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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    Neuronal regulation of the gut immune system and neuromodulation for treating inflammatory bowel disease
    Populin, L ; Stebbing, MJ ; Furness, JB (WILEY, 2021-08-27)
    The gut immune system in the healthy intestine is anti-inflammatory, but can move to a pro-inflammatory state when the gut is challenged by pathogens or in disease. The nervous system influences the level of inflammation through enteric neurons and extrinsic neural connections, particularly vagal and sympathetic innervation of the gastrointestinal tract, each of which exerts anti-inflammatory effects. Within the enteric nervous system (ENS), three neuron types that influence gut immune cells have been identified, intrinsic primary afferent neurons (IPANs), vasoactive intestinal peptide (VIP) neurons that project to the mucosa, and cholinergic neurons that influence macrophages in the external muscle layers. The enteric neuropeptides, calcitonin gene-related peptide (CGRP), tachykinins, and neuromedin U (NMU), which are contained in IPANs, and VIP produced by the mucosa innervating neurons, all influence immune cells, notably innate lymphoid cells (ILCs). ILC2 are stimulated by VIP to release IL-22, which promotes microbial defense and tissue repair. Enteric neurons are innervated by the vagus, and, in the large intestine, by the pelvic nerves. Vagal nerve stimulation reduces gut inflammation, which may be both by stimulation of efferent (motor) pathways to the ENS, and stimulation of afferent pathways that connect to integrating centers in the CNS. Efferent pathways from the CNS have their anti-inflammatory effects through either or both vagal efferent neurons and sympathetic pathways. The final neurons in sympathetic pathways reduce gut inflammation by the action of noradrenaline on β2 adrenergic receptors expressed by immune cells. Activation of neural anti-inflammatory pathways is an attractive option to treat inflammatory bowel disease that is refractory to other treatments. Further investigation of the ways in which enteric reflexes, vagal pathways and sympathetic pathways integrate their effects to modulate the gut immune system and gut inflammation is needed to optimize neuromodulation therapy.
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    Analysis of Bioavailability and Induction of Glutathione Peroxidase by Dietary Nanoelemental, Organic and Inorganic Selenium
    Ringuet, MT ; Hunne, B ; Lenz, M ; Bravo, DM ; Furness, JB (MDPI, 2021-04-01)
    Dietary organic selenium (Se) is commonly utilized to increase formation of selenoproteins, including the major antioxidant protein, glutathione peroxidase (GPx). Inorganic Se salts, such as sodium selenite, are also incorporated into selenoproteins, and there is evidence that nanoelemental Se added to the diet may also be effective. We conducted two trials, the first investigated inorganic Se (selenite), organic Se (L-selenomethionine) and nanoelemental Se, in conventional mice. Their bioavailability and effectiveness to increase GPx activity were examined. The second trial focused on determining the mechanism by which dietary Se is incorporated into tissue, utilising both conventional and germ-free (GF) mice. Mice were fed a diet with minimal Se, 0.018 parts per million (ppm), and diets with Se supplementation, to achieve 0.07, 0.15, 0.3 and 1.7 ppm Se, for 5 weeks (first trial). Mass spectrometry, Western blotting and enzymatic assays were used to investigate bioavailability, protein levels and GPx activity in fresh frozen tissue (liver, ileum, plasma, muscle and feces) from the Se fed animals. Inorganic, organic and nanoelemental Se were all effectively incorporated into tissues. The high Se diet (1.7 ppm) resulted in the highest Se levels in all tissues and plasma, independent of the Se source. Interestingly, despite being ~11 to ~25 times less concentrated than the high Se, the lower Se diets (0.07; 0.15) resulted in comparably high Se levels in liver, ileum and plasma for all Se sources. GPx protein levels and enzyme activity were significantly increased by each diet, relative to control. We hypothesised that bacteria may be a vector for the conversion of nanoelemental Se, perhaps in exchange for S in sulphate metabolising bacteria. We therefore investigated Se incorporation from low sulphate diets and in GF mice. All forms of selenium were bioavailable and similarly significantly increased the antioxidant capability of GPx in the intestine and liver of GF mice and mice with sulphate free diets. Se from nanoelemental Se resulted in similar tissue levels to inorganic and organic sources in germ free mice. Thus, endogenous mechanisms, not dependent on bacteria, reduce nanoelemental Se to the metabolite selenide that is then converted to selenophosphate, synthesised to selenocysteine, and incorporated into selenoproteins. In particular, the similar efficacy of nanoelemental Se in comparison to organic Se in both trials is important in the view of the currently limited cheap sources of Se.
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    Dopamine and ghrelin receptor co-expression and interaction in the spinal defecation centers
    Furness, JB ; Pustovit, R ; Syder, AJ ; Ringuet, MT ; Yoo, EJ ; Fanjul, A ; Wykosky, J ; Fothergill, LJ ; Whitfield, EA ; Furness, SGB (WILEY, 2020-12-02)
    BACKGROUND: Dopamine receptor 2 (DRD2) and ghrelin receptor (GHSR1a) agonists both stimulate defecation by actions at the lumbosacral defecation center. Dopamine is in nerve terminals surrounding autonomic neurons of the defecation center, whereas ghrelin is not present in the spinal cord. Dopamine at D2 receptors generally inhibits neurons, but at the defecation center, its effect is excitatory. METHODS: In vivo recording of defecation and colorectal propulsion was used to investigate interaction between DRD2 and GHSR1a. Localization studies were used to determine sites of receptor expression in rat and human spinal cord. KEY RESULTS: Dopamine, and the DRD2 agonist, quinpirole, directly applied to the lumbosacral cord, caused defecation. The effect of intrathecal dopamine was inhibited by the GHSR1a antagonist, YIL781, given systemically, but YIL781 was not an antagonist at DRD2. The DRD2 agonist, pramipexole, administered systemically caused colorectal propulsion that was prevented when the pelvic nerves were cut. Drd2 and Ghsr were expressed together in autonomic preganglionic neurons at the level of the defecation centers in rat and human. Behaviorally induced defecation (caused by water avoidance stress) was reduced by the DRD2 antagonist, sulpiride. We had previously shown it is reduced by YIL781. CONCLUSIONS AND INFERENCES: Our observations imply that dopamine is a transmitter of the defecation pathways whose actions are exerted through interacting dopamine (D2) and ghrelin receptors on lumbosacral autonomic neurons that project to the colorectum. The results explain the excitation by dopamine agonists and the conservation of GHSR1a in the absence of ghrelin.