Robert C. Ritter - US grants

The controls of food selection and ingestion are of major importance to human and animal health. Disturbances of the controls of ingestion occur in many human illnesses and may be pathogenic for some. The brain is ultimately responsible for controlling food intake. Nevertheless, the forebrain is the only brain area where control of ingestion has been studied extensively. The overall goal of this work is to better understand the roles of the most caudal part of the brain (hindbrain) in control of ingestion. The proposal focuses on understanding the role of the area postrema and adjacent solitary nucleus in the control of ingestion. Specifically, the experiments proposed will 1) determine which neural connections of this region are responsible for the individual ingestive changes observed after damage to the area postrema and solitary nucleus and 2) attempt to identify chemically discrete neuronal populations which mediate the effects of this brain region on food intake. These studies will be among the first to analyze the role of the hindbrain in the control of hunger and appetite. Such an analysis is important because some controls of appetite seem to arise from the hindbrain and all controls of appetite must be executed via the hindbrain.

DESCRIPTION (ADAPTED FROM THE APPLICANT'S ABSTRACT): The long term goals of this work are to identify the neuronal systems through which peptides influence food intake, to characterize these systems both anatomically and functionally, and ultimately to appreciate how peptides and the neurons that mediate their actions participate in physiologically and/or pathologically-induced changes in food intake. The Principal Investigator (PI) has concentrated on neural systems that mediate suppression of food intake by cholecystokinin (CCK), a peptide found in the intestine and in the brain. He has found that CCK mediates reductions of food intake that occur when some nutrients are placed in the small intestine. The proposed experiments are designed to determine where the CCK that mediates reduction of food intake acts and at what type of receptors it acts. To accomplish these ends the experiments address the three following specific aims: 1) use pharmacological and immunological methods together with restricted drug application to identify the general location and subtype of the CCK receptors involved in suppression of sham feeding by intestinal nutrients; 2) utilize intraintestinal infusions of agents that desensitize gut neurons to assess the role of the local intestinal innervation in suppression of food intake by intestinal nutrients and exogenous CCK; and 3) use immuno-histochemical techniques to anatomically identify central and peripheral neurons that are activated by intestinal stimuli and exogenous peptides.

Although ethanol is a drug with significant effects in virtually all physiological systems, it is also a source of calories. Its voluntary consumption could, therefore, be considered eating. consequently, investigations into the interaction of alcohol with the controls of food intake may provide important information concerning the control of ethanol ingestion. Convincing experimental evidence indicates that the small intestine, its innervation and endocrine cells, play an important role in the control of food intake. For example, the termination of ingestion is mediated in part by the detection of specific nutrients in the small intestine. Furthermore, nutrients in the small intestine release gastrointestinal hormones which appear to be involved in the reduction of food intake. The potential interaction of ethanol with gastrointestinal controls of food intake has not been explored. The goal of the work proposed in this application is to determine whether intestinal controls of food intake are influenced by ethanol and whether these controls may be involved in the control of ethanol intake.

Dysfunction of the GI innervation appears to be involved in a variety of debilitating diseases involving abnormal secretion, motility and inflammation in GI structures. In addition, the GI innervation is an important participant in the control of food intake and body weight. There is abundant evidence that sensory neurons in the vagus nerve respond to chemical changes in the lumen of the gut, including changes in specific nutrient content of the food. This nutrient responsiveness permits the brain to alter GI function and control food intake according to nutrient content in the gut lumen. In addition it is known that the intestinal wall contains about ten million enteric neurons, providing local control of gut function. Recent results suggest that certain peptides, may be released from enteric neurons, to activate vagal sensory nerves. However, there have been no efforts to identify the structural or chemical nature of connections between enteric neurons and vagal sensory neurons. Furthermore, nutrient responsiveness of enteric neurons is based on circumstantial evidence. Electrical responses to nutrients have not yet been recorded in enteric neurons. Therefore, one aim of this work is to examine the intestine for histochemically specific synaptic contacts between enteric neurons and vagal sensory neurons. The second aim is to record, intracellularly, from enteric neurons to determine their sensitivity to intestinal nutrients. The results should point the way toward more selective means of intervention in certain GI diseases and control of appetite.

DESCRIPTION (provided by applicant): The overall goal of this work is to define the neural mechanisms through which gastrointestinal hormones, especially cholecystokinin (CCK) and glucagon-related peptide (CCK), control food intake and to appreciate how these substrates are involved in the physiology and pathology of food intake and body weight. CCK is a peptide hormone secreted by enteroendcrine "I cells" of the upper small intestine, while GLP-1 is secreted by the "L-cells" of the distal jejunum, ileum and colon. Evidence from our lab and others suggests that while CCK and GLP-1 both alter short-term food intake, they also may influence the long-term control of food intake and body weight by interacting with other signals, such as fat cell hormone, leptin. The neural substrates through which CCK and GLP-1 interact with leptin to influence long-term control of food intake and body weight are not understood. Furthermore, it is not known whether hormones like CCK and GLP-1 activate the same or distinct peripheral neural substrates. Finally, while nearly all investigations of CCK's involvement in control of food intake have used the C-terminal 8 amino acid CCK fragment, CCK-8, CCK-8 is not a physiological form of circulating CCK in the rat. Rather, it is now clear that the 58 amino acid form of CCK, CCK-58, is the only circulating form of CCK. Because physiological responses to CCK-58 differ both quantitatively and qualitatively from responses to CCK-8, it now is important to specifically evaluate CCK-58's effect on feeding and body weight control, as well as CCK-58's ability to interact with leptin in control of food intake and body weight. The experiments proposed in this application will 1) characterize the effects of CCK-58, alone and in combination with leptin, on the activation of vagal afferents in culture and on meal parameters and body weight gain of rats, 2) use surgical and neurotoxin-induced vagal afferent destruction to determine the role of vagal afferents in control of 24h food intake by CCK and leptin and 3) use retrograde neuronal labeling, ratiometric calcium imaging, and near arterial infusions to determine the relative distribution of GLP-1 and CCK responsiveness of vagal afferents innervating the jejuno-ileum, duodenum and stomach, and to determine whether leptin modulates activation of vagal afferents and control of food intake by GLP-1. The results of these experiments will expand our understanding, specifically with regard to the role of visceral afferents in cooperative control of food intake and body weight by enteroendocrine signals and leptin. The substrates of such cooperative control are likely to be critical components in the control of food intake and body weight during health and disease. PUBLIC HEALTH RELEVANCE: This research is intended to characterize the interaction of gastrointestinal hormones with other signals, such as leptin, and to determine the extent to which visceral afferents participate in the cooperative control of food intake and body weight by gastrointestinal hormones and leptin.

ABSTRACT This application for renewal continues an enduring interest in neural mechanisms by which gastrointestinal (GI) satiation signals are communicated to the hindbrain nucleus of the solitary tract (NTS) by vagal afferents and are integrated with other controls of food intake. Glutamate is the principal neurotransmitter released by vagal afferent terminals in the nucleus tractus solitarius (NTS). As such, glutamate receptors in the NTS are pivotal to the transmission and processing of satiation signals. Our published results, demonstrating that hindbrain injections of N-methyl-D-aspartate-type glutamate receptor (NMDAR) antagonists increase meal size and prevent reduction of food intake by cholecystokinin (CCK) support this assertion. Although vagal afferent activation by GI stimuli reduce meal size, central neuropeptides and circulating hormones also control food intake by controlling meal size, suggesting that they may modulate or imitate the effects of GI stimuli. We postulate that brain peptides interact with NMDAR to control meal size by modulating the strength of glutamatergic vagal afferent synapses in the NTS. Our preliminary results suggest that interaction of vagal afferent NMDAR and melanocortin 4 receptors (MC4R) triggers long-lasting changes in vagal afferent synapsin phosphorylation that are consistent with strengthened vagal afferent synaptic function. Experiments of Aim 1 utilize pharmacological, immunochemical, chemogenetic and electrophysiological approaches to determine the nature of NMDAR participation in MC4R effects on vagal afferent synaptic function and control of meal size. Vagal afferents express type 1 and type 2 NPY receptors (Y1R and Y2R) as well as MC4R and NMDAR. We find that NTS injection of NPY or the Y2R agonist, PYY 3-36, increases food intake, an effect that is attenuated by NTS co-injection of SP-cAMP. In Aim 2 of the application we test the hypothesis that vagal afferent Y2R control food intake by antagonizing MC4R effects on PKA activation, synapsin phosphorylation and vagal afferent synaptic strength. Our long- term goal is to determine how NMDAR participate in modulation of vagal afferent synaptic strength to reduce food intake. A detailed appreciation of the mechanisms by which peptides and hormone interact with NMDAR to control of food intake is of significance to human health because it may provide avenues for therapeutic intervention in eating disorders and obesity.