Anatomy and Neuroscience - Research Publications

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    Expression of the relaxin family peptide 4 receptor by enterochromaffin cells of the mouse large intestine
    Koo, A ; Pustovit, R ; Woodward, ORM ; Lewis, JE ; Gribble, FM ; Hossain, MA ; Reimann, F ; Furness, JB (SPRINGER, 2022-05-21)
    The gastrointestinal hormone, insulin-like peptide 5 (INSL5), is found in large intestinal enteroendocrine cells (EEC). One of its functions is to stimulate nerve circuits that increase propulsive activity of the colon through its receptor, the relaxin family peptide 4 receptor (RXFP4). To investigate the mechanisms that link INSL5 to stimulation of propulsion, we have determined the localisation of cells expressing Rxfp4 in the mouse colon, using a reporter mouse to locate cells expressing the gene. The fluorescent signal indicating the location of Rxfp4 expression was in EEC, the greatest overlap of Rxfp4-dependent labelling being with cells containing 5-HT. In fact, > 90% of 5-HT cells were positive for Rxfp4 labelling. A small proportion of cells with Rxfp4-dependent labelling was 5-HT-negative, 11-15% in the distal colon and rectum, and 35% in the proximal colon. Of these, some were identified as L-cells by immunoreactivity for oxyntomodulin. Rxfp4-dependent fluorescence was also found in a sparse population of nerve endings, where it was colocalised with CGRP. We used the RXFP4 agonist, INSL5-A13, to activate the receptor and probe the role of the 5-HT cells in which it is expressed. INSL5-A13 administered by i.p. injection to conscious mice caused an increase in colorectal propulsion that was antagonised by the 5-HT3 receptor blocker, alosetron, also given i.p. We conclude that stimuli that excite INSL5-containing colonic L-cells release INSL5 that, through RXFP4, excites 5-HT release from neighbouring endocrine cells, which in turn acts on 5-HT3 receptors of enteric sensory neurons to elicit propulsive reflexes.
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    Morphologies and distributions of 5-HT containing enteroendocrine cells in the mouse large intestine
    Kuramoto, H ; Koo, A ; Fothergill, LJ ; Hunne, B ; Yoshimura, R ; Kadowaki, M ; Furness, JB (SPRINGER, 2021-02-06)
    Serotonin (5-HT)-containing gastrointestinal endocrine cells contribute to regulation of numerous bodily functions, but whether these functions are related to differences in cell shape is not known. The current study identified morphologies and localization of subtypes of 5-HT-containing enteroendocrine cells in the mouse large intestine. 5-HT cells were most frequent in the proximal colon compared with cecum and distal colon. The large intestine harbored both open (O) cells, with apical processes that reached the lumen, and closed (C) cells, not contacting the lumen, classified into O1, O2, and O3 and C1, C2, and C3 cells, by the lengths of their basal processes. O1 and C1 cells, with basal processes sometimes longer that 100 µm, were most common in the distal colon. Their long basal processes ran against the inner surfaces of the mucosal epithelial cells and were strongly immunoreactive for 5-HT; these processes are ideally placed to communicate with the epithelium and to react to mechanical forces. O2 and C2 cells that had similar but shorter basal processes were also most common in the distal colon. O3 and C3 cells had no or very short basal processes. The O3 open type 5-HT cells were abundant in the proximal colon, particularly at the luminal surface, where they could release 5-HT into the lumen to act on luminal 5-HT receptors. Numerous O3 type 5-HT cells occurred in the lower (submucosal) region of the crypts in all segments and might release 5-HT to influence cell renewal in the crypt proliferative zones.
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    5-HT containing enteroendocrine cells characterised by morphologies, patterns of hormone co-expression, and relationships with nerve fibres in the mouse gastrointestinal tract
    Koo, A ; Fothergill, LJ ; Kuramoto, H ; Furness, JB (SPRINGER, 2021-02-19)
    5-HT containing enteroendocrine cells (EEC), the most abundant type of EEC in the gut, regulate many functions including motility, secretion and inflammatory responses. We examined the morphologies of 5-HT cells from stomach to rectum, patterns of hormone co-expression in the stomach and colon, and the relationship of 5-HT cells with nerve fibres. We also reviewed some of the relevant literature. The morphologies of 5-HT cells were distinct, depending on their location in the gut. A noticeable feature of some 5-HT cells in the antrum and colon was their long basal processes, which resembled processes of neurons, whereas 5-HT cells in the small intestinal mucosa lacked basal processes. In the stomach, numerous 5-HT cells, including cells with basal processes, were identified as enterochromaffin-like cells by their expression of histidine decarboxylase. In the colon, we observed a small number of 5-HT cells that were in close contact with, but distinct from, oxyntomodulin (OXM) and PYY immunoreactive EEC. We did not find specific relationships between nerve fibres and the processes of colonic 5-HT cells. We conclude that five major features, i.e., gut region, morphology, hormone content, receptor repertoire and cell lineage, can be used to define 5-HT cells.
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    A Novel Antagonist Peptide Reveals a Physiological Role of Insulin-Like Peptide 5 in Control of Colorectal Function
    Pustovit, R ; Zhang, X ; Liew, JJM ; Praveen, P ; Liu, M ; Koo, A ; Oparija-Rogenmozere, L ; Ou, Q ; Kocan, M ; Nie, S ; Bathgate, RAD ; Furness, JB ; Hossain, MA (AMER CHEMICAL SOC, 2021-08-30)
    Insulin-like peptide 5 (INSL5), the natural ligand for the relaxin family peptide receptor 4 (RXFP4), is a gut hormone that is exclusively produced by colonic L-cells. We have recently developed an analogue of INSL5, INSL5-A13, that acts as an RXFP4 agonist in vitro and stimulates colorectal propulsion in wild-type mice but not in RXFP4-knockout mice. These results suggest that INSL5 may have a physiological role in the control of colorectal motility. To investigate this possibility, in this study we designed and developed a novel INSL5 analogue, INSL5-A13NR. This compound is a potent antagonist, without significant agonist activity, in two in vitro assays. We report here for the first time that this novel antagonist peptide blocks agonist-induced increase in colon motility in mice that express RXFP4. Our data also show that colorectal propulsion induced by intracolonic administration of bacterial products (short-chain fatty acids, SCFAs) is antagonized by INSL5-A13NR. Therefore, INSL5-A13NR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrheas.
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    Surgical method to prevent early death of neonatal rat pups with Hirschsprung disease, thus permitting development of long-term therapeutic approaches
    Stamp, LA ; Lei, E ; Liew, JJM ; Pustovit, R ; Hao, MM ; Croaker, DH ; Furness, JB ; Adams, CD (OXFORD UNIV PRESS, 2022-01-10)
    Hirschsprung disease occurs when children are born with no intrinsic nerve cells in varying lengths of the large intestine. In the most severe cases, neurons are also missing from the distal part of the small intestine. Nerve-mediated relaxation of the aganglionic bowel fails and fecal matter accumulates in the more proximal regions of the intestine. This is life threatening. Perforation of the bowel can ensue, causing sepsis and in some cases, death of the infant. Repopulation of the colon with neural stem cells is a potential therapy, but for this to be successful the patient or experimental animal needs to survive long enough for neural precursors to differentiate and make appropriate connections. We have developed a surgical procedure that can be applied to rats with Hirschsprung disease. A stoma was created to allow the normal bowel to empty and a second stoma leading to the aganglionic bowel was also created. This allowed homozygous mutants that would usually die at less than 3 weeks of age to survive into adulthood. During this time, the rats also required post-operative care of their stomas. The interventions we describe provide an animal model of Hirschsprung disease that is suited to assess the effectiveness of cell therapies in the treatment of this condition.
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    Morphologies, dimensions and targets of gastric nitric oxide synthase neurons
    Di Natale, MR ; Hunne, B ; Liew, JJM ; Fothergill, LJ ; Stebbing, MJ ; Furness, JB (SPRINGER, 2022-02-11)
    We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.
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    ATH434 Reverses Colorectal Dysfunction in the A53T Mouse Model of Parkinson's Disease
    Diwakarla, S ; McQuade, RM ; Constable, R ; Artaiz, O ; Lei, E ; Barnham, KJ ; Adlard, PA ; Cherny, RA ; Di Natale, MR ; Wu, H ; Chai, X-Y ; Lawson, VA ; Finkelstein, D ; Furness, JB (IOS PRESS, 2021-01-01)
    BACKGROUND: Gastrointestinal (GI) complications, that severely impact patient quality of life, are a common occurrence in patients with Parkinson's disease (PD). Damage to enteric neurons and the accumulation of alpha-synuclein in the enteric nervous system (ENS) are thought to contribute to this phenotype. Copper or iron chelators, that bind excess or labile metal ions, can prevent aggregation of alpha-synuclein in the brain and alleviate motor-symptoms in preclinical models of PD. OBJECTIVE: We investigated the effect of ATH434 (formally PBT434), a small molecule, orally bioavailable, moderate-affinity iron chelator, on colonic propulsion and whole gut transit in A53T alpha-synuclein transgenic mice. METHODS: Mice were fed ATH434 (30 mg/kg/day) for either 4 months (beginning at ∼15 months of age), after the onset of slowed propulsion ("treatment group"), or for 3 months (beginning at ∼12 months of age), prior to slowed propulsion ("prevention group"). RESULTS: ATH434, given after dysfunction was established, resulted in a reversal of slowed colonic propulsion and gut transit deficits in A53T mice to WT levels. In addition, ATH434 administered from 12 months prevented the slowed bead expulsion at 15 months but did not alter deficits in gut transit time when compared to vehicle-treated A53T mice. The proportion of neurons with nuclear Hu+ translocation, an indicator of neuronal stress in the ENS, was significantly greater in A53T than WT mice, and was reduced in both groups when ATH434 was administered. CONCLUSION: ATH434 can reverse some of the GI deficits and enteric neuropathy that occur in a mouse model of PD, and thus may have potential clinical benefit in alleviating the GI dysfunctions associated with PD.
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    The first brain: Species comparisons and evolutionary implications for the enteric and central nervous systems
    Furness, JB ; Stebbing, MJ (WILEY, 2018-02-01)
    BACKGROUND: The enteric nervous system (ENS) and the central nervous system (CNS) of mammals both contain integrative neural circuitry and similarities between them have led to the ENS being described as the brain in the gut. PURPOSE: To explore relationships between the ENS and CNS across the animal kingdom. We found that an ENS occurs in all animals investigated, including hydra, echinoderms and hemichordates that do not have a CNS. The general form of the ENS, which consists of plexuses of neurons intrinsic to the gut wall and an innervation that controls muscle movements, is similar in species as varied and as far apart as hydra, sea cucumbers, annelid worms, octopus and humans. Moreover, neurochemical similarities across phyla imply a common origin of the ENS. Investigation of extant species suggests that the ENS developed in animals that preceded the division that led to cnidaria (exemplified by hydra) and bilateria, which includes the vertebrates. The CNS is deduced to be a bilaterian development, later than the divergence from cnidaria. Consistent with the ENS having developed independent of the CNS, reciprocal connections between ENS and CNS occur in mammals, and separate neurons of ENS and CNS origin converge on visceral organs and prevertebral ganglia. We conclude that an ENS arose before and independently of the CNS. Thus the ENS can be regarded as the first brain.
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    Heterogeneity of enterochromaffin cells within the gastrointestinal tract
    Diwakarla, S ; Fothergill, LJ ; Fakhry, J ; Callaghan, B ; Furness, JB (WILEY, 2017-06-01)
    Enterochromaffin cells were the first endocrine cells of the gastrointestinal tract to be chemically distinguished, almost 150 years ago. It is now known that the chromaffin reaction of these cells was due to their content of the reactive aromatic amine, 5-hydroxytryptamine (5-HT, also known as serotonin). They have commonly been thought to be a special class of gut endocrine cells (enteroendocrine cells) that are distinct from the enteroendocrine cells that contain peptide hormones. The study by Martin et al. in the current issue of this journal reveals that the patterns of expression of nutrient receptors and transporters differ considerably between chromaffin cells of the mouse duodenum and colon. However, even within regions, chromaffin cells differ; in the duodenum there are chromaffin cells that contain both secretin and 5-HT, cholecystokinin and 5-HT, and all three of secretin, cholecystokinin, and 5-HT. Moreover, the ratios of these different cell types differ substantially between species. And, in terms of function, 5-HT has many roles, including in appetite, motility, fluid secretion, release of digestive enzymes and bone metabolism. The paper thus emphasizes the need to define the many different classes of enterochromaffin cells and relate this to their roles.