Influence of Cholecystokinin-8 on compound nerve action potentials from ventral gastric vagus in rats

Objective: Vagus Nerve Stimulation (VNS) has shown great promise as a potential therapy for a number of conditions, such as epilepsy, depression and for Neurometabolic Therapies, especially for treating obesity. The objective of this study was to characterize the left ventral subdiaphragmatic gastric trunk of vagus nerve (SubDiaGVN) and to analyze the influence of intravenous injection of gut hormone cholecystokinin octapeptide (CCK-8) on compound nerve action potential (CNAP) observed on the same branch, with the aim of understanding the impact of hormones on VNS and incorporating the methods and results into closed loop implant design. Methods: The cervical region of the left vagus nerve (CerVN) of male Wistar rats was stimulated with electric current and the elicited CNAPs were recorded on the SubDiaGVN under four different conditions: Control (no injection), Saline, CCK1 (100pmol/kg) and CCK2 (1000pmol/kg) injections. Results: We identified the presence of

Investigating the physiological and pharmacological effects of the gut hormone peptide YY (PYY)

The obesity epidemic is a critical and global public health burden. Drugs that safely promote weight loss are urgently needed to halt the rising prevalence of obesity and its associated complications, such as type 2 diabetes (T2D). Gut hormones are important regulators in metabolism and have therapeutic potential as treatments for obesity and T2D. The gut hormone peptide YY (PYY) is released from the intestine after a meal. Exogenous PYY3–36 suppresses food intake in both rodents and humans, including in the obese state. PYY3–36 suppresses appetite by acting on its receptor, the Y2R. Y2R is expressed in brain appetite centres but also in the afferent vagus nerve, the main neuroanatomical link carrying information from the gut to the brain. However, the relevant contribution of the afferent vagus to the overall effects of PYY3–36 is unknown. Chemogenetic activation of vagal afferent neurones results in reduced food intake (surpassing the effects of PYY) and might have altered the immune landscape of the gastrointestinal tract. To dissect the role of the Y2R expressed in the afferent vagus, we have developed a novel microsurgical technique in the mouse. Our work suggests that vagal Y2R mediates the anorectic effect of low dose and endogenous PYY3–36 and that this vagal signalling pathway regulates short-term feeding. This anorectic effect was not caused by an aversive response. In vitro calcium imaging confirmed that PYY3–36 directly activates vagal afferents. Chronic treatment of diet-induced obese (DIO) mice with a long-acting PYY3–36 analogue, Y242, did not cause a significant body weight loss. Longitudinal tracking of individual islet function using a novel imaging platform allowed to study the effect of diet and Y242 treatment. Chronic Y242 did not improve or worsen islet function in obese mice. Therefore, PYY-based treatments might not be suitable as a single agent but have potential in combination with other gut-hormones. Vagus nerve neuromodulation has shown potential as an anti-obesity therapy and the work in this thesis adds to a better understanding of vagal afferent function which will help optimise therapeutic interventions.Open Acces

Oleoylethanolamide in the gut-brain axis

Oleoylethanolamide (OEA), a PPAR-α agonist, is a mediator of satiety. After peripheral administration, OEA induces Fos expression and activation in areas of the CNS involved in the control of feeding behavior and energy homeostasis, such as the nucleus of the solitary tract (NST) and in the area postrema (AP) in the brainstem, the hypothalamic paraventricular (PVN), supraoptic (SON) and ventral tuberomammillary (vTMN) nuclei. Moreover, it is known to increase the noradrenergic trasmission in the NST and AP, by increasing the expression of the dopamine-β-hydroxylase (DBH). Visceral ascending fibers were hypothesized to mediate such effects, but recent findings demonstrate that abdominal vagal afferents are not necessary for the anorectic effect of OEA. In fact, OEA is able to decrease food intake both in rats that underwent a subdiaphragmatic vagal deafferentation (SDA), a surgical procedure that eliminates all abdominal vagal afferents but spares about 50% of the vagal efferents, and in SHAM controls. Thus, the aim of the present work was to better elucidate the role of abdominal vagal afferents in mediating OEA's effects on the CNS. To meet this aim, we subjected rats to SDA surgery, using SHAM rats as control. By using immunohistochemistry, Fos and DBH expression patterns were investigated in the NST, in the AP, and in the hypothalamus after OEA administration (10 mg kg -1). Consistently with the behavioral results, OEA increases Fos expression in the NST and in the AP. Moreover, in these nuclei, SDA did not cause any alteration of DBH expression. In the hypothalamus, in line with the behavioral results, OEA is able to increase Fos expression in the PVN and the vTMN, even though in the latter does not reach statistical significance. Overall, our findings indicate that vagal afferents are not strictly necessary for the satiety effect of OEA at both behavioral and neurochemical levels

The mechanism of action of capsaicin on sensory C-type neurones and their axons in vitro

The mechanism of action of the sensory neurotoxin, capsaicin, on visceral afferent fibres and ganglion cells has been studied using electrophysiological and histological techniques. Extracellular in vitro recording from adult vagus nerves revealed a depolarization and a reduced C-spike amplitude. These probably reflect effects on unmyelinated sensory fibres, since no such action was detected in fibre trunks lacking sensory fibres, such as preganglionic sympathetic nerves and ventral spinal roots. Ion substitution experiments indicated that the capsaicin-induced depolarization is mediated by a mechanism that involves sodium (Na+) calcium (Ca2+) and, to a lesser extent chloride, (Cl-) ions. In vitro intracellular recordings from sensory neurone perikarya, showed that capsaicin depolarizes 70% of the C-type neurones located within the rat nodose ganglion. The capsaicin-induced depolarization was primarily mediated by an increase by an in membrane conductance to Na+ and Ca2+. An additional membrane conductance increase to potassium (K+) was also induced. However, this depended on an influx of calcium via the primary conductance mechanism. Histological experiments using light and electron-microscopic techniques indicated that capsaicin can induce substantial cytotoxic damage to a subpopulation of nodose sensory neurones and vagus nerve unmyelinated fibres. Moreover, the cytotoxic effects could be induced by short applications (< 10 mins) and low concentrations (1-10 μM) of capsaicin. The entry of calcium ions into the cells appeared to play a major role in the cytotoxic process, as the replacement of extracellular calcium with magnesium minimised the cytotoxic damage. The failure of calcium channel-blockers to reduce the calcium-dependent neurotoxic effect indicated that calcium entry through capsaicin-activated channels, rather than voltage-gated calcium channels, initiates the cytotoxicity. It is suggested that capsaicin opens cationic channels and that calcium entry through these channels might not only modify cell excitability but also prime the neurotoxic process which can lead to cell death

Appetite and Satiety Control-Gut Mechanisms

The prevalence of obesity and its comorbidities, particularly type 2 diabetes, cardiovascular and hepatic disease and certain cancers, continues to rise worldwide. Paradoxically, despite an increasingly obesogenic environment, particularly in Western societies, undernutrition is also extremely common. The application of novel, sophisticated techniques, particularly related to imaging and molecular biology, has substantially advanced our understanding of the mechanisms controlling appetite and energy intake. This has led to a redefinition of many concepts, including the relative importance of central versus peripheral mechanisms, recognising that the gastrointestinal (GI) tract, particularly gut hormones, plays a critical role. Given the major advance in knowledge in the field, this Special Issue provides a comprehensive overview of the GI mechanisms underlying the regulation of appetite and energy intake, as a series of definitive reviews by international authorities. The reviews address gut-related mechanisms, including nutrient sensing, gut hormones and GI motility, gut-brain communication, including the roles of the vagus and the modulation of reward perception, the roles of diet and the microbiota, as well as the abnormalities associated with eating disorders, specifically obesity and anorexia of ageing, and the beneficial effects of bariatric surgery. The reviews cover both preclinical research and studies in humans, and are complemented by a number of important original papers

The regulation of appetite by gut hormones

Food intake is essential to life, and thus the drive to eat is a priority. Hunger and satiety are governed by homeostatic and hedonic pathways. The homeostatic control of food intake is primarily mediated by nuclei of the hypothalamus and brainstem, while non-homeostatic control is predominantly afforded by the mesocortical and mesolimbic pathways. Hedonic drive to eat may override homeostatic control, leading to increased food intake. The increasing intake of calorie-dense, highly palatable food has contributed to escalating levels of obesity, which now represents a major public health burden. Thus, the development of appetite-reducing agents to combat the obesity epidemic is a priority. Non-specific appetite inhibitors often result in side-effects such as alterations in blood pressure, locomotor activity and disrupted eating patterns. If an appetite-reducing agent is observed to be acting specifically, it may represent a better target for the development of anti-obesity drugs. The anorectic gut hormones, peptide YY (PYY) and glucagon-like-peptide-1 (GLP-1), reduce food intake by peripheral mechanisms, and also have effects on central homeostatic and hedonic pathways. However, exogenous administration of these peptides results in nausea in humans and aversion in rodents at higher doses. This project investigated the effects of peripheral administration of GLP-1 and PYY on food intake, cardiovascular parameters and behaviour in rats. Feeding studies in fasted animals identified 1.5 nmol/kg as the minimally effective anorectic dose of PYY, while conditioned taste aversion (CTA) was present from doses of 2.5 nmol/kg PYY. Peripheral administration of 300 nmol/kg PYY significantly decreased food intake and led to significant changes in blood pressure. This dose also produced a trend for increased latency to feeding, and decreased activity. In c-Fos studies, peripheral administration of 300 nmol/kg PYY increased neuronal activation in several nuclei of the mesocorticolimbic pathways, and the area postrema (AP). Signalling in these pathways may mediate the aversive properties of PYY, while the AP may detect concurrent alterations in cardiovascular parameters. Feeding studies in fasted animals identified 10 nmol/kg as the minimally effective anorectic dose of GLP-1. Food intake was significantly reduced by 300 nmol/kg GLP-1, including decreased intake in the first feeding bout and a trend for increased latency to feeding. The same dose significantly depressed ambulatory activity and increased heart rate. A CTA was not established following peripheral GLP-1 administration at any dose tested, though patterns in activity and feeding would suggest that aversion was present at high doses. A dose of 300 nmol/kg GLP-1 increased neuronal activation in several areas important in the acquisition of aversion, including the central nucleus of the amygdala (CeA), nucleus of the solitary tract (NTS) and the mesocorticolimbic system. Activation of brain regions by high doses of PYY and GLP-1 correspond to neuronal activation by administration of LiCl. However, 32 mg/kg LiCl increased activation to a far greater degree, suggesting a distinction between substances that reduce food intake purely by aversion, and those that have endogenous homeostatic functions. The effects of GLP-1 and PYY on appetite and aversion are complex, but likely represent separate systems that are activated differentially by different circulating levels of these hormones. By collaborating with the Mathematical Department, Imperial College London, we hope to develop a mathematical model that distinguishes between specific satiety and aversive behaviours. Further work is now required to determine the utility of such modelling in detecting specific appetite inhibitors and reducing animal use.Open Acces

The effect of spinal cord injury on vagal afferents.

Spinal cord injury (SCI) is a significant public health concern that leaves patients with a multitude of life-long disabilities. Major complications of SCI apart from paralysis, include deficits in bladder and bowel function. Lower urinary tract dysfunction continues to remain a top priority issue affecting quality of life for this population. The majority of visceral organs receive a dual sensory innervation from both spinal nerves as well as the vagus nerve. Following SCI, the vagus nerve is a potential pathway through which information from regions below the level of a spinal injury can travel directly to the brainstem, bypassing the spinal cord. The effect of SCI on the vagus nerve and the tissue it supplies has not been thoroughly examined. In order to advance bladder management after SCI, a thorough understanding of its neural control following chronic injury is needed to ultimately improve existing therapeutic options, as well as develop novel interventions that take advantage of this extraspinal route. The objective of this project was to describe the anatomical, neurochemical, and electrophysiological profiles of vagal innervation of the rat urinary bladder. Initially, the first study identified both single and double-labeled vagal afferents supplying the rat bladder and distal colon in the nodose ganglion (NG). The degree of neural innervation to the colon also was assessed, as a single axon that dichotomizes and innervates both organs can serve an important role for mediating both normal physiological and pathological reflexes. Following chronic SCI, we evaluated potential plasticity in subsets of NG neurons which contain projections that bypass the spinal cord from visceral organs, including those projections that specifically supply the bladder. Vagal sensory cell bodies displayed an increase in P2X3 expression and a decrease in IB4 binding, which also held true for many neurons innervating the bladder. Bladder-innervating neurons also displayed altered membrane electrophysiological properties, suggesting they are responsive to a chronic spinal injury. Even though SCI does not directly sever the vagus nerve, our results indicate vagal afferents, including those innervating the bladder, exhibit neurochemical plasticity post-injury that may have implications for visceral homeostatic mechanisms and nociceptive signaling

Pathophysiology of Respiratory Failure Following Acute Organophosphate Poisoning : A Dissertation

Organophosphate (OP) poisoning is a health issue worldwide with over 200,000 deaths per year. Although not a problem in most developed countries, in some third world countries, one third of a hospital’s population could be patients with OP exposure. Even with the most aggressive therapy, 10-40% of patients admitted to an intensive care unit will die. Research into the best practice for treating OP poisoning is lacking, due somewhat to a lack of detailed understanding of the physiology of OP poisoning. Our research uses animal models of acute OP poisoning to explore the mechanism of OP-induced respiratory failure. Our research shows that animals poisoned with dichlorvos demonstrated a uniformly fatal central apnea that, if prevented, was followed immediately by a variable pulmonary dysfunction. Potential mechanisms for dichlorvos-induced central apnea can be divided into direct effects on the central respiratory oscillator (CRO) and feedback inhibition of the CRO. Two afferent pathways that can induce apnea include vagal feedback pathways and feed-forward pathways from the cerebral hemispheres. In our studies we found that vagal feedback and feed forward inhibition from the cerebral hemispheres were not required for OP-induced central apnea. The pre-Botzinger complex in the brainstem is thought to be the kernel of the CRO, but exposure of the pre-Botzinger complex to dichlorvos was not sufficient for apnea. Although OP induced central apnea was uniformly fatal, partial recovery of the CRO occurred post apnea with mechanical ventilation. Central apnea was ubiquitous in our rat poisoning model, but pulmonary dysfunction was extremely variable, with a range of pulmonary effects from fulminate pulmonary failure with prominent pulmonary secretions to no pulmonary dysfunction at all. Vagal efferent activity is involved in neural control of pulmonary tissue but the vagus was not involved in OP-induced pulmonary dysfunction. Anti-muscarinic medications are the mainstay of clinical therapy and are commonly dosed by their effects on pulmonary secretions. Our studies found that atropine (the most common therapeutic agent for OP poisoning) resulted in a ventilation-perfusion mismatch secondary to effects on the pulmonary vasculature

A projection from the hypothalamic paraventricular nucleus to the nucleus tractus solitarii is critical for cardiorespiratory responses to hypoxia

The arterial chemoreflex is an essential protective mechanism for adaptive responses to hypoxia. Stimulation of peripheral chemoreceptors initiates a reflex response that generates compensatory physiological responses, including increased ventilation, arterial pressure and sympathetic nerve activity. However, chemoreflex dysfunction, including over-excitation of chemoreflex pathways, leads to respiratory instability and increased sympathetic nerve activity (SNA) in disease states including heart failure, hypertension and obstructive sleep apnea (170, 199, 232). Determining the mechanisms involved in the central chemoreflex neurocircuitry and its plasticity in health and disease may lead to the development of targeted therapies in cardiorespiratory disease. This dissertation seeks to provide new insight into the neural circuits that drive chemoreflex function. Compensatory responses to chemoreflex stimulation are generated through coordinated interactions between nuclei in the brainstem, forebrain and spinal cord. However, the underlying neurocircuitry, including relevant connections between these nuclei, and the signaling mechanisms that take place within each region are not completely understood. The nucleus tractus solitarii (nTS) and the paraventricular nucleus (PVN) are two central nuclei known to drive chemoreflex function and are implicated in altered cardiorespiratory responses resulting from chemoreflex dysfunction. These two regions form reciprocal connections but the extent to which these connections influence cardiorespiratory regulation and specifically chemoreflex function is unclear. The overarching goal of this dissertation is to examine whether a population of PVN neurons that project to the nTS is involved in shaping cardiorespiratory responses to chemorefle activation by hypoxia. The experiments performed in the three studies (Chapters 2-4) test the overall hypothesis that a descending PVN-nTS projection is an essential component of chemoreflex neurocircuitry; chemoreflex-evoked activation of this pathway is critical for compensatory cardiorespiratory responses to hypoxia.Includes bibliographical reference