Dual purinergic synaptic transmission in the human enteric nervous system

Based on findings in rodents, we sought to test the hypothesis that purinergic modulation of synaptic transmission occurs in the human intestine. Time series analysis of intraneuronal free Ca(2+) levels in submucosal plexus (SMP) from Roux-en-Y specimens was done using Zeiss LSM laser-scanning confocal fluo-4 AM Ca(2+) imaging. A 3-s fiber tract stimulation (FTS) was used to elicit a synaptic Ca(2+) response. Short-circuit current (I(sc) = chloride secretion) was recorded in mucosa-SMP in flux chambers. A distension reflex or electrical field stimulation was used to study I(sc) responses. Ca(2+) imaging was done in 1,222 neurons responding to high-K(+) depolarization from 61 surgical cases. FTS evoked synaptic Ca(2+) responses in 62% of recorded neurons. FTS caused frequency-dependent Ca(2+) responses (0.1-100 Hz). FTS Ca(2+) responses were inhibited by Omega-conotoxin (70%), hexamethonium (50%), TTX, high Mg(2+)/low Ca(2+) (< or = 100%), or capsaicin (25%). A P2Y(1) receptor (P2Y(1)R) antagonist, MRS-2179 or PLC inhibitor U-73122, blocked FTS responses (75-90%). P2Y(1)R-immunoreactivity occurred in 39% of vasoactive intestinal peptide-positive neurons. The selective adenosine A(3) receptor (AdoA(3)R) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IBMECA) caused concentration- and frequency-dependent inhibition of FTS Ca(2+) responses (IC(50) = 8.5 x 10(-8) M). The AdoA(3)R antagonist MRS-1220 augmented such Ca(2+) responses; 2-Cl-IBMECA competed with MRS-1220. Knockdown of AdoA(1)R with 8-cyclopentyl-3-N-(3-{[3-(4-fluorosulphonyl)benzoyl]-oxy}-propyl)-1-N-propyl-xanthine did not prevent 2-Cl-IBMECA effects. MRS-1220 caused 31% augmentation of TTX-sensitive distension I(sc) responses. The SMP from Roux-en-Y patients is a suitable model to study synaptic transmission in human enteric nervous system (huENS). The P2Y(1)/Galphaq/PLC/inositol 1,3,5-trisphosphate/Ca(2+) signaling pathway, N-type Ca(2+) channels, nicotinic receptors, and extrinsic nerves contribute to neurotransmission in huENS. Inhibitory AdoA(3)R inhibit nucleotide or cholinergic transmission in the huENS.     

Granulocyte-colony stimulating factor: a new player for the enteric nervous system

The enteric nervous system (ENS) controls and modulates gut motility and responds to food intake and to internal and external stimuli such as toxins or inflammation. Its plasticity is maintained throughout life by neural progenitor cells within the enteric stem cell niche. Granulocyte-colony stimulating factor (G-CSF) is known to act not only on cells of the immune system but also on neurons and neural progenitors in the central nervous system (CNS). Here, we demonstrate, for the first time, that G-CSF receptor is present on enteric neurons and progenitors and that G-CSF plays a role in the expansion and differentiation of enteric neural progenitor cells. Cultured mouse ENS-neurospheres show increased expansion with increased G-CSF concentrations, in contrast to CNS-derived spheres. In cultures from differentiated ENS- and CNS-neurospheres, neurite outgrowth density is enhanced depending on the amount of G-CSF in the culture. G-CSF might be an important factor in the regeneration and differentiation of the ENS and might be a useful tool for the investigation and treatment of ENS disorders.     

Alternative processing pathways for preprovasoactive intestinal peptide in the enteric nervous system of the rat

In order to study biosynthetic processing of preprovasoactive intestinal peptide (prepro VIP) we have raised antisera to sequences that flank the biologically active peptides VIP and PHI (peptide with N-terminal His and C-terminal Ile). We have used these antisera in radioimmunoassays to identify the N-terminal flanking peptide (NFP) and C-terminal flanking peptide (CFP)-like immunoreactivities in rat brain and gastrointestinal tract. Concentrations of NFP-LI were similar to those of VIP in brain and throughout the gut. Concentrations of CFP-LI were 10-20% those of VIP-LI but could be increased 5-fold by digestion with carboxypeptidase B, suggesting that the C-terminal lysine residue of prepro VIP is not normally removed during processing. In rat stomach the NFP-LI was of higher molecular weight and greater hydrophobicity than the intestinal component. The data are consistent with alternative processing pathways for prepro VIP in enteric nerves of rat stomach and intestine.     

Development of the enteric nervous system

The mature enteric nervous system (ENS) is characterized by a degree of neuronal phenotypic diversity and independence of central nervous system control unequaled by any other region of the peripheral nervous system. Studies that have utilized the immunocytochemical demonstration of neurofilament protein and explanation of primordial gut with subsequent growth in culture have indicated that the neural crest precursors of enteric neurons are already committed to the neuronal lineage when they colonize the bowel; however, neuronal phenotypic expression occurs within the gut itself. It is likely that precursors able to give rise to each type of neuron found in the mature ENS are present among the earliest neural crest émigrés to reach the bowel. In mice a proximodistal wave of neuronal phenotypic expression occurs that does not appear to reflect the descent of neuronal precursors. This observation, the known plasticity of developing neural crest-derived neurons, and the demonstration of a persistent population of proliferating neuroblasts in the gut raise the possibility that enteric neuronal phenotypic expression is influenced by the enteric microenvironment.     

Evidence for neural progenitor cells in the human adult enteric nervous system

Putative neural stem cells have been identified within the enteric nervous system (ENS) of adult rodents and cultured from human myenteric plexus. We conducted studies to identify neural stem cells or progenitor cells within the submucosa of adult human ENS. Jejunum tissue was removed from adult human subjects undergoing gastric bypass surgery. The tissue was immunostained, and confocal images of ganglia in the submucosal plexus were collected to identify protein gene product 9.5 (PGP 9.5) - immunoractive neurons and neuronal progenitor cells that coexpress PGP 9.5 and nestin. In addition to PGP-9.5-positive/nestin-negative neuronal cells within ganglia, we observed two other types of cells: (1) cells in which PGP 9.5 and nestin were co-localized, (2) cells negative for both PGP 9.5 and nestin. These observations suggest that the latter two types of cells are related to a progenitor cell population and are consistent with the concept that the submucosa of human adult ENS contains stem cells capable of maintenance and repair within the peripheral nervous system.     

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.     

Cellular changes in the enteric nervous system during ageing

The intrinsic neurons of the gut, enteric neurons, have an essential role in gastrointestinal functions. The enteric nervous system is plastic and continues to undergo changes throughout life, as the gut grows and responds to dietary and other environmental changes. Detailed analysis of changes in the ENS during ageing suggests that enteric neurons are more vulnerable to age-related degeneration and cell death than neurons in other parts of the nervous system, although there is considerable variation in the extent and time course of age-related enteric neuronal loss reported in different studies. Specific neuronal subpopulations, particularly cholinergic myenteric neurons, may be more vulnerable than others to age-associated loss or damage. Enteric degeneration and other age-related neuronal changes may contribute to gastrointestinal dysfunction that is common in the elderly population. Evidence suggests that caloric restriction protects against age-associated loss of enteric neurons, but recent advances in the understanding of the effects of the microbiota and the complex interactions between enteric ganglion cells, mucosal immune system and intestinal epithelium indicate that other factors may well influence ageing of enteric neurons. Much remains to be understood about the mechanisms of neuronal loss and damage in the gut, although there is evidence that reactive oxygen species, neurotrophic factor dysregulation and/or activation of a senescence associated phenotype may be involved. To date, there is no evidence for ongoing neurogenesis that might replace dying neurons in the ageing gut, although small local sites of neurogenesis would be difficult to detect. Finally, despite the considerable evidence for enteric neurodegeneration during ageing, and evidence for some physiological changes in animal models, the ageing gut appears to maintain its function remarkably well in animals that exhibit major neuronal loss, indicating that the ENS has considerable functional reserve.     

Cell death and the developing enteric nervous system

This review discusses current knowledge about cell death in the developing enteric nervous system (ENS). It also includes findings about the molecular mechanisms by which such death is mediated. Additional consideration is given to trophic factors that contribute to survival of the precursors and neurons and glia of the ENS, as well to genes that, when mutated or deleted, trigger their death. Although further confirmation is needed, present observations support the view that enteric neural crest-derived precursor cells en route to the gut undergo substantial levels of apoptotic death, but that once these cells colonize the gut, there is relatively little death of precursor cells or of neurons and glia during the fetal period. There are also indications that normal neuron loss occurs in the ENS, but at times beyond the perinatal stage. Taken together, these findings suggest that ENS development is similar is some ways, but different in others from extra-enteric areas of the vertebrate central and peripheral nervous systems, in which large-scale apoptotic death of precursor neurons and glia occurs during the fetal and perinatal periods. Potential reasons for these differences are discussed such as a fetal enteric microenvironment that is especially rich in trophic support. In addition to the cell death that occurs during normal ENS development, this review discusses mechanisms of experimentally-induced ENS cell death, such as those that are associated with defective glial cell-line derived neurotrophic factor/Ret signaling, which are an animal model of human congenital megacolon (aganglionosis; Hirschsprung’s disease). Such considerations underscore the importance of understanding cell death in the developing ENS, not just from a curiosity-driven point of view, but also because the pathophysiology behind many disorders of human gastrointestinal function may originate in abnormalities of the mechanisms that govern cell survival and death during ENS development.     

A role for the enteric nervous system in the response to helminth infections

The enteric nervous system (ENS) in the gut contains a particularly high concentration of nerve cells, and effectively functions as an independent 'minibrain'. Interactions between nerve, endocrine, immune and other cell types allow the sophisticated regulation of normal gut physiology. They can also bring about a co-ordinated response to parasitic infection, possibly leading to expulsion of the parasite. In this review, Derek McKay and Ian Fairweather will consider, in brief, data pertaining to changes in the ENS following intestinal helminth infections and speculate on the role that these alterations may have in the expulsion of the parasite burden and the putative ability of the parasite to modulate these events.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Development of the mammalian enteric nervous system.

The mammalian enteric nervous system is derived from neural crest cells which invade the foregut and hindgut mesenchyme. It has been established that signalling molecules produced by the mesenchyme of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have characterised further the role of such molecules and have identified novel extracellular and intracellular signals that are critical for enteric ganglia formation.     

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.     

Expression of leukaemia inhibitory factor during the development of the human enteric nervous system

Leukaemia inhibitory factor (LIF) is a neuropoietic cytokine, which promotes the development of enteric neurons in vitro, particularly when administered together with neurotrophin-3 (NT-3). The purpose of this study was to map the LIF immunoreactivity in the human enteric nervous system in foetuses, children, adults, and in patients with Hirschsprung's disease. Normal bowel specimens were obtained at postmortem examination of 13 foetuses, at 13–31 weeks of gestation, and at surgery in five children and two adults. Bowel resected in seven patients with Hirschsprung's disease was also investigated. Immunohistochemical analysis was performed on material fixed in formalin and embedded in paraffin. The specimens were exposed to antibodies raised against LIF. The ABC-complex method was used to visualise binding of antibodies to the corresponding antigen. LIF immunoreactivity was disclosed in the myenteric and submucous ganglion cells at 13–31 weeks of gestation, in childhood cases, and adults. LIF-immunoreactive ganglion cells were absent in aganglionic bowel, where the ganglia in the intermuscular layer were replaced by hypertrophic nerve bundles. These morphological findings indicate that LIF may play a role in the development of the enteric nervous system.