An important feature of the enteric nervous system (ENS) is its capability to respond to mechanical stimulation which, as currently suggested for the guinea-pig ileum, is encoded by specialized intrinsic primary afferent neurons (IPANs). We used von Frey hairs or intraganglionic volume injections to mimic ganglion deformation as observed in freely contracting preparations. Using fast voltage-sensitive dye imaging we identified rapidly adapting mechanosensitive enteric neurons (RAMEN, 25% of all neurons) in the myenteric plexus of the guinea pig ileum. RAMEN responded with phasic spike discharge to dynamic changes during ganglion deformation. This response was reproducible and increased with increasing forces. Deformation-evoked spike discharge was not changed by synaptic blockade with hexamethonium, ω-conotoxin or low Ca 2+ /high Mg 2+ , defunctionalization of extrinsic afferents with capsaicin or muscle paralysis with nifedipine, suggesting direct activation of RAMEN. All RAMEN received hexamethonium-sensitive fast EPSPs, which were blocked by ω-conotoxin and low Ca 2+ /high Mg 2+ . Seventy-two per cent of RAMEN were cholinergic, 22% nitrergic, and 44% were calbindin and NeuN negative, markers used to identify IPANs. Mechanosensitivity was observed in 31% and 47% of retrogradely traced interneurons and motor neurons, respectively. RAMEN belong to a new population of mechanosensitive neurons which differ from IPANs. We provided evidence for multifunctionality of RAMEN which may fulfil sensory, integrative and motor functions. In light of previously identified mechanosensitive neuron populations, mechanosensitivity appears to be a property of many more enteric neurons than generally assumed. The findings call for a revision of current concepts on sensory transmission within the ENS. Abbreviations ChAT, choline acetyltransferase; ENS, enteric nervous system; IPANs, intrinsic primary afferent neurons; NOS, nitric oxide synthase; RAMEN, rapidly adapting mechanosensitive enteric neurons.

BackgroundWithin the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions.Methodology/Principal FindingsWe deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca++/high Mg++. Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin.Conclusions/SignificanceWe showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions.

Non-technical summary

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrP(Sc)). Special attention has been paid to the follicles of Peyer's patches (PPs), which are major sites of PrP(Sc) accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrP(Sc) entry into the central nervous system. Thus, PrP(Sc) might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine beta hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrP(Sc) to the central nervous system.

Key points• We investigated altered colonic functions in high fat diet-induced obesity in pre-diabetic mice.• After feeding an adipogenic diet for 12 weeks, accelerated colonic transit was associated with upregulation and enhanced signalling of acetylcholine and serotonin, two key mediators in the enteric nervous system (ENS). Importantly, these changes occurred without signs of impaired mucosal integrity or immune cell infiltration in the gut wall. Neuronal sensitization was not observed in obese mice fed for 4 weeks.• Weight gain correlated positively with the level of adipocyte markers and the degree of neuronal sensitization.• We conclude that enhanced neural excitation in the colon by acetylcholine and serotonin is a key feature of a later phase of obesity and is involved in altered ENS functions and abnormal colonic transit.• Furthermore, the results suggest that the occurrence of altered gut functions in obesity is independent of inflammation in the gut wall.Abstract Colonic transit and mucosal integrity are believed to be impaired in obesity. However, a comprehensive assessment of altered colonic functions, inflammatory changes and neuronal signalling of obese animals is missing. In mice, we studied the impact of diet-induced obesity (DIO) on: (i) in vivo colonic transit; (ii) signalling in the myenteric plexus by recording responses to nicotine and 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), together with the expression of tryptophan hydroxylase (TPH) 1 and 2, serotonin reuptake transporter, choline acetyltransferase and the paired box gene 4; and (iii) expression of proinflammatory cytokines, epithelial permeability and density of macrophages, mast cells and enterochromaffin cells. Compared with controls, colon transit and neuronal sensitivity to nicotine and 2-methyl-5-HT were enhanced in DIO mice fed for 12 weeks. This was associated with increased tissue acetylcholine and 5-hydroxytryptamine (5-HT) content, and increased expression of TPH1 and TPH2. In DIO mice, upregulation of proinflammatory cytokines was found in fat tissue, but not in the gut wall. Accordingly, mucosal permeability or integrity was unaltered without signs of immune cell infiltration in the gut wall. Body weight showed positive correlations with adipocyte markers, tissue levels of 5-HT and acetylcholine, and the degree of neuronal sensitization. DIO mice fed for 4 weeks showed no neuronal sensitization, had no signs of gut wall inflammation and showed a smaller increase in leptin, interleukin-6 and monocyte chemoattractant protein 1 expression in fat tissue. DIO is associated with faster colonic transit and impacts on acetylcholine and 5-HT metabolism with enhanced responsiveness of enteric neurones to both mediators after 12 weeks of feeding. Our study demonstrates neuronal plasticity in DIO prior to the development of a pathological histology or abnormal mucosal functions. This questions the common assumption that increased mucosal inflammation and permeability initiate functional disorders in obesity.

No abstract

The specific patterns of gastrointestinal motility in large herbivores may relate to differences in the organization of enteric nerve circuits, compared with other mammals. To investigate this possibility, we characterized the morphologies, chemical phenotypes, and projections of myenteric plexus (MP) neurons of the sheep ileum. Morphologies and projections were investigated after application of the carbocyanine dye (1,1 0 , di-octadecyl-3,3,3 0 ,3 0 ,-tetramethylindo-carbocyanine perchlorate, DiI) to fixed tissues. To study chemical phenotypes, the fluorescent tracer Fast Blue (FB) was injected into the wall of the ileum, in vivo, 12-14 cm oral to the ileo-caecal junction. Over 80% of the descending and ascending DiI-labeled neurons had typical Dogiel type I morphology, whereas only a few Dogiel type II neurons were observed. Nevertheless, there were long projections (up to 10 cm) of Dogiel type II neurons in both directions. Both type II and type I neurons were neurofilament immunoreactive (IR). We observed long projections of descending (up to 18 cm) and ascending (up to 12-14 cm) FB-labeled MP neurons. Nitric oxide synthase (NOS)-IR, peripheral choline acetyltransferase (pChAT)-IR, and substance P (SP)-IR occurred in both descending and ascending myenteric neurons. NOS-IR was in approximately 60% of FB-labeled descending and ascending neurons, whereas those expressing pChAT-IR were 67 6 15% and 60 6 14%, respectively. Descending neurons expressing SP-IR were 48 6 15% and ascending were 56 6 12%. NOS-IR and pChAT-IR, and SP-IR and pChAT-IR were commonly colocalized in both ascending and descending pathways. In descending pathways, almost all SP-IR neurons were also pChAT-IR (98 6 3%) and NOS-IR (99 6 2 NOS 1 /SP 1 /pChAT 2 ). Many FB-labeled descending neurons showed both NOS-and pChAT-IR. Descending neurons may represent inhibitory motor neurons (NOS 1 /SP 1 /pChAT 2 ) and two classes of interneurons (pChAT 1 /NOS 2 , and pChAT 1 /NOS

Until now, significant differences in the neurochemical pattern of enteric neurons have been demonstrated in all species studied; however, some strong similarities also occur across species, such as the occurrence of nitric oxide synthase immunoreactivity (NOS-IR) in inhibitory motor neurons to muscle. In consideration of the insufficient data regarding the enteric nervous system (ENS) of sheep, we investigated the myenteric plexus and submucosal plexus of the ovine ileum. Since the pivotal role of the ENS in the early pathogenesis of sheep scrapie, the “prototype” of prion diseases, has been suggested, we have focused our observations also on the host's PrP genotype. We have studied the morphology and distribution of NOS-IR neurons and their relationships with the enteric glia in whole-mount preparations and in cryostat sections. NOS-IR neurons, always encircled by glial processes, were located in both plexuses. Many NOS-IR fibers were seen in the circular muscle layer, in the submucosa, and in the mucosa. In the submucosa they were close to the lymphoid tissue. No differences in the distribution and percentage of NOS-IR fibers and neurons were observed among sheep carrying different PrP genotype, thus making unlikely their contribution in the determinism of susceptibility/resistance to scrapie infection.