Alan C. Spector - US grants

Impairments in the sense of taste are debilitating, adversely affecting the quality of life and jeopardizing health. Specific and unequivocal impairments in taste responsiveness in rats result from gustatory nerve injury. This strongly suggests that information transmitted in the various nerves is to some extent differentially channeled through neural circuits in the brain that are involved with different taste functions. When the transected chorda tympani nerve (CT) or glossopharyngeal nerve (GL) regenerate to reinnervate their appropriate receptor fields, taste functions that are normally impaired by their respective transection entirely recover despite a reduction in the number of regenerated taste buds. For the GL, this includes the recovery of the spatially distinct pattern of neural activity in the nucleus of the solitary tract [NST] (as assessed by Fos-like immunoreactivity) stimulated by oral application of quinine. The purpose of the research here is to extend these findings in new and important ways to help clarify the peripheral organization of the gustatory system. A-response operant discrimination procedure will be modified so that it can be used to measure just-noticeable-differences (JNDs) in intensity in a rat models. This will provide animal researchers with a tool to quantitatively assess stimulus intensity processing in the suprathreshold range and provide a more challenging task to discern the consequences of taste nerve transection and regeneration. The capacity of the gustatory system to maintain function when input from taste receptor fields is channeled through atypical central gustatory circuits will also be tested. This will be accomplished by the use of sophisticated psychophysical procedures to behaviorally test rats that have cross- regenerated nerves in which the taste input of the anterior tongue is channeled through the GL or the taste input of the posterior tongue is channeled through the CT. Through the use of c-Fos immunohistochemistry, the extent to which the topography or quinine- induced neural activity in the NST and parabrachial nucleus is altered by cross regeneration of the CT and GL will be examined. These experiments will provide a useful functional framework to help guide the search for the neural circuits underlying taste-related behavior as well as begin to define the functional boundaries of neural plasticity in the gustatory system.

Dr. Spector's long-term goal is to conduct sophisticated research on taste and feeding that is explicitly coordinated with anatomical and electrophysiological approaches under a unified experimental framework. The overall goal of the proposed research plan is to specify formal correspondences between the measured psychophysical characteristics of the animal and the electrophysiological properties characterizing peripheral taste afferents. These experiments are designed to make refined psychophysical assessments of taste function before and after selective gustatory nerve transection in rats. The behavioral tasks are designed to measure the detectability, the suprathreshold taste functions, and the between-stimulus discriminability associated with an array of theoretically relevant taste compounds. The specific aims of this research are: 1) to specify the relative contribution of the various gustatory nerves to taste sensibility in an effort to reveal the peripheral organization of the gustatory system; 2) to identify significant features of the neural coding process by comparing psychophysically measured changes in sensory function as a result of specific nerve transection with the known response properties of afferents from both the removed and remaining fields; and 3) to provide a consistent parametric psychophysical data base for rat gustation to guide the analysis and interpretation of neurophysiological findings in both the peripheral and central nervous system. A better understanding of the neural organization of taste processes should facilitate the diagnosis and treatment of chemosensory and neurological disorders.

? DESCRIPTION (provided by applicant): Despite the importance of complex carbohydrates and amino acids in the diet of omnivores including humans, our understanding of the peripheral and central mechanisms underlying the detection and processing of these critical dietary compounds by the gustatory system remains in its infancy. Since the initial discovery and characterization of the T1R family of receptor proteins, evidence has accumulated supporting the T1R2+3 heterodimer as the primary taste receptor for sweet-tasting ligands. Likewise, the T1R1+3 heterodimer has been suggested to be critical in the perception of the prototypical umami compound, L-glutamate, when in the presence of the 5'-ribonucleotide, inosine monophosphate (IMP). At the same time, however, research from this lab and others has suggested that additional, potentially T1R-independent receptors may be involved in signaling the presence of at least some amino acids, including L-glutamate when mixed with IMP. Such conclusions are based, in part, on residual behavioral and neural responsiveness in single knock-out (KO) mice missing either T1R1 or T1R3. Similarly, mice lacking either T1R2 or T1R3, while displaying severely impaired psychophysical responsiveness to common sugars are only mildly impaired in responsiveness to glucose polymer mixtures such as Polycose. The taste function spared following deletion of single T1R subunits of the heterodimers may be due to the ability of the remaining subunit to form a homodimer or combine with an unidentified protein to serve as a partially effective receptor. Alternatively, such maintained function could reflect T1R-independent mechanisms. Accordingly, with the use of rigorous psychophysical methodology, T1R2+3 double KO and newly generated T1R1+3 double KO mice along with wild type (WT) mice will tested in the proposed studies to determine whether T1R-independent receptors contribute to the detectability of glucose polymer solutions and select amino acids such as L-glutamate (+IMP), glycine, and L-lysine. Nerve transection studies will isolate the critical oral receptor field(s) responsible for the maintained behavioral responsiveness to the relevant stimuli in the double KO mice. Finally, because it is possible that any lack of behavioral competence in a given taste task does not reflect the absence of a peripheral signal reaching the brain, single-unit recording in the rostral nucleus of the solitary tract (rNST), the first central taste relay, f WT and double KO mice will be performed. This will determine whether signals generated from these taste stimuli reach the brain and from what oral field they originate. Moreover, whether the loss of the relevant T1R heterodimeric receptors differentially affects evoked activity in particulr functional neuronal types, as defined by response profile and anatomical projection status, will be determined. These integrated behavioral and electrophysiological studies will not only reveal whether T1R-independent taste signals for glucose polymers and select amino acids exist, but will provide insight into their neural channeling and their functional significance.

DESCRIPTION (provided by applicant): Injury to structures in the central gustatory system such as the insula and operculum, the site of primary gustatory cortex in humans, can alter the flow of ascending and descending taste-related information through the brain which has great potential to disrupt normal gustatory processing and can have detrimental consequences on health and quality of life. As more advances are made in discerning the pharmacology, connectivity, and physiological phenotype of taste-responsive neurons in the brain, it is becoming increasingly critical for the field to develop and apply appropriate behavioral assays in animal models to explicitly link neurobiological processes to taste function. There is evidence in the literature that patients with damage involving primary gustatory cortex display hypogeusia and have difficulty identifying and recognizing tastes. In rats, lesions in the gustatory cortex retard, but do not eliminate, the acquisition of a conditioned taste aversion, retard acquisition of taste-based anticipatory contrast, and impair the expression of taste neophobia. In contrast, these lesions cause little, if any, effect on unconditioned taste preference and avoidance and do not disrupt taste preference conditioning. The prevailing view in the literature is that animals with gustatory cortex lesions display normal taste detection and discriminability, but, in actuality, this remains to be explicitly tested. Virtually all of the experiments on the effects of gustatory cortex lesions in rats have involved assessment of either conditioned or unconditioned affective/hedonic responsiveness to taste compounds. Moreover, with few exceptions, taste palatability has been assessed with intake and preference tests, which can be heavily influenced by postingestive factors and provide only a partial analysis of affective responsiveness. Thus, conclusions regarding the functional role of gustatory cortex are based on a relatively constrained set of behavioral observations. The goal of the proposed series of experiments is to fill this interpretive void by explicitly testing the necessity of the gustatory cortex in the maintenance of sensory-discriminative vs. affective taste function. Using a technically sophisticated apparatus (gustometer), we plan to test rats with and without ibotenic acid-induced lesions in the gustatory cortex in a variety of behavioral tasks designed to measure taste sensitivity (Exp. 1), taste quality discrimination (Exp.2 & 3), the qualitative specificity of conditioned taste aversions (Exp. 3), and unconditioned affective responsiveness to taste stimuli as assessed by brief access licking tests (Exp. 4) and oromotor taste reactivity (Exp. 5). We predict that these lesions will blunt sensitivity and compromise the animal's ability to discriminate among taste stimuli that are used as sensory signals in psychophysical tasks, without affecting the palatability of the compounds. Regardless of the specific outcomes of these experiments, the data generated should provide a functional context in which to understand central gustatory processing. PUBLIC HEALTH RELEVANCE: Impairments in the sense of taste can be very debilitating, adversely affecting the quality of life in such patients and jeopardizing their health. Moreover, given the role of taste in feeding and drinking, damage to the gustatory system can potentially contribute to more complex clinical disorders involving nutritional status, hydromineral balance, and obesity. The development of animal models, in which the gustatory system can be experimentally manipulated and the perceptual consequences assessed, is essential to gain an understanding of the underlying neurobiology of normal and abnormal taste function - a first step in refining the clinical management of patients suffering damage to gustatory brain sites and potentially leading to the development of therapeutic interventions.

DESCRIPTION (provided by applicant): Roux-en-Y gastric bypass (RYGB) is considered by many to be the most effective treatment available for weight loss maintenance. Patients lose, on average, 25% of their body weight postoperatively for the long term, and this is accompanied by an attenuation of appetite leading to decreased caloric intake. Importantly alterations in food selection have been reported. Indeed, there is some evidence that patients decrease their preference for and intake of high sweet and high fat foods and possibly increase their preference for fruits and vegetables. Moreover, there are many anecdotal reports from patients that they experience changes in taste sensibility. Taste indisputedlty guides food choices and the effect of RYGB on gustatory processes may promote a shift to a lower glycemic index diet and potentially contribute to the weight loss and health gain. However, the literature, which is dominated by measures of verbal report such as dietary recall, is mixed, with some studies finding changes and others not. Our strategy to studying RYGB-induced taste and motivational changes has been to take a coordinated approach to the problem by conducting parallel experiments in both our established rat model and our human bariatric clinical research program at Imperial College London. Our view is that disparities in the literature could be more effectivel resolved by complementing the collection of existing findings with more direct measures of target behaviors in humans that can also be applied to our animal model. Accordingly, our first objective is to use our animal model to follow up our promising preliminary findings suggesting that RYGB in humans decreases the motivation to work for food reward as demonstrated by a decrease in breakpoint (the point at which animals cease working for reward) in a progressive ratio task (in which each subsequent reward delivery requires a progressively greater number of responses). We wish to establish whether rats will display similar modulation of breakpoint after RYGB and, importantly, whether this effect depends on the orosensory characteristics of the reward. The progressive ratio task is a pure measure of appetitive behavior and is designed for applications in which the reinforcer delivery needs to be limited to obviate satiation. As such, itis well suited for assessing changes in the reward value of simple and complex taste stimuli after RYGB, while rendering the potential effects of the postingestive load negligible. Our second objective is to begin to study potential underlying physiological mechanisms. After RYGB, circulating levels of key anorexogenic gastrointestinal hormones such as peptide YY and glucagon-like peptide-1 increase. Systemic administration of the somatostatin analog, octreotide, which blocks the release of these and other gut hormones, doubles short-term chow intake in rats after RYGB, but has no effects in SHAM-operated controls. Thus, we will test whether changes in the reward value of specific nutritionally relevant stimuli might be reversed by octreotide, paving the way for more selective mechanistic manipulations in future work.

? DESCRIPTION (provided by applicant): After Roux-en-Y gastric bypass (RYGB), patients decrease: appetite, caloric intake, body weight, and glycemia, all of which are maintained long-term. This is a large reason why it has become a popular treatment for morbid obesity and Type 2 diabetes mellitus (T2DM). However, a major unresolved issue is whether, after RYGB, patients choose to eat less foods that are high in fat and sugar in favor of lower energy dense alternatives such as vegetables. If true, this could conceivably contribute to improved body weight and glycemia. Disparities among studies on food selection and intake are likely due to the almost complete reliance on self- reported food intake which is vulnerable to inaccuracy. This controversy can best be resolved by complementing existing findings with direct measures of target behaviors in humans that can also be applied to animal models and vice-versa. The proposed experiments will extend the published and preliminary findings suggesting that RYGB alters food preferences without ostensibly changing food palatability. Such changes in food selection alone have been postulated to benefit patients with obesity and/or T2DM and this may be an under-investigated means by which RYGB improves maintenance of body weight and glycemic control. Direct measures in both rats and humans after RYGB will be used to test the hypothesis that the selection and intake of foods varying in fat content and glycemic index, as well as the pattern of ingestion within and across meals, changes in a manner that leads to beneficial outcomes on body weight. While the apparent progressive changes in food preference after RYGB in the rat model strongly suggest learning, such experience-based changes could be driven by either food aversion (changed palatability) or food avoidance (unchanged palatability). Evidence disambiguating these learning processes is lacking, and the proposed experiments are designed to explicitly distinguish between these important and conceptually distinct behavioral mechanisms. The exaggerated pleiotropic gut hormone response to RYGB has been the most promising lead to a physiologic mechanism underlying the changes in feeding, weight loss, and glycemic control. One gut hormone in particular, glucagon like peptide-1 (GLP-1) has been a target in pharmacological interventions in the management of T2DM and is implicated in feeding satiation. Accordingly, the somatostatin analogue Octreotide will be used to block the pleiotropic gut hormone response to RYGB in translational experiments to interrogate the role of these endocrine processes in the progressive changes in ingestive behavior observed. In other experiments, the GLP-1 receptor will be selectively targeted by the antagonist Exendin-9. This scientific alliance is made possible through the US-Ireland R&D Partnership Program which is allowing this established international research team to continue to apply their complementary expertise in the service of addressing fundamental questions that can help guide future research in the treatment and management of obesity and T2DM.

PROJECT SUMMARY Cephalic phase responses (CPRs) are autonomic and endocrine events triggered by stimulation of ?head? receptors, especially those of the gustatory system. These are widely considered as the first preparatory steps required for the optimal digestion, absorption, and utilization of nutrients. Although postoral mechanisms are clearly essential for this purpose, the significance of oral stimulation during eating for maintaining normal metabolic function should not be underestimated. One of the most extensively studied CPRs is the early rise in insulin that is stimulated by oral glucose. However, the insulin CPR literature is uneven, most likely because of methodological limitations and variations in blood sampling sites. To clarify how CPRs and their underlying neural mechanisms are organized we have developed a unique and sensitive rat preparation. It combines in a single animal intraoral and intragastric cannulae that precisely deliver test solutions, with a hepatic portal vein (HPV) sampling catheter. We have used this preparation in male rats to find strikingly early rises in HPV insulin and GLP-1 levels that are significantly greater after oral compared to gastric delivery of glucose. Responses to fructose or water showed no such oral/gastric differences. These insulin and GLP-1 increases were robust and rapid, reaching peak levels within 3 min of orally delivering 180 mg of glucose in less than 1 min. This is the first report of significant GLP-1 release triggered by oral stimulation with glucose. Impressively, its peak is substantially greater than that seen after a normal meal. Considering the rapidity of both the GLP-1 CPR and its subsequent degradation in blood, one hypothesis that we will test is whether oral glucose-driven GLP-1 release from enteroendocrine cells acts as an incretin that mediates the insulin CPR neurally through a vago-vagal reflex. Our design employs two experimental approaches organized in three Specific Aims to reveal the neural mechanisms and circuits responsible for these GLP-1 and insulin CPRs. One approach uses explicitly controlled intraoral or intragastric infusions of glucose, fructose, and control taste solutions followed by HPV measurements of plasma GLP-1, insulin, and glucose responses. It will test whether insulin and GLP-1 CPRs can be conditioned, and whether CPRs are recapitulated in females. The other approach will use transneuronal viral tracing techniques combined with state-of-the-art neuroinformatics methods to map the neural pathways through which gustatory signals control pancreatic and enteroendocrine secretions. Furthermore, both approaches use functional nerve transections to define the organization of the neural pathways driving GLP-1 and insulin CPRs. The project is a scientific alliance of highly experienced investigators who have complementary expertise. Its outcomes will provide new insights into the neural control of insulin and GLP-1 secretion. It will also define mechanisms that could be therapeutically targeted to facilitate treatment strategies for patients requiring enteral or parenteral nutrition, as well as promoting healthier eating and nutrient assimilation in the general population.