Brian D. Gulbransen, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Nasal trigeminal chemosensitivity in mice and rats is mediated in part by solitary chemoreceptor cells (SCCs) in the nasal epithelium. SCCs express the G-protein a-gustducin in addition to other elements of the "bitter"- taste signaling cascade including PLCp2, TrpM5 and T2R "bitter"-taste receptors. Many noxious substances elicit the sensation of bitter taste in the oral cavity but are irritating or painful in the nasal cavity. The proposed experiments fall into 2 aims: first to describe what type of trigeminal nociceptive fibers innervate SCCs. We will utilize immunocytochemistry to assay nerve fibers innervating SCCs for neuropeptide content, the ability to bind isolectin B4, and the expression of P2X3 and kainate receptors, both of which are commonly expressed by nociceptive fibers. Second, we will investigate which part of the SCC response is TrpM5-mediated. TrpMS is an obligatory component of T2R-mediated responses in taste cells and TrpM5- KO mice lack taste responses to T2R ligands. We will use calcium imaging to visualize SCC responses to test stimuli from a panel of classic bitter and trigeminal stimuli. Responses will be recorded in SCCs from both wild type and TrpM5-KO mice to separate TrpM5 and non-TrpM5-mediated responses. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Reflex behaviors of the intestine including peristalsis are orchestrated by the enteric nervous system (ENS); a complex neural network embedded in the gut wall. Inflammation profoundly alters ENS circuits controlling motility by promoting enteric ganglionitis; an inflammatory neuropathy characterized by the death of enteric neurons. Neuropathy is increasingly recognized as a trigger for persistent gut dysfunction in gastrointestinal (GI) motility and functional bowel disorders but the mechanisms that regulate neuropathy are not understood. This proposal investigates the role of enteric glial cells, astrocyte-like cells that surround neurons in the ENS, in the regulation of enteric neuropathy. The proposed studies will use in vivo models of GI inflammation, transgenic mice, immunohistochemistry, live-cell imaging with fluorescent probes, biosensing assays and functional tests to study neuron-glia interactions. The central hypothesis is that purinergic activation of enteric glial cells differentially regulates neuron survival depending on glial activation by ADP or adenosine. There are 2 specific aims in this proposal, each with three sub-aims. Each aim will link in vitro mechanistic studies in tissue from humans and mice with in vivo functional studies in transgenic mice. Aim 1 will test the hypothesis that glial Ca2+ responses driven by ADP cause reactive gliosis, neuron death and gut dysfunction. Specific aim 1A will test how activation of glial Ca2+ responses in GFAP:hM3Dq mice or human tissue transduced with glial- specific vectors affects the induction of reactive gliosis and neuron death. Aim 1B wil test whether glial cells directly drive neuron death by releasing neurotoxic substances or if glial driven neuron death requires immune cell recruitment. Mice with an inducible ablation of connexin-43 or MHC-II in glia will be used to specifically interfere with gliotransmitter release o immune cell recruitment, respectively. Aim 1C will test how manipulation of gliosis using the transgenic mice listed above affects in vivo and ex vivo intestinal function. Aim 2 will test the hypothesis that adenosine inhibits reactive gliosis and stimulates protective mechanisms in glia to preserve ENS function. Aim 2A will use drugs and CD73 null mice to test if activation of glial adenosine receptors is necessary and/or sufficient to reverse reactive gliosis. Aim 2B will test whether the neuroprotective actions of glial A2BR activation are mediated by altering the release of glial mediators or by decreasing the inflammatory infiltrate following in vivo inflammation in CD73 null mice. Aim 2C will use in vivo inflammation, drugs and CD73 null mice to determine how manipulation of glial adenosine signaling impacts in vivo and ex vivo assays of gut function following acute inflammation. Significance: Intestinal inflammation can drive enteric neuropathy, leading to persistent gut dysfunction in GI motility disorders. Understanding how glial mechanisms both promote, and limit enteric neuropathies is important because it could lead to the discovery of novel therapeutic targets and a common causative mechanism of neuron death in GI motility disorders, functional bowel disorders and inflammatory bowel disease.

ABSTRACT Abdominal pain is the most common gastrointestinal issue and is a major cause of suffering in functional gastrointestinal disorder such as irritable bowel syndrome (IBS) and in the inflammatory bowel diseases (IBD). Despite extensive efforts, there is still no consensus regarding the mechanisms responsible for producing abdominal pain. This lack of progress is reflected by the poor efficacy of current therapies and the stalled progress toward novel, targeted therapies for abdominal pain. The overall goal of this proposal is to understand mechanisms that sensitize visceral nerve fibers and the specific focus of this proposal is on mechanisms regulated by enteric glia. Glia play a central role in the regulation of pain transmission in the central nervous system, but how interactions between glia and nerve fibers in the intestine contribute to the generation of visceral pain is unknown. This proposal tests the central hypothesis that enteric glia contribute to visceral hypersensitivity by direct interactions with nociceptors, and indirectly by modulating immune responses. This dual hypothesis will be tested in two specific aims that utilize targeted genetic models to manipulate glia, established animal models of visceral hypersensitivity, and optogenetic recordings, immunohistochemical assays, electrophysiology, and visceromotor reflex recordings to study the impact on sensory neuron activity in health and disease. Aim 1 will test the hypothesis that enteric glia directly modulate the activity of visceral nociceptors. Specific Aim 1a will use chemogenetic and knockout mouse models to study the role of gliotransmitter release and Specific Aim 1b will use selective drugs and knockout mice to study the role of glial ectoenzymes that regulate ATP and histamine availability. Aim 2 will test the hypothesis that enteric glia indirectly modulate visceral nociceptors through interactions with immune cells. Specific Aim 2a will use transgenic mice and monoclonal antibodies to study glial interactions with macrophages mediated by the release of mediators including ATP and M-CSF. Specific Aim 2b will use knockout mice and monoclonal antibodies to study interactions that are secondarily dependent on interactions between glia and lymphocytes mediated by antigen presentation. Immune responses in Aim 2 will be analyzed by microarrays, immunohistochemistry, and flow cytometry. The results of this study will identify novel effects of glial?immune interactions on sensory neurons in the periphery. This new insight into mechanisms that sensitize visceral nerves will facilitate the development of new therapies for abdominal pain in functional gastrointestinal disorders, such as IBS and IBD.

PROJECT SUMMARY Reflexive motor behaviors of the intestine including peristalsis are controlled by the enteric nervous system (ENS); a complex neural network embedded in the gut wall. Perturbations within the ENS contribute to the development of dysmotility in irritable bowel syndrome, inflammatory bowel disease, and severe motility disorders such as chronic intestinal pseudo-obstruction, but the mechanisms responsible for persistent changes in enteric neural circuitry are unknown. Recent data show that enteric glia, non-neuronal cells that surround enteric neurons, regulate neuronal excitability and contribute to neuroinflammation. The overall goal of this proposal is to define how specialized interactions between enteric glia and neurons regulate motility and how alterations in those mechanisms contribute to disease. This proposal tests the central hypothesis that enteric glia are specialized to potentiate the activity of ascending excitatory neural pathways involved in normal contractile motility, and that disruption of this regulatory system by inflammation contributes to neuronal hyperexcitability. This dual hypothesis will be tested in two specific aims that utilize genetically encoded calcium indicators to study neuron-glia interactions, glial chemogenetic actuators to study how glia modulate specific types of enteric neurons, and a post-inflammatory model of enteric neuroplasticity to study how glia contribute to neuronal hyperexcitability following inflammation. Aim 1 will test the hypothesis that enteric glia are specialized to sense excitatory neurons and potentiate ascending neural pathways involved in the contractile phase of motility. Aim 1.1 will use genetically encoded calcium indicators to study glial recruitment by polarized neural pathways in motility reflexes. Aim 1.2 will combine the chemogenetic activation of enteric glia with neuronal and glial imaging using genetically encoded calcium indicators to test the hypothesis that glia differentially affect subsets of enteric neurons. Aim 2 will test the hypothesis that glia contribute to neuronal hyperexcitability following colitis by increasing positive feedback to excitatory neurons and by reducing inhibitory feedback from inhibitory neurons. Aim 2.1 will study how altered interactions between glia and excitatory neurons contribute to neuronal hyperexcitability following colitis. Aim 2.2 will use mutant mice and selective drugs to study how glia contribute to neuronal hyperexcitability through interactions with inhibitory neurons. The results of this study will provide novel insight into glial mechanisms that regulate the excitability of enteric neural circuits. A better understanding of the glial mechanisms that regulate motility will facilitate the development of therapeutics for dysmotility by revealing novel targets to modify gastrointestinal reflexes.