Physiology - Theses

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    The effect of early life antibiotic exposure on the development of the gut microbiota and enteric nervous system
    Hung, Lin Yung ( 2019)
    Early postnatal life is a critical stage of microbiota establishment and ENS development. While the initial postnatal stage from birth is fundamental for the development of the gut microbiota and ENS, weaning is another key developmental period where there are major changes in diet, behaviour and physiology, and notably, microbiota. Antibiotics are frequently administered to infants and young children, however, recent studies have identified prospective long-term health consequences of early life antibiotic exposure on the developing gut microbiota. Yet, how antibiotic influences short and long-term ENS development remains unclear. Vancomycin is given as a prophylactic to preterm babies and paediatric patients to treat and prevent infections. It is also one of the most commonly used antibiotics on its own or as part of a cocktail in research to induce dysbiosis in mice. The aim of my PhD was to examine how early life exposure to vancomycin during two critical developmental periods affects microbiota and ENS development and whether changes observed during early postnatal life have long-term repercussions. In chapter 3, I investigated if acute administration of vancomycin, during the early postnatal period, influenced gut microbiota and ENS development. A single regimented dose of either water or vancomycin was administered daily to Wnt1-Cre;R26R-GCaMP3 mouse pups from postnatal (P) day 0 to P10/11. These mice contain a genetically-encoded fluorescent Ca2+ indicator in all enteric neurons and glia. At P10/11, vancomycin-fed pups showed significant dysbiosis, reduced myenteric neuron density and altered nNOS and calbindin neuronal subtype proportions compared to water-fed littermates. Using Ca2+ imaging, I showed that vancomycin-fed pups had more neurons responding to electrical stimulation applied to interganglionic connectives and larger amplitudes of train-evoked [Ca2+]i transients. These changes in the ENS contributed to dysmotility of the colon of vancomycin-fed pups. In contrast to the colon, the structure of the ENS and motility patterns of the duodenum were not affected by vancomycin, ruling out drug toxicity effects. P10/11 vancomycin-fed pups also had lower numbers of serotonin (5-HT) positive cells in the colonic mucosa. Altered 5-HT metabolism in these animals were confirmed by performing mass spectrometry on 5-HT biosynthesis intermediates, showing reduced concentrations of the 5-HT metabolite, 5-HIAA and droplet digital PCR (ddPCR) revealing increased gene expression of the 5-HT transporter, SERT. Bypassing tryptophan hydroxylase, by supplementing vancomycin-fed pups with 5-HTP, restored 5-HIAA levels in the colonic mucosa and prevented some of the vancomycin-induced effects on myenteric neurons, colonic motility and gut microbiota. Therefore, vancomycin exposure during the neonatal period induced significant developmental changes to both the gut microbiota and ENS. Some of these changes could be mediated by altered mucosal serotonergic signalling. In Chapter 4, I examined if vancomycin-induced changes on the gut microbiota and ENS observed at P10 were long-lasting. Newborn mouse pups were only treated with water and vancomycin till P10, then pups were left to grow to adulthood. 6-week-old mice given neonatal vancomycin had enlarged caeca, which is an indication of dysbiosis. This suggests that the gut microbiota of vancomycin-fed mice was not fully recovered despite cessation of antibiotic treatment. Adult mice treated with neonatal vancomycin had sustained reduction in myenteric neuron density. However, alterations in the proportions of nNOS+ and calbindin+ neurons observed during the neonatal periods was now restored. In contrast to the heightened [Ca2+]i activity at P10s, adult mice given neonatal vancomycin had lower numbers of neurons responding to electrical stimulation and no change in the amplitudes of electrically-evoked [Ca2+]i transients in their myenteric neurons compared to water-fed controls. Furthermore, there were no treatment-induced changes in colonic motility. Interestingly, faecal water content, which was unaffected in vancomycin-fed pups at P10, was lower in adult mice given neonatal vancomycin compared to controls. These findings indicate that although vancomycin treatment is terminated, the gut microbiota is not fully recovered and significant re-modelling of the ENS occurs, some of which are distinct to changes observed during the neonatal period. In Chapter 5, I explored the effects of vancomycin exposure between weaning and adulthood. From the day of weaning, mice were administered vancomycin or sterile water in their drinking bottles for three weeks. At 6-weeks of age, vancomycin-treated mice had dysbiosis accompanied with enlarged caeca. Similar to vancomycin-treated neonates in Chapter 3, increased synaptic activity exhibited by enteric neurons were mainly observed by larger amplitudes of train-evoked [Ca2+]i transients and increased number of neurons responding to electrical stimulation. However, in contrast to antibiotic exposure during the neonatal period, vancomycin-treated mice displayed significantly slower colonic motility, increased faecal water content and a decrease in the proportions of ChAT+ cholinergic neurons including calbindin and neurofilament-M subtypes in the myenteric plexus of the colon. Moreover, vancomycin treatment between weaning and adulthood had no effects on the serotonergic system in the colonic mucosa. Collectively, these findings suggest that vancomycin exposure from weaning had differential effects on the gut microbiota and ENS compared to administration of the antibiotic during the neonatal period. Together, my study is the first to identify and compare effects of antibiotic exposure on the gut microbiota and ENS during two critical stages of development. While vancomycin did not deplete bacterial diversity and abundance, it caused profound shifts in microbial composition in both developmental periods. Additionally, acute vancomycin exposure in both periods, resulted in dysmotility and alterations of the neuronal circuitry. Although the effects on colonic motility for mice given neonatal antibiotic treatment did not appear to be long-lasting, changes in the ENS and disrupted faecal and caeca weights, which manifested only in adulthood, suggests that early life exposure to antibiotics can have other long-term consequences on microbiota and host gut physiology.
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    Neural mechanisms involved in enterotoxin- induced intestinal hypersecretion
    Koussoulas, Katerina ( 2017)
    Exotoxins of the bacteria Vibrio cholerae (cholera toxin, CT) and Clostridium difficile (C. diff., TcdA) induce rampant disease in the form of irresolvable diarrhoea causing rapid dehydration and potential death if left untreated. Both bacterial toxins affect the nervous system of the gut, the enteric nervous system (ENS), but the types of enteric neurons involved are still indistinct. Additionally, preliminary work from a collaborator showed that specific bacterial metabolites, particularly GABA produced by the microorganisms residing in the gut, exacerbate pathophysiological effects of C. diff. GABA and its receptors are expressed in several parts of the gut wall, including enteric neurons. While studies have proposed GABA to be a putative neurotransmitter in the ENS, physiological roles of GABA in the gut remain unclear. It is unknown how enterotoxins and increased GABA at the level of the gut mucosa activate underlying enteric circuitry; my PhD aimed to elucidate these mechanisms. In Chapter 3, I investigated the enteric neural pathways underlying CT effects via in vitro incubations of CT in guinea pig jejunum. Previous work highlighted the impacts of CT on secretomotor neurons; I endeavoured to expand this by examining other key neuronal subtypes. I recorded neuronal activity in the myenteric plexus (MP) up to 6 hours after CT incubation via intracellular electrophysiology. A colleague undertook similar recordings in the submucosal plexus (SMP). We found that CT induced hyperexcitability in myenteric, but not submucosal, sensory neurons. The effect was neurally mediated and required activation of NK3 tachykinin receptors, but was independent of activation of 5-HT3 receptors or NK1 tachykinin receptors, suggesting that the effects of CT on myenteric sensory neurons are likely to be indirect and via a pathway independent of 5-HT release. In Chapter 4, I determined the effects of luminal incubations TcdA and GABA on myenteric sensory neurons via electrophysiology. I found that in vitro incubations of guinea pig jejuna with TcdA or GABA also increased the excitability of myenteric sensory neurons, highlighting the key role of these neurons as a common point through which enterotoxins and GABA operate. The GABA-induced effects were inhibited by GABAB and GABAC receptor antagonists, but enhanced by a GABAA antagonist, indicating involvement of at least two distinct GABA activated pathways. The GABAA antagonist enhanced excitability on its own suggesting that tonic release of endogenous GABA may play a role in suppressing the excitability of these neurons. In Chapter 5, I explored the role of endogenous GABA in the ENS of mouse small intestine. I employed Wnt1-Cre;R26R-GCaMP3 mice, which express a fluorescent calcium indicator in the ENS, for use in Ca2+ imaging. Neurons responded to GABA exposure via activation of GABAA, GABAB and GABAC receptors in myenteric ganglia. Further, I showed that the effects of GABA were neuronal subtype specific, for example neurons immunoreactive for neuronal nitric oxide synthase rarely responded to GABA. I also demonstrated that endogenous release of GABA may inhibit activation of myenteric neurons by activation of GABAC receptors, despite such receptors exciting myenteric neurons when activated by exogenous GABA. My data also suggest that neither GABAA nor GABAB receptors contribute to synaptic transmission in this system. Further I also demonstrated the expression of GABA in neurons and varicosities surrounding specific enteric neurons within the MP. This study clarifies the complex nature of GABAergic transmission in the ENS. In Chapter 6 to further examine the effects of enterotoxins on the enteric circuitry, I made intracellular recordings from myenteric neurons following in vivo incubations of CT in mouse ileal loops. A lab member previously showed that CT increases calcium responses in the submucosal but not myenteric, neurons. In undertaking electrophysiological recordings, a striking sampling bias was revealed with exclusion of largely descending interneurons and inhibitory motor neurons being markedly underrepresented in the data set and low sampling from sensory neurons meant that significant effects in excitability may have been missed. Nevertheless in concordance with the results from Ca2+ imaging, no significant changes in excitability of myenteric neurons were found at their resting membrane potential. However, CT induced spontaneous synaptic activity in specific myenteric neurons, but the sources of this input could not be identified due to the technical difficulty of maintaining impalements, the relative rarity of myenteric sensory neurons and the sampling bias. The data suggest a minor role for myenteric neurons in CT-induced hypersecretion in vivo. In Chapter 7 I employed the high throughput assay of Ca2+ imaging to perform a more extensive examination of the effects of TcdA on the ENS. I utilized the well-established ileal loop mouse model and incubated TcdA in vivo. Spontaneous and neurally-stimulated calcium responses were reduced in submucosal neurons, and myenteric neuronal activity was unchanged. However enteric neurons in regions of the gastrointestinal tract off-target from the site of acute toxin exposure were activated during the incubation as indicated by expression of activity dependent markers. This off target effect could possibly be due to release of inflammatory cytokines into the circulation or extrinsic neural pathways. In all, I have demonstrated a generality in the actions of enterotoxins and GABA as the pathways they activate converge to excite myenteric sensory neurons which may lead to activation of submucosal secretomotor neurons. I have extended our understanding of the role of GABA in the ENS as a means to elucidate the mechanisms through which microbial metabolites act and contribute to disease. Using the mouse ileal loop model, I further defined the effects of enterotoxins on the enteric circuitry. In this way, my thesis highlights neural elements involved in the mechanisms underlying enterotoxin-induced hypersecretion and identifies potential avenues for future research.
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    The role of submucosal neurons in physiological and pathophysiological intestinal secretion
    Fung, Candice ( 2016)
    The enteric nervous system (ENS) has two major types of secretomotor neurons, which contain either acetylcholine (i.e. are cholinergic) or vasoactive intestinal peptide (VIP). These are well conserved across species, and are key mediators of physiological and pathophysiological intestinal secretion. Notably, VIP is a potent secretogogue implicated in cholera toxin (CT)-evoked hypersecretion – the most widely-studied model of bacterial toxin-induced diarrhoea. As diarrhoeal disease remains a significant health problem worldwide, a better understanding of these secretomotor pathways and mediators in physiological and pathophysiological secretion is crucial for advancing therapeutic treatments. While acetylcholine is an established enteric neurotransmitter, VIP has long been considered a putative transmitter. VIP acts via VPAC1 and VPAC2 receptors, but their physiological role within the enteric circuitry remains unclear. Despite previous efforts to better characterize the role of VIP, a number of discrepancies arose from these studies. These issues were mainly due to the lack of selective pharmacological tools, as well as technical limitations. Furthermore, although the secretory effects of CT have been studied extensively, the degree of enteric neural involvement, and the relative contribution of cholinergic and VIP secretomotor neurons remains debatable. Thus, the aim of my PhD studies was to address these longstanding questions which have impeded our understanding of the control of intestinal secretion by employing specific agonists and antagonists for VPAC receptors and more advanced experimental techniques that are now available. In Chapter 3, I localized VPAC1 receptors to a subset of cholinergic secretomotor neurons and cholinergic excitatory longitudinal muscle motor neurons. Further, I demonstrated that VIP acts via VPAC1 receptors on cholinergic secretomotor neurons to stimulate secretion and longitudinal muscle contraction. This was done using a combination of molecular, immunohistochemical and functional studies, as well as selective antagonists. There was no evidence of VPAC2 receptor involvement. In Chapter 4, I identified a novel role of VIP and VPAC receptors in modulating neuro-glial communication in the mouse submucosal plexus. This was achieved by performing Ca2+ imaging on transgenic Wnt1-Cre;R26R-GCaMP3 mice, which express a fluorescent Ca2+ indicator in their ENS (enteric neurons and glia). VIP application to submucosal ganglia exclusively evoked Ca2+ responses in neurons, but surprisingly, using specific agonists and antagonists for VPAC1 and 2 revealed a role for VIP as a transmitter in signaling from neurons to glia. Activating VPAC1 receptors initiates neuron-to-glia signaling by stimulating purine release. Whereas, activating VPAC2 receptors suppresses this signaling pathway. Thus, VIP may have a dual role through its activation of VPAC1 and 2 receptors in fine-tuning purinergic neuron-glia communication. In Chapter 5, I used an in vivo mouse ileal loop model of CT-incubation, followed by a series of in vitro analysis to investigate the relative contributions of cholinergic and VIP secretomotor neurons to CT-evoked hypersecretion. Following the incubation, I found an increased fluid accumulation in CT-treated loops which corresponded to an increase in basal secretion measured using Ussing chambers. This hypersecretory effect did not appear to have a significant neuronal component. However, I found that CT induced a sustained increase in the excitability of cholinergic submucosal neurons by utilizing Wnt1-Cre;R26R-GCaMP3 mice to examine Ca2+ activity within the enteric circuitry. Increased spontaneous activity and enhanced responses to neural stimulation was observed in cholinergic submucosal neurons following CT-exposure. Further, CT was found to activate most submucosal neurons with the increased expression of an activity-dependent marker pCREB. I also found that the myenteric plexus did not significantly contribute to the sustained hypersecretion. Collectively, I showed that CT induced sustained hypersecretion by a direct effect on the mucosa, but also partly by increasing the excitability of cholinergic submucosal neurons.
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    Mathematical and computer modelling of the enteric nervous system
    Thomas, Evan Alexander ( 2001)
    The enteric nervous system (ENS) runs within the intestinal wall and is responsible for initiating and enacting several reflexes and motor patterns, including peristalsis and the complex interdigestive motor programs, known as migrating motor complexes (MMCs). The ENS consists of several neuron types including intrinsic sensory neurons, interneurons and motor neurons. A great deal is known about the anatomy, pharmacology and electrophysiology of the ENS, yet there is almost no understanding of how enteric neural circuits perform the functions that they do and how they switch from one function to another. The ENS contains intrinsic sensory neurons (ISNs) that connect to every neuron type in the ENS, including making recurrent connections amongst themselves. Thus, they are likely to play a key role, not just in sensory transduction, but in coordination of reflexes and motor patterns. This thesis has explored how these functions are performed by developing and analysing mathematical and computer models of the network of ISNs. (For complete abstract open document)
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    Enteric serotonin interneurons: connections and role in intestinal movement
    NEAL, KATHLEEN BRONWYN ( 2008)
    5-HT powerfully affects gastrointestinal function. However, the study of these effects is complicated because 5-HT from both mucosa and a subset of enteric neurons acts on multiple receptor subtypes in enteric tissues. The role of neural 5-HT has been difficult to isolate with current techniques. This thesis aimed to elucidate the role of 5-HT neurons in motility using anatomical and functional methods. In Chapter 2, confocal microscopy was used to examine over 95% of myenteric neurons in guinea pig jejunum, categorized neurochemically, to identify neurons that received anatomically-defined input from 5-HT interneurons. The data showed that cholinergic secretomotor neurons were strongly targeted by 5-HT interneurons. In another key finding, excitatory motor neurons were surrounded by 5-HT terminals; this could provide an anatomical substrate for the descending excitation reflex. Subgroups of ascending interneurons and neurons with immunoreactivity for NOS, were also targeted by 5-HT interneurons. Thus, subtypes of these neurons might act in separate reflex pathways. Despite strong physiological evidence for 5-HT inputs to AH/Dogiel type II neurons, few contacts were identified. In Chapter 3, the confocal microscopy survey was extended to the three other interneuron classes (VIP/NOS and SOM descending interneurons; calretinin ascending interneurons) of guinea pig small intestine. A high degree of convergence between the otherwise polarized ascending and descending interneuron pathways was identified.
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    Insights into intestinal fed-state motor patterns
    Chambers, Jordan David ( 2010)
    After a meal, the duodenum and jejunum exhibit a specific set of contractile patterns, collectively known as the fed-state, which facilitate the functions of the gastrointestinal tract. Non-propagating contractions observed during the fed-state are known as segmentation. In the guinea pig small intestine, nutrient induced motor activity consists of a number of discrete motor patterns including propulsive contractions and rhythmic stationary contractions (segmentation) that occur episodically at specific locations along the intestine. In addition to rhythmic stationary contractions, during segmentation episodes of activity incorporating all contractions last 40-60s and are separated by quiescent periods lasting 40-200s. The enteric nervous system (ENS) regulates segmentation, but the exact circuit is unknown. Possible circuits were investigated using a combination of computer models and video recordings of segmentation in vitro using guinea-pig jejunum, in which segmentation was induced with luminal fatty acid. A simple computer model simulated the mean neuron firing rate in the feedforward ascending and descending reflex pathways. A stimulus evoked pacemaker was located in the afferent pathway or in a feedforward pathway. Output of the feedforward pathways was fed into a simple model to determine the response of the muscle. In this computational model, local stimuli produced an oral contraction and anal dilation, similar to in vitro responses to local distension, but did not produce segmentation. When the stimulus was distributed, representing a nutrient load, the result was either a tonic response or globally synchronized oscillations. However, when local variations were introduced, stationary contractions occurred around these locations. This predicts that severing the ascending and descending pathways will induce stationary contractions. An acute lesion of the longitudinal muscle, myenteric plexus and circular muscle around the entire circumference of the intestine significantly increased the number of stationary contractions immediately oral and anal to the lesion in vitro. These results suggest spatially localised rhythmic contractions arise from a local imbalance between ascending excitatory and descending inhibitory muscle inputs. Also, these results require a distributed stimulus and a rhythm generator in the afferent pathway. A possible rhythm generator in the afferent pathway was targeted pharmacologically by blocking after-hyperpolarising potentials (AHPs) in sensory neurons in vitro. Blocking AHPs did not affect properties of rhythmic stationary contractions, but increased the duration of activity episodes without affecting quiescent periods. This indicates that there are at least two separate rhythm generators operating concurrently during segmentation, which determine the rhythms of stationary contraction and episodes of activity. A computer model was developed to explain the changes in activity episodes without affecting the quiescent periods. The model described activity in sensory neurons, excitatory and inhibitory motor neurons, and feedback to sensory neurons. The model could reproduce the pharmacological data provided there was feedback to sensory neurons via contraction induced serotonin release. It also required fast transmission from sensory neurons to excitatory motor neurons and slow transmission from sensory neurons to inhibitory motor neurons. Therefore, the model identified plausible functions for known mechanisms and predicted relative strength of synaptic transmission in the ENS.