Anatomy and Neuroscience - Research Publications
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ItemEarly Development of Electrical Excitability in the Mouse Enteric Nervous System(SOC NEUROSCIENCE, 2012-08-08)Neural activity is integral to the development of the enteric nervous system (ENS). A subpopulation of neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development (at E10.5 in the mouse). However, the electrical activity of these cells has not been previously characterized, and it is not known whether all cells expressing neuronal markers are capable of firing action potentials (APs). In this study, we examined the activity of "neuron"-like cells (expressing pan-neuronal markers or with neuronal morphology) in the gut of E11.5 and E12.5 mice using whole-cell patch-clamp electrophysiology and compared them to the activity of neonatal and adult enteric neurons. Around 30-40% of neuron-like cells at E11.5 and E12.5 fired APs, some of which were very similar to those of adult enteric neurons. All APs were sensitive to tetrodotoxin (TTX), indicating that they were driven by voltage-gated Na+ currents. Expression of mRNA encoding several voltage-gated Na+ channels by the E11.5 gut was detected using RT-PCR. The density of voltage-gated Na+ currents increased from E11.5 to neonates. Immature active responses, mediated in part by TTX- and lidocaine-insensitive channels, were observed in most cells at E11.5 and E12.5, but not in P0/P1 or adult neurons. However, some cells expressing neuronal markers at E11.5 or E12.5 did not exhibit an active response to depolarization. Spontaneous depolarizations resembling excitatory postsynaptic potentials were observed at E12.5. The ENS is one of the earliest parts of the developing nervous system to exhibit mature forms of electrical activity.
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ItemNo Preview AvailableDevelopment of enteric neuron diversity(WILEY, 2009-07)The mature enteric nervous system (ENS) is composed of many different neuron subtypes and enteric glia, which all arise from the neural crest. How this diversity is generated from neural crest-derived cells is a central question in neurogastroenterology, as defects in these processes are likely to underlie some paediatric motility disorders. Here we review the developmental appearance (the earliest age at which expression of specific markers can be localized) and birthdates (the age at which precursors exit the cell cycle) of different enteric neuron subtypes, and their projections to some targets. We then focus on what is known about the mechanisms underlying the generation of enteric neuron diversity and axon pathfinding. Finally, we review the development of the ENS in humans and the etiologies of a number of paediatric motility disorders.
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ItemEffects of NGF, NT-3 and GDNF family members on neurite outgrowth and migration from pelvic ganglia from embryonic and newborn mice(BMC, 2008-07-25)BACKGROUND: Pelvic ganglia are derived from the sacral neural crest and contain both sympathetic and parasympathetic neurons. Various members of the neurotrophin and GDNF families of neurotrophic factors have been shown to play important roles in the development of a variety of peripheral sympathetic and parasympathetic neurons; however, to date, the role of these factors in the development of pelvic ganglia has been limited to postnatal and older ages. We examined the effects of NGF, NT-3, GDNF, neurturin and artemin on cell migration and neurite outgrowth from explants of the pelvic ganglia from embryonic and newborn mice grown on collagen gels, and correlated the responses with the immunohistochemical localization of the relevant receptors in fixed tissue. RESULTS: Cell migration assays showed that GDNF strongly stimulated migration of tyrosine hydroxylase (TH) cells of pelvic ganglia from E11.5, E14.5 and P0 mice. Other factors also promoted TH cell migration, although to a lesser extent and only at discrete developmental stages. The cells and neurites of the pelvic ganglia were responsive to each of the GDNF family ligands--GDNF, neurturin and artemin--from E11.5 onwards. In contrast, NGF and NT-3 did not elicit a significant neurite outgrowth effect until E14.5 onwards. Artemin and NGF promoted significant outgrowth of sympathetic (TH+) neurites only, whereas neurturin affected primarily parasympathetic (TH-negative) neurite outgrowth, and GDNF and NT-3 enhanced both sympathetic and parasympathetic neurite outgrowth. In comparison, collagen gel assays using gut explants from E11.5 and E14.5 mice showed neurite outgrowth only in response to GDNF at E11.5 and to neurturin only in E14.5 mice. CONCLUSION: Our data show that there are both age-dependent and neuron type-dependent differences in the responsiveness of embryonic and neo-natal pelvic ganglion neurons to growth factors.
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ItemAll Is Not Normal in "Normoganglionic" Bowel Regions of Hirschsprung Disease Model Mice(ELSEVIER INC, 2015-01)
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ItemSurgical Intervention to Rescue Hirschsprung Disease in a Rat Model(KOREAN SOC NEUROGASTROENTEROLOGY & MOTILITY, 2015-10)BACKGROUND/AIMS: Rats with a spontaneous null mutation in endothelin receptor type B or Ednrb (sl/sl; spotting lethal) lack enteric neurons in the distal bowel and usually die within the first week after birth. This early postnatal lethality limits their use for examining the potential of cell therapy to treat Hirschsprung disease, and for studies of the influence of EDNRB on the mature CNS and vascular systems. METHODS: We have developed a surgical intervention to prolong the life of the spotting lethal sl/sl rat, in which we perform a colostomy on postnatal (P) day 4-6 rats to avoid the fatal obstruction caused by the lack of colonic enteric neurons. RESULTS: The stomas remained patent and functional and the rats matured normally following surgery. Weight gains were comparable between control and Hirschsprung phenotype (sl/sl) rats, which were followed until 4 weeks after surgery (5 weeks old). We confirmed the absence of enteric neurons in the distal colon of rats whose lives were saved by the surgical intervention. CONCLUSIONS: This study provides a novel approach for studying EDNRB signalling in multiple organ systems in mature rats, including an animal model to study the efficacy of cell therapy to treat Hirschsprung disease.
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ItemNo Preview AvailableMyenteric neurons of the mouse small intestine undergo significant electrophysiological and morphological changes during postnatal development(WILEY, 2012-05)Organized motility patterns in the gut depend on circuitry within the enteric nervous system (ENS), but little is known about the development of electrophysiological properties and synapses within the ENS. We examined the electrophysiology and morphology of myenteric neurons in the mouse duodenum at three developmental stages: postnatal day (P)0, P10–11, and adult. Like adults, two main classes of neurons could be identified at P0 and P10–11 based on morphology: neurons with multiple long processes that projected circumferentially (Dogiel type II morphology) and neurons with a single long process. However, postnatal Dogiel type II neurons differed in several electrophysiological properties from adult Dogiel type II neurons. P0 and P10–11 Dogiel type II neurons exhibited very prominent Ca(2+)-mediated after depolarizing potentials (ADPs) following action potentials compared to adult neurons. Adult Dogiel type II neurons are characterized by the presence of a prolonged after hyperpolarizing potential (AHP), but AHPs were very rarely observed at P0. The projection lengths of the long processes of Dogiel type II neurons were mature by P10–11. Uniaxonal neurons in adults typically have fast excitatory postsynaptic potentials (fEPSPs, ‘S-type' electrophysiology) mainly mediated by nicotinic receptors. Nicotinic-fEPSPs were also recorded from neurons with a single long process at P0 and P10–11. However, these neurons underwent major developmental changes in morphology, from predominantly filamentous neurites at birth to lamellar dendrites in mature mice. Unlike Dogiel type II neurons, the projection lengths of neurons with a single long process matured after P10–11. Slow EPSPs were rarely observed in P0/P10–11 neurons. This work shows that, although functional synapses are present and two classes of neurons can be distinguished electrophysiologically and morphologically at P0, major changes in electrophysiological properties and morphology occur during the postnatal development of the ENS.
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ItemNo Preview AvailableEnteric neural progenitors are more efficient than brain-derived progenitors at generating neurons in the colon(AMER PHYSIOLOGICAL SOC, 2014-10-01)Gut motility disorders can result from an absent, damaged, or dysfunctional enteric nervous system (ENS). Cell therapy is an exciting prospect to treat these enteric neuropathies and restore gut motility. Previous studies have examined a variety of sources of stem/progenitor cells, but the ability of different sources of cells to generate enteric neurons has not been directly compared. It is important to identify the source of stem/progenitor cells that is best at colonizing the bowel and generating neurons following transplantation. The aim of this study was to compare the ability of central nervous system (CNS) progenitors and ENS progenitors to colonize the colon and differentiate into neurons. Genetically labeled CNS- and ENS-derived progenitors were cocultured with aneural explants of embryonic mouse colon for 1 or 2.5 wk to assess their migratory, proliferative, and differentiation capacities, and survival, in the embryonic gut environment. Both progenitor cell populations were transplanted in the postnatal colon of mice in vivo for 4 wk before they were analyzed for migration and differentiation using immunohistochemistry. ENS-derived progenitors migrated further than CNS-derived cells in both embryonic and postnatal gut environments. ENS-derived progenitors also gave rise to more neurons than their CNS-derived counterparts. Furthermore, neurons derived from ENS progenitors clustered together in ganglia, whereas CNS-derived neurons were mostly solitary. We conclude that, within the gut environment, ENS-derived progenitors show superior migration, proliferation, and neuronal differentiation compared with CNS progenitors.
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ItemNo Preview AvailableChanges in Nicotinic Neurotransmission during Enteric Nervous System Development(SOC NEUROSCIENCE, 2015-05-06)Acetylcholine-activating pentameric nicotinic receptors (nAChRs) are an essential mode of neurotransmission in the enteric nervous system (ENS). In this study, we examined the functional development of specific nAChR subtypes in myenteric neurons using Wnt1-Cre;R26R-GCaMP3 mice, where all enteric neurons and glia express the genetically encoded calcium indicator, GCaMP3. Transcripts encoding α3, α4, α7, β2, and β4 nAChR subunits were already expressed at low levels in the E11.5 gut and by E14.5 and, thereafter, α3 and β4 transcripts were the most abundant. The effect of specific nAChR subtype antagonists on evoked calcium activity in enteric neurons was investigated at different ages. Blockade of the α3β4 receptors reduced electrically and chemically evoked calcium responses at E12.5, E14.5, and P0. In addition to the α3β4 antagonist, antagonists to α3β2 and α4β2 also significantly reduced responses by P10-11 and in adult preparations. Therefore, there is an increase in the diversity of functional nAChRs during postnatal development. However, an α7 nAChR antagonist had no effect at any age. Furthermore, at E12.5 we found evidence for unconventional receptors that were responsive to the nAChR agonists 1-dimethyl-4-phenylpiperazinium and nicotine, but were insensitive to the general nicotinic blocker, hexamethonium. Migration, differentiation, and neuritogenesis assays did not reveal a role for nAChRs in these processes during embryonic development. In conclusion, there are significant changes in the contribution of different nAChR subunits to synaptic transmission during ENS development, even after birth. This is the first study to investigate the development of cholinergic transmission in the ENS.
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ItemIon Channel Expression in the Developing Enteric Nervous System(PUBLIC LIBRARY SCIENCE, 2015-03-23)The enteric nervous system arises from neural crest-derived cells (ENCCs) that migrate caudally along the embryonic gut. The expression of ion channels by ENCCs in embryonic mice was investigated using a PCR-based array, RT-PCR and immunohistochemistry. Many ion channels, including chloride, calcium, potassium and sodium channels were already expressed by ENCCs at E11.5. There was an increase in the expression of numerous ion channel genes between E11.5 and E14.5, which coincides with ENCC migration and the first extension of neurites by enteric neurons. Previous studies have shown that a variety of ion channels regulates neurite extension and migration of many cell types. Pharmacological inhibition of a range of chloride or calcium channels had no effect on ENCC migration in cultured explants or neuritogenesis in vitro. The non-selective potassium channel inhibitors, TEA and 4-AP, retarded ENCC migration and neuritogenesis, but only at concentrations that also resulted in cell death. In summary, a large range of ion channels is expressed while ENCCs are colonizing the gut, but we found no evidence that ENCC migration or neuritogenesis requires chloride, calcium or potassium channel activity. Many of the ion channels are likely to be involved in the development of electrical excitability of enteric neurons.
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ItemNo Preview AvailableTransplanted progenitors generate functional enteric neurons in the postnatal colon(AMER SOC CLINICAL INVESTIGATION INC, 2013-03)Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut; however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons; they received synaptic inputs; and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.