influences metabolic, nutritional, and immunologic processes and has been associated with a broad range of adverse health outcomes including asthma, obesity and Type 2 diabetes. Early life exposures may alter the course of gut microbial colonization leading to differences in metabolic and immune regulation throughout life. Although approximately 50 % of low-risk full-term infants born in Canada are exposed to intrapartum antibiotics, little is known about the influence of this common prophylactic treatment on the developing neonatal intestinal microbiota. The purpose of this study is to describe the intestinal microbiome over the first 3 years of life among healthy, breastfed infants born to women with low-risk pregnancies at full term gestation and to determine if at 1 year of age, the intestinal microbiome of infants exposed to intrapartum antibiotics differs in type and quantity from the infants that are not exposed. METHODS: A prospectively followed cohort of 240 mother-infant pairs will be formed by enrolling eligible pregnant women from midwifery practices in the City of Hamilton and surrounding area in Ontario, Canada. Participants will be followed until the age of 3 years. Women are eligible to participate in the study if they are considered to be low-risk, planning a vaginal birth and able to communicate in English. Women are excluded if they have a multiple pregnancy or a preterm birth. Study questionnaires are completed, anthropometric measures are taken and biological samples are acquired including eight infant stool samples between 3 days and 3 years of age. DISCUSSION: Our experience to date indicates that midwifery practices and clients are keen to participate in this research. The midwifery client population is likely to have high rates of breastfeeding and low rates of intervention, allowing us to examine the comparative development of the microbiome in a relatively healthy and homogenous population. Results from this study will make an important contribution to the growing understanding of the patterns of intestinal microbiome colonization in the early years of life and may have implications for best practices to support the establishment of the microbiome at birth.

Vagal Fibers Form Associations With Interstitial Cells of Cajal During Fetal Development.

Vagal intramuscular arrays (IMAs) have been shown to form complexes with intramuscular interstitial cells of Cajal (ICC). We tested the hypothesis that associations between vagal nerve endings and ICC arise in fetal development. Intraganglionic laminar endings (IGLEs) and IMAs were identified by applying 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate (DiI) to vagal nerve trunks and myenteric plexus (MP) and intramuscular (IM) ICC were immunolabeled with antibodies to c-Kit in fetal and early postnatal mice (E16-P7). At E16, c-Kit immunoreactive cells were abundant in the primordial smooth muscle, with early ICC networks discernable by E18 and ongoing organization at P1 and P7. The distribution of vagal endings was found to change during the course of development, with significantly more putative IGLEs in the prenatal compared to the postnatal period and less IMAs in the prenatal compared to postnatal period. Associations of ICC with both IGLEs and IMAs were detected as early as E16 and were maintained into postnatal life. These findings suggest that vagal fibers begin to associate with ICC during prenatal development. Future studies will be needed to determine the mechanisms through which vagal endings and ICC interact.

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.

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.

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.