The mucosa of the small intestine is clearly able to discriminate specific chemical components of ingested meals to stimulate gastrointestinal feedback pathways and reduce further food intake. Luminal carbohydrates delay gastric emptying and initiate satiation, which are mediated by reflexes via the vagus nerve upon activation of vagal afferent endings in the mucosa. Nutrients activate these nerve fibres through intermediary epithelial cells, which release neuromediators upon transduction of luminal signals through the apical membrane. 5-hydroxytryptamine (5-HT) and glucagon-like peptide-1 (GLP-1) are released from enteroendocrine cells in response to luminal carbohydrates and both slow gastric emptying and inhibit food intake via vagal afferent pathways. The molecular mechanisms for carbohydrate detection and transduction leading to 5-HT and GLP-1 release are unknown. However molecules key to transduction of taste by receptor cells in the lingual epithelium are expressed in the gastrointestinal mucosa. The studies in this thesis aimed to investigate 1) the possibility that taste molecules expressed in the intestine form part of the carbohydrate sensing pathway that leads to 5-HT and GLP-1 release, which in turn activate mucosal vagal afferents and 2) to gauge any alterations in taste molecule expression that may relate to adaptation of carbohydrate-induced gastric motility reflexes that occurs in dietary and disease states. Firstly these studies show key taste molecules, including sweet taste receptors T1R2 and T1R3, the Gprotein gustducin (alpha-subunit Gαgust), and the taste transduction channel TRPM5, are expressed in the mouse gastrointestinal mucosa shown by RT-PCR and were further localised to individual epithelial ‘taste’ cells using immunohistochemistry. Quantification of transcript levels by real time RT-PCR revealed the proximal small intestine as the preferential site of sweet taste receptor expression along the gastrointestinal tract. This finding was also confirmed in humans using gastric and intestinal mucosal biopsies obtained at enteroscopy with significantly higher transcript expression levels in the small intestine compared to stomach. In the mouse, double label immunohistochemistry with Gα[subscript]gust antibody, as a marker of intestinal taste cells, was performed using lectin UEA-1, a marker of intestinal brush cells, and 5-HT or GLP-1 to link intestinal taste transduction to 5-HT and GLP-1 release. Results show Gα[subscript]gust is expressed within a subset of all three cell types in the small intestine but predominantly within UEA-1-expressing cells. Although Gα[subscript]gust, 5-HT and GLP-1 are largely expressed in mutually exclusive cells, within the jejunum a portion Gαgust positive cells coexpressed 5-HT or GLP-1. This Indicates a subpopulation of intestinal taste cells may be dedicated to carbohydrate-evoked gastrointestinal reflexes through 5-HT and GLP-1 mediated pathways, however, taste transduction within the small intestine appears to predominantly link to alternate mediators. After nutrient detection at the luminal surface, activation of mucosal afferents by 5-HT released from enterochromaffin cells is well documented, however although vagal afferents express GLP-1 receptors direct activation has not been demonstrated. For this purpose the effects of GLP-1 on gastrointestinal vagal afferents were investigated through single fibre recordings in in vitro tissue preparations. GLP-1 had no effect on the activity of mouse gastroesophageal vagal afferents but a rat duodenal preparation proved too problematic to be able to test GLP-1 specifically on duodenal vagal afferents. Altered gastric motility in response to carbohydrate meals due to prior dietary patterns and diabetes mellitus suggest adaptation in feedback mechanisms. Towards the second aim of this thesis taste molecule expression was quantified in fed and fasted mice by real time RT-PCR and revealed taste gene transcription is altered with the changing luminal environment, specifically transcription of taste genes was significantly decreased after feeding compared to the fasted state. Studies comparing expression in the duodenum of type 2 diabetics and non-diabetic controls show no significant difference in taste transcript levels between the two groups. However taste molecule expression was correlated to blood glucose levels in diabetics suggesting transcription of these signal molecules is adapted to both luminal and systemic carbohydrate levels. Findings in both the mouse and human gastrointestinal tract in terms of intestinal chemosensing are discussed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363582 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2009

The mucosa of the small intestine is clearly able to discriminate specific chemical components of ingested meals to stimulate gastrointestinal feedback pathways and reduce further food intake. Luminal carbohydrates delay gastric emptying and initiate satiation, which are mediated by reflexes via the vagus nerve upon activation of vagal afferent endings in the mucosa. Nutrients activate these nerve fibres through intermediary epithelial cells, which release neuromediators upon transduction of luminal signals through the apical membrane. 5-hydroxytryptamine (5-HT) and glucagon-like peptide-1 (GLP-1) are released from enteroendocrine cells in response to luminal carbohydrates and both slow gastric emptying and inhibit food intake via vagal afferent pathways. The molecular mechanisms for carbohydrate detection and transduction leading to 5-HT and GLP-1 release are unknown. However molecules key to transduction of taste by receptor cells in the lingual epithelium are expressed in the gastrointestinal mucosa. The studies in this thesis aimed to investigate 1) the possibility that taste molecules expressed in the intestine form part of the carbohydrate sensing pathway that leads to 5-HT and GLP-1 release, which in turn activate mucosal vagal afferents and 2) to gauge any alterations in taste molecule expression that may relate to adaptation of carbohydrate-induced gastric motility reflexes that occurs in dietary and disease states. Firstly these studies show key taste molecules, including sweet taste receptors T1R2 and T1R3, the Gprotein gustducin (alpha-subunit Gαgust), and the taste transduction channel TRPM5, are expressed in the mouse gastrointestinal mucosa shown by RT-PCR and were further localised to individual epithelial ‘taste’ cells using immunohistochemistry. Quantification of transcript levels by real time RT-PCR revealed the proximal small intestine as the preferential site of sweet taste receptor expression along the gastrointestinal tract. This finding was also confirmed in humans using gastric and intestinal mucosal biopsies obtained at enteroscopy with significantly higher transcript expression levels in the small intestine compared to stomach. In the mouse, double label immunohistochemistry with Gα[subscript]gust antibody, as a marker of intestinal taste cells, was performed using lectin UEA-1, a marker of intestinal brush cells, and 5-HT or GLP-1 to link intestinal taste transduction to 5-HT and GLP-1 release. Results show Gα[subscript]gust is expressed within a subset of all three cell types in the small intestine but predominantly within UEA-1-expressing cells. Although Gα[subscript]gust, 5-HT and GLP-1 are largely expressed in mutually exclusive cells, within the jejunum a portion Gαgust positive cells coexpressed 5-HT or GLP-1. This Indicates a subpopulation of intestinal taste cells may be dedicated to carbohydrate-evoked gastrointestinal reflexes through 5-HT and GLP-1 mediated pathways, however, taste transduction within the small intestine appears to predominantly link to alternate mediators. After nutrient detection at the luminal surface, activation of mucosal afferents by 5-HT released from enterochromaffin cells is well documented, however although vagal afferents express GLP-1 receptors direct activation has not been demonstrated. For this purpose the effects of GLP-1 on gastrointestinal vagal afferents were investigated through single fibre recordings in in vitro tissue preparations. GLP-1 had no effect on the activity of mouse gastroesophageal vagal afferents but a rat duodenal preparation proved too problematic to be able to test GLP-1 specifically on duodenal vagal afferents. Altered gastric motility in response to carbohydrate meals due to prior dietary patterns and diabetes mellitus suggest adaptation in feedback mechanisms. Towards the second aim of this thesis taste molecule expression was quantified in fed and fasted mice by real time RT-PCR and revealed taste gene transcription is altered with the changing luminal environment, specifically transcription of taste genes was significantly decreased after feeding compared to the fasted state. Studies comparing expression in the duodenum of type 2 diabetics and non-diabetic controls show no significant difference in taste transcript levels between the two groups. However taste molecule expression was correlated to blood glucose levels in diabetics suggesting transcription of these signal molecules is adapted to both luminal and systemic carbohydrate levels. Findings in both the mouse and human gastrointestinal tract in terms of intestinal chemosensing are discussed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363582 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2009

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VTP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.

The nucleus of the solitary tract (NTS) receives viscerosensory information from the vagus nerve to regulate diverse homeostatic reflex functions. The NTS projects to a wide network of other brain regions, including the paraventricular nucleus of the hypothalamus (PVN). Here we examined the synaptic characteristics of primary afferent pathways to PVN-projecting NTS neurons in rat brainstem slices. Expression of the Transient Receptor Potential Vanilloid receptor (TRPV1+ ) distinguishes C-fiber afferents within the solitary tract (ST) from A-fibers (TRPV1-). We used resiniferatoxin (RTX), a TRPV1 agonist, to differentiate the two. The variability in the latency (jitter) of evoked excitatory postsynaptic currents (ST-EPSCs) distinguished monosynaptic from polysynaptic ST-EPSCs. Rhodamine injected into PVN was retrogradely transported to identify PVN-projecting NTS neurons within brainstem slices. Graded shocks to the ST elicited all-or-none EPSCs in rhodamine-positive NTS neurons with latencies that had either low jitter (<200 µs – monosynaptic), high jitter (>200 µs - polysynaptic inputs) or both. RTX blocked ST-evoked TRPV1 + EPSCs whether mono- or polysynaptic. Most PVN-projecting NTS neurons (17/21 neurons) had at least one input polysynaptically connected to the ST. Compared to unlabeled NTS neurons, PVN-projecting NTS neurons were more likely to receive indirect inputs and be higher order. Surprisingly, sEPSC rates for PVN-projecting neurons were double that of unlabeled NTS neurons. The ST synaptic responses for PVN-projecting NTS neurons were either all TRPV1+ or all TRPV1-, including neurons that received both direct and indirect inputs. Overall, PVN-projecting NTS neurons received direct and indirect vagal afferent information with strict segregation regarding TRPV1 expression.

Endomorphin 1 (10, 30, 100 nmol/kg) administered intravenously (i.v.) to urethane-anesthetized rats consistently and dose-dependently lowered heart rate (HR) and mean arterial pressure (MAP); the decrease in blood pressure recovered faster as compared to the HR. The effects of endomorphin 2 were qualitatively similar. Naloxone (2 mg/kg, i.v.) completely antagonized the bradycardia and hypotension caused by endomorphin 1. Pretreatment of the rats with atropine methylnitrate, atropine sulfate (2 mg/kg, i.v.) or bilateral vagotomy nearly abolished the bradycardia and attenuated the hypotensive effect of endomorphin 1. Our studies suggest that the bradycardia effect following systemic administration of the new opioid peptide may be explained by activation of vagal afferents and the hypotensive effect may be secondary to a reduction of cardiac output and/or a direct vasodilation.

Véritable enjeu de santé publique, l’obésité et ses complications seraient la conséquence d’un état inflammatoire chronique de bas-grade qui pourrait résulter de la présence dans le sang de composés bactériens, les lipopolysaccharides (LPS), état appelé endotoxémie métabolique. Le premier objectif de cette thèse était de comprendre pourquoi les LPS, initialement contenus dans le microbiote, sont capables de traverser l’intestin et d’entrer dans le système sanguin. Mon second objectif était d’étudier l’impact de la composition du microbiote dans le contrôle de la satiété par le nerf vague, lien de communication entre l’intestin et le cerveau. Pour cela, un modèle de rats soumis à un régime obésogène a été utilisée.Mes travaux ont montré que la consommation d’un régime obésogène induisait une perte de la fonction de barrière intestinale au niveau de l’iléon caractérisée par une baisse des défenses mucosales et une augmentation de la perméabilité au LPS. L’obésité est également caractérisée par une altération du comportement alimentaire, avec notamment une réduction de la sensibilité aux signaux de satiété. Nous avons montré que ni l’obésité ni le pourcentage de lipides du régime n’étaient responsables de cette perte de sensibilité aux signaux de satiété mais que l’altération du microbiote en serait le contributeur principal. Ainsi, l’endotoxémie métabolique serait le résultat d’une augmentation du passage transepithelial de LPS, qui, une fois dans le sang, pourraient atteindre, entre autres, le nerf vague où ils perturberaient les signaux intestinaux de satiété. / A real public health issue, obesity and its associated metabolic and behavioral disorders are the consequences of a state of low grade chronic inflammation that might originate from the presence in host plasma of gut-derived bacteria components, lipopolysaccharides (LPS). This present state is called metabolic endotoxemia. The first aim of my thesis was to understand why, in diet-induced obesity (DIO), LPS initially contained in the gut lumen, are able to cross the intestine and enter into the circulatory system. My second aim was to investigate the effect of gut microbiota composition and LPS on the satiety regulation by the vagus nerve, the main communication pathway between the gut and the brain. To answer these questions, we have mainly used a DIO rat model.We showed that consumption of WD induced a loss of ileal barrier function characterized by a reduction in mucosal defenses associated to elevated LPS permeability. Obesity is also characterized by an alteration in feeding behavior including a decreased sensitivity to intestinal satiety signals. We showed that neither obesity nor the lipid percentage of the diet triggers loss of sensitivity to satiety signals but that gut microbiota alterations could rather be the main driver. Hence, metabolic endotoxemia could result from an increased transepithelial passage of LPS, which once spread in the blood could reach, among other things, the vagus nerve where they could disrupt intestinal signals of satiety.