Bret N. Smith - US grants

DESCRIPTION: Regulation of gastrointestinal function by the brain is an important but inadequately understood component of energy homeostasis in humans. The long-term objective of this project is to understand the local neuronal connectivity in the brainstem solitary complex. As the site of first central synaptic contact for sensory afferent fibers of the vagus nerve from the digestive tract, neurons in the nucleus tractus solitarius (NTS) process digestive system information prior to further integration via higher brain areas and eventual activation of motor output to the stomach. Included in the neuronal circuits controlling autonomic function are local synaptic circuits in the NTS, inputs from CNS regions outside the solitary complex, and reflex-circuit interactions with vagal motor neurons in the dorsal motor nucleus of the vagus nerve (DMV). Although the neuronal circuitry of the caudal solitary complex is the functional substrate for visceral sensory-motor integration relating to feeding behavior, local synaptic connectivity in the area has not been adequately described. We will use whole-cell patch-clamp recordings in brain slices to investigate the functional organization of the caudal solitary complex. We will identify neurons on the basis of their functional connections with the proximal stomach using in vivo labeling methods. We will also correlate synaptic responses with quantitative morphological features using biocytin labeling techniques. Both electrical stimulation of primary viscerosensory input and photoactivation of caged glutamate to stimulate discrete sites within the slice will be used to investigate the local amino acid-mediated synaptic circuitry of the region. The specific aims of the proposal are designed to test hypotheses regarding the synaptic circuitry of the solitary complex. The specific hypotheses for the proposed research are: 1) Gastric-related principal neurons in the NTS are inhibited by a convergent system of local GABAergic neurons (i.e., lateral inhibition); 2) small, putative inhibitory NTS neurons are activated by local excitatory NTS neurons; and 3) stomach-projecting neurons in the DMV are directly inhibited by NTS input. These experiments will result in an understanding of neuronal connectivity within the NTS and between regions of the solitary complex. This information is highly relevant to the mechanistic understanding of feeding behaviors such as receptive relaxation and accommodation in the stomach. It will also enhance understanding of the mechanisms of action for treatments of digestive system-related disorders.

[unreadable] DESCRIPTION (provided by applicant): A principal goal of epilepsy research is to identify and develop novel treatment strategies for seizure control that may be relatively selective for modulation of the synaptically reorganized circuitry in the epileptic brain. The objective of this Exploratory/Developmental (R21) research proposal is to identify the effects of exogenous and endogenous cannabinoids on neurons of the dentate gyrus in mice with pilocarpine-induced temporal lobe epilepsy (TLE), characterized by chronic spontaneous seizures, mossy fiber sprouting, and increased neuronal excitability and synchrony. The use of cannabinoids for treating seizure disorders has been proposed, but results of case study analyses have been mixed. The goal of this proposal is to determine how cannabinoids modulate activity in the dentate gyrus in a murine model of TLE in order to understand effects of the substance on a system that has undergone synaptic reorganization (versus effects on normal neural circuits). Most known central effects of cannabinoids are mediated by cannabinoid type 1 receptors (CB1R), usually located on presynaptic terminals. In the hippocampus, cannabinoids suppress synaptic input-especially feedback inhibition--to principal neurons, including dentate gyrus granule cells. Although cannabinoids appear to suppress some types of seizures, the cellular "disinhibitory" properties appear to be proconvulsive in normal brains. Recent analyses suggest that cannabinoids may suppress seizure activity in the epileptic brain. However, no studies at the cellular level have been performed in the dentate gyrus in an animal model of TLE (i.e., having cell loss, axon sprouting, and synaptic reorganization). Preliminary data presented here suggest that, in the absence of GABAA receptor-mediated inhibition, cannabinoids can inhibit epileptiform discharges that are attributable to activation of newly-formed recurrent excitatory circuitry in the dentate gyrus in mice with pilocarpine-induced TLE. Using electrophysiological, anatomical, and molecular biological techniques, the hypotheses that: 1) cannabinoids inhibit new recurrent excitatory circuitry; 2) cannabinoids inhibit granule cells directly, and 3) CB1R expression is upregulated in animals with TLE and synaptic reorganization will be tested in a murine model of TLE developed in this lab. Understanding these effects may promote more effective treatment strategies designed specifically for TLE patients, whose brains have undergone a degree of synaptic reorganization. [unreadable] [unreadable]

The aims of this project are to advance understanding about the activity of a specific set of nerve cells, called inhibitory interneurons, in the brain stem that are responsible for controlling the functions of most organ systems (e.g., cardiac, respiratory, digestive systems). Although their importance in brain function is evident, inhibitory interneurons have been difficult to study because they resemble other nearby nerve cells; little is known about how they are regulated. A strain of genetically modified "transgenic" mice has been developed, in which inhibitory interneurons fluoresce under ultraviolet light. These transgenic mice will be utilized to target the inhibitory interneurons for study in relative isolation from the rest of the brain and to separate them from other types of nearby cells. The area of the brain stem to be studied receives input from most organ systems, and these inhibitory interneurons play critical roles in processing that sensory information, helping to integrate it with information from the rest of the brain before sending command signals to the nerve cells that control organ function. Some of the most abundant chemicals made in the brain are a class of opioid peptides (i.e., the brain's natural morphine). These peptides have profound effects on organ function, but the specific effects on inhibitory interneurons in the brain stem are unknown. Part of this study aims to determine the effects of one class of these opioid peptides on the specific activities of inhibitory interneurons involved in controlling digestion.
The broader impacts of the research include advancing our understanding of this important brain region and scientific training for both graduate and undergraduate students. Results of these studies will impact several scientific fields of study, including physiology, endocrinology, digestion, and cardiovascular biology. Results will be disseminated by publishing the data and by presentation at national meetings and symposia.

? DESCRIPTION (provided by applicant): Diabetes mellitus is a major health concern, affecting nearly 26 million people in the United States. Serious complications resulting from diabetes including include heart disease, stroke, hypertension, blindness, nervous system damage, and autonomic dysfunction. A major impediment to developing successful diabetes treatments (versus treating symptoms) is the relative knowledge gap regarding the multifaceted and redundant systems that contribute to control of metabolic homeostasis. This proposal investigates disease-related plasticity of central neural circuitry involved in autonomic control, including control of blood glucose homeostasis. Experiments utilize murine models of type 1 and type 2 diabetes. Preautonomic neurons of the dorsal vagal complex, which contains second-order viscerosensory neurons in the nucleus tractus solitarius (NTS) and preganglionic parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV), are glucosensors and also contribute significantly to autonomic regulation of glucose homeostasis. Vagal motor output is suppressed in diabetes, leading to autonomic dysregulation, including excess hepatic glucose production and gastric motility dysfunction. Preliminary results show that GABA neurons in the NTS in particular are responsive to elevated glucose. Paradoxically, GABAA receptor-mediated responses in the DMV are persistently enhanced in a model of type 1 diabetes, in a manner consistent with maintenance of prolonged hyperglycemia. Some, but not all of these responses are preserved in a type 2 diabetes model, suggesting a form of GABA receptor plasticity that mediates the decreased vagal output seen in diabetes. In addition, modulation of GABA receptors in the dorsal vagal complex has a significant effect on blood glucose levels, and this effect is hypothesized to be enhanced in diabetic mice versus controls. This proposal aims to determine the causes and underlying features of the recently-discovered, diabetes-induced plasticity of the GABAergic system in the vagal complex. Electrophysiological recordings from vagal complex neurons in slices from control and diabetic mice will be used to obtain functional cellular data related to altered GABAergic inhibition changes associated with diabetes development in the streptozotocin-treated mouse, a model of type 1 diabetes, and the TallyHo mouse, a model of type 2 diabetes. Aim 1 will determine insulin- and glucose- dependence of enhanced tonic GABA currents in diabetic mice, aim 2 will identify cellular mechanisms contributing to diabetes-associated GABA receptor plasticity in the DMV, and aim 3 will determine the effects of GABA receptor modulation in the dorsal vagal complex on systemic glucose homeostasis. Results will guide future studies aimed at disease-modifying therapies from a systemic standpoint, based on modulating specific inhibitory neural functions in the brainstem to address diabetes-related autonomic dysregulation in patients.

DESCRIPTION (provided by applicant): The experiments in this research grant proposal will identify the cellular and synaptic effects of endogenous cannabinoid (eCB) ligands and of glucocorticoid-induced release of eCBs in the dorsal motor nucleus of the vagus (DMV). Neurons in the DMV regulate parasympathetic output to most of the subdiaphragmatic viscera and therefore critically control feeding, digestion, glucose and insulin secretion, and other metabolic functions. Their activity is largely controlled by synaptic input to the DMV, which is modulated by locally released chemicals and circulating hormones. Regulation of DMV neurons by cannabinoids, vanilloids, and glucocorticoids has been suggested;when applied centrally these compounds profoundly alter parasympathetic function. Several eCB ligands, which are thought to be released from cell membranes in a retrograde fashion, activate both cannabinoid type 1 receptors (CB1R) and transient receptor potential vanilloid type 1 (TRPV1). In the DMV, activation of TRPV1 enhances neurotransmitter release, whereas CB1R tends to inhibit synapses. Both effects occur by activation of receptors on presynaptic terminals. Preliminary evidence suggests that eCB ligands are released from DMV neurons, and that glucocorticoids or depolarization can induce this release. Thus, eCB activity in the DMV may modulate both TRPV1 and CB1R activity. Neither the type(s) of eCB ligands released, the effects of most eCB ligands on synaptic activity, nor the trigger or mechanism of eCB release in the DMV are known. We will use whole-cell patch-clamp recordings from DMV neurons in brainstem slices to identify effects of eCB ligands on cellular activity in the DMV, and will also identify the compounds released by cells in the area using pharmacological and biochemical methods. The experiments will be guided by three specific aims: 1) Differentiate effects of eCB ligands on CB1R and TRPV1 in the DMV;2) Determine the eCB involvement in mediating rapid effects of glucocorticoids on local circuitry in the DMV;and 3) Identify the cellular pathway of the glucocorticoid effect. We will test the hypotheses that eCBs alter TRPV1 and CBR1 activity in the DMV in specific and predictable spatial, temporal, functionally relevant, and activity-dependent patterns, and that glucocorticoids induce eCB release from DMV cells by acting at membrane-bound G protein-coupled receptors on DMV neurons. Drugs based on the eCB system are being investigated for therapeutic use in a variety of nervous system pathologies, including disorders related to feeding, digestion, and obesity. Glucocorticoids, which release eCBs in some systems, are widely prescribed, and are also released by stressful stimuli. Results of these studies will be critical to predicting and understanding how these compounds interact with each other and affect parasympathetic function. Possible translational benefits also exist because of the benefit in controlling eCB levels in the vagal system of patients with elevated glucocorticoids. PUBLIC HEALTH RELEVANCE Endogenous cannabinoid compounds have particularly complex actions in the area of the brainstem most responsible for maintaining functions of organ systems, and a number of drugs therapies based on altering these compounds in the brain are being developed without knowledge about their effects in this critical brain area. The experiments in this research grant proposal will identify the characteristics of these drugs, how they are released by glucocorticoid hormones, and what actions they have on the neurons. Results of these studies will be critical for predicting and understanding how these compounds interact to affect digestive functions, and they will also be applicable to multiple other neuronal systems.

Project summary Diabetes mellitus is a major health concern, affecting nearly 26 million people in the United States. Serious complications resulting from diabetes including heart disease, stroke, hypertension, blindness, nervous system damage, and autonomic dysfunction. A major impediment to developing successful diabetes treatments (versus treating symptoms) is the relative knowledge gap regarding the multifaceted and redundant systems that contribute to control of metabolic homeostasis. This proposal investigates disease-related plasticity of central neural circuitry involved in autonomic control, including control of blood glucose homeostasis. Experiments utilize a model of type 1 diabetes, but findings will more broadly apply to other forms of diabetes, since autonomic dysfunction increases the risk of developing type 2 diabetes. Preautonomic neurons of the dorsal vagal complex, which contains second-order viscerosensory neurons in the nucleus tractus solitarius (NTS) and preganglionic parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV), are glucosensors and also contribute significantly to autonomic regulation of glucose homeostasis. Vagal motor output is suppressed in diabetes, leading to autonomic dysregulation, including excess hepatic glucose production and gastric motility dysfunction. Preliminary results show that glutamate release in the DMV is persistently enhanced in a model of type 1 diabetes, in a manner consistent with development of a possible compensatory response to prolonged hyperglycemia that suggests homeostatic plasticity that mitigates against the decreased vagal output seen in diabetes. In addition, NMDA receptor modulation has a relatively larger effect on glutamate release in diabetic mice versus controls. This exploratory proposal aims to determine the causes and underlying features of the recently-discovered, diabetes-induced enhancement of tonic excitatory drive to DMV neurons. Electrophysiological recordings from vagal complex neurons in slices from control and diabetic mice will be used to obtain functional cellular data related to NMDA receptor sensitivity changes associated with diabetes development in the streptozotocin- treated mouse, a model of type 1 diabetes. Aim 1 will determine if recently-identified NMDA receptors located on presynaptic terminals contacting DMV cells are upregulated in diabetes. Aim 2 will determine if postsynaptic NMDA receptors on the somadendritic portion of identified glutamatergic NTS neurons that project to the DMV are upregulated in diabetes. The sensitivity of these changes to glucose and insulin will also be identified. Results will guide future studies aimed at disease-modifying therapies from a systemic standpoint, based on modulating specific neural functions in the brainstem to eventually address diabetes- related autonomic dysregulation in patients.

? DESCRIPTION (provided by applicant): Over 2 million people in the United States have experienced unprovoked seizures or been diagnosed with epilepsy. In approximately 25% of cases, seizures are refractory to medical therapies. Inability to effectively treat epilepsy reflecs a lack of understanding of the basic mechanisms of this disorder. Up to 50% of traumatic brain injury survivors develop epilepsy. Posttraumatic epilepsy (PTE) is associated with alterations in hippocampal circuits including cell loss and reactive plasticity. In the mammalian brain, there is continual generation of new neurons in a few key brain regions throughout adulthood. This process, referred to as adult neurogenesis, represents a form of experience-dependent plasticity that is believed to support normal brain function. Brain insults including traumatic bran injury, seizures, and stroke are associated with increases in hippocampal adult neurogenesis, and abnormal integration of adult-born neurons within hippocampal circuitry may provide a substrate for hyperexcitable circuits that contribute to seizures. Epilepsy is associated with the emergence of adult-born dentate granule cells (DGCs) that display abnormal dendritic fields and axons that may project to unexpected targets. Adult-born DGCs are associated with spontaneous seizures in experimental epilepsy and blockade of adult neurogenesis reduces spontaneous seizure expression in an animal model of PTE. Despite these reports, inherent limitations of the techniques used have prevented the characterization of functional cellular connections formed by adult-born neurons with their synaptic targets. This proposal aims to develop a new technique to selectively label and stimulate newly-born neurons in the adult brain and then use this technique to assess functional outputs of hippocampal adult-born neurons in a mouse model of PTE. Work here aims to: 1) Selectively target expression of channelrhodopsin (ChR2) in adult-born progenitor cells based on their tamoxifen-inducible expression of nestin using Nestin-Cre mice. Nestin-Cre mice will be administered with a Cre-inducible adeno-associated virus (DIO-AAV) with double- floxed reverse cassettes containing channelrhodopsin (ChR2) and the fluorescent report mCherry (abbreviation: ChR2-mCherry; construct: pAAV-Ef1a-DIO-hChR2(H134R)-mCherry-WPRE-pA); and 2) Use blue-light stimulation parameters to activate adult-born neurons and drive signaling to their postsynaptic targets. Whole-cell patch-clamp recordings will be performed on DGCs in hippocampal slices from Nestin-Cre mice that have received injections of the ChR2-mCherry construct to describe the functional projections formed by adult-born neurons after brain injury. Improved understanding of how adult-born neurons incorporate into neural networks and signal during normal and PTE states will help define their relevance as therapeutic targets and will also provide new context for evaluation of clinically available drugs that have documented effects on adult-born cells.

Project Summary This is a resubmission of a T32 application requesting funding for a new Graduate Training in Integrative Physiology training program for 4 predoctoral fellows/year. Training of the predoctoral fellows will be carried out by participating faculty with proven track records of cross-disciplinary research on a variety of health issues. The overall goal of the proposed program is to provide broad-based training in modern research concepts required to study quantitatively the nature of physiological processes in both healthy and disease states, which is necessary to drive discovery of new therapeutic strategies for treating the underlying causes of diseases affecting multiple organs and systems. The primary objectives are to: 1) provide a comprehensive, quantitative training program for early stage trainees in physiology with a particular emphasis on cardiovascular, aging, neurophysiology, and muscle biology research; and 2) promote interdisciplinary learning teams involving basic scientists, clinicians and trainees to foster rigorous innovative translational science. Predoctoral fellows will be trained in the practical issues involved in the design and conduct of basic and translational research to enhance their future ability to make therapeutic discoveries for diseases of broad etiology as independent investigators. To accomplish this, the predoctoral fellows will engage in cutting-edge, quantitative, physiologically-based research. In addition to specialized didactic coursework, they will participate in a comprehensive Experimental Design Workshop and attend weekly seminars and grand rounds presentations focused on cardiovascular, aging-related, neurological, or musculoskeletal diseases. Although most of the trainees are anticipated to pursue careers in laboratory-based discovery research, they will also receive training in conveying knowledge to other scientists through grant-writing and communications skills workshops. The combination of these training avenues will provide skills to disseminate their knowledge and provide them with an understanding of how to design and conduct research in a manner that will enable the translation of promising therapies into the clinic.

Project Summary More than one million people are treated medically each year in the United States after sustaining a brain injury and traumatic brain injury (TBI) is often accompanied by the delayed development of posttraumatic epilepsy (PTE), for which there are few effective therapies. Although clinical association between TBI and epilepsy is well documented, treatments designed to prevent PTE have been largely unsuccessful. Among the most promising antiepileptogenic treatments reported to date center on inhibition of the mammalian (mechanistic) target of rapamycin (mTOR) pathway. mTOR is activated after TBI and seizures, and it's activity regulates a variety of cellular activities, including growth and proliferation, especially in developing neurons. Inhibiting mTOR activity has shown promise for altering the progression of epileptogenesis in rodent models of epilepsy, including PTE, but several caveats have also been acknowledged, specifically: Suppression of mTOR post-TBI has been proposed to prevent epileptogenesis, whereas mTOR activation has been proposed as a means of improving cognitive recovery after TBI in patients. The mechanisms by which mTOR modulation exerts its anti-epileptogenic effects are not known, and the contribution of newborn neurons and synaptic reorganization in the dentate gyrus to epileptogenesis and cognition are controversial. Preventing PTE is hampered by these fundamental knowledge gaps. This proposal will use the controlled cortical impact (CCI) model of TBI, which results in cell loss, increased neurogenesis and synaptic reorganization in the dentate gyrus, and delayed development of spontaneous seizures (i.e., epileptogenesis) to study the impact of newborn neurons on synaptic excitability changes in the dentate gyrus. The effects of both negative and positive regulation of mTOR on epileptogenesis and cognitive recovery will also be determined in the context of neurogenesis after brain injury. The overarching hypotheses are that adult born neurons contribute to synaptic reorganization after TBI and that mTOR activity-dependent regulation of neurogenesis alters epileptogenesis and post-TBI cognitive recovery. A combination of electrophysiological, histological, and behavioral techniques utilizing optogenetic and chemogenetic modification of adult born neurons will be used to address three aims: 1) Determine the functional synaptic organization of adult born DGCs after TBI; 2) Determine effects of mTOR modulation on neurogenesis and synaptic connectivity in the dentate gyrus after TBI; and 3) Determine how adult born DGCs contribute to functional recovery and seizures after TBI. A mechanistic understanding of how adult born neurons contribute to DGC circuitry and how mTOR modulation alters the circuitry of these neurons after CCI will be developed in the context of both cognitive recovery after TBI and development of PTE. A better understanding of the contribution of adult born neurons to recovery and epileptogenesis after TBI will facilitate the development of treatments to prevent PTE.

Project Summary Diabetes mellitus is a major health concern, affecting over 30 million people in the United States. Serious complications resulting from diabetes including include heart disease, stroke, hypertension, blindness, nervous system damage, and autonomic dysfunction. A major impediment to developing successful diabetes treatments (versus treating symptoms) is the relative knowledge gap regarding the multifaceted and redundant systems that are affected by and contribute to control of metabolic homeostasis. This proposal investigates disease-related plasticity of central neural circuitry controlling autonomic function. Experiments utilize murine models of type 1 and type 2 diabetes. Second-order viscerosensory neurons in the nucleus tractus solitarius (NTS) are glucosensors and contribute significantly to autonomic regulation of glucose homeostasis by signaling integrated visceral and humoral signals to brain areas that directly regulate systemic glucose levels, including the dorsal motor nucleus of the vagus nerve (DMV), which contains vagal motor neurons. Vagal motor function is altered in diabetes, leading to autonomic dysregulation, including excess hepatic glucose production and gastric motility dysfunction. We have found that changes in activity of GABA neurons or altering glucose pathways in the NTS affect systemic [glucose]. Glutamate and GABA receptors are reorganized, and synaptic excitation of NTS GABA neurons is persistently increased in the vagal complex after a few days of hyperglycemia in a model of type 1 diabetes. The majority of GABA neurons in the NTS is responsive to elevated [glucose], being either excited or inhibited, but glucose-excitatory responses are blunted in diabetic mice. Vertical sleeve gastrectomy rapidly improves glycemic index in patients and animal models of diabetes, independent of weight loss; convergent data suggest the brainstem dorsal vagal complex (DVC) is integral to this response. Electrophysiological recordings from NTS neurons in slices, chemogenetic and pharmacological manipulation of NTS neuron activity, and direct glutamate and glucose measurements from the NTS of control and diabetic mice will be used to obtain functional cellular and molecular data relevant to the contribution of the NTS to glucose metabolism in the streptozotocin-treated mouse and the BKS-db mouse, models of type 1 and type 2 diabetes, respectively. The broad hypothesis of this proposal is that altered neural function in the vagal complex reflects a neurogenic component of diabetic pathology. The experiments in this proposal aim to: 1) Identify cellular outcomes of glucose responsiveness in the caudal DVC associated with diabetes; 2); Determine effects of DVC manipulation on systemic glucose metabolism; and 3) Determine effects of bariatric surgery on diabetes-related neuroplasticity in the vagal complex. Results will guide future development of novel disease-modifying therapies, based on modulating specific neural functions in the vagal system to address diabetes-related glycemic dysregulation in patients.