1988 — 1990 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Spinal Cord Regeneration @ University of Illinois At Chicago
Recently, a number of studies have shown that mammalian central nervous system axons can regrow following lesion if given the proper environment (Richardson, 1980; David and Aguayo, 1981; Bently and Aguayo, 1982; Guth et al., 1981; Silver and Ogawa, 1983; Goldberg et al., 1986). These findings have renewed interest in regeneration of central nervous system neurons with much of the attention focused on inducing regeneration in species that under normal conditions exhibit little capacity for recovery of function, e.g., rats. While these experiments have produced dramatic and exciting results, they have not revealed the requirements for, or the mechanisms of, successful regeneration. The experiments described in this proposal will provide a detailed description of naturally occurring regeneration in an adult system, the salamander spinal cord. These studies will complement the mammalian studies of regeneration and provide a basis for future studies of the molcular biology of regeneration. The salamander spinal cord has been a model system for the study of regeneration for several decades because of the salamander's ability to recover function after transection of large ablation (e.g., Piatt, 1955a,b; Holtzer, 1951; Butler and Ward, 1967; Stensaas, 1983). Despite this long history many important questions remain unanswered: 1) what percentage of cells regenerate following ablation or transaction of the spinal cord and what is the origin of these cells, 2) where are the somata that give rise to the axons found in the regenerated spinal cord and how far do regenerated axons grow pass the lesion site into the undamaged spinal cord, 3) do regenerated axons make synapses which are appropriate, and 4) how does the repopulation of the damaged spinal cord by neurons and fiber tracts relate to recovery of function. Horseradish peroxidase (HRP) pathway tracing techniques and 3H-thymidine labeling will be utilized to examine these questions in order to produce a complete description of the events involved in the regeneration of the thoracic and lumber regions of the salamander spinal cord.
|
0.961 |
1993 — 1996 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Effects of Overexpression of Nerve Growth Factor
NGF is a neurotrophic factor known to play a central role in neuron survival, axon growth and gene expression during development and in the adult. NGF can also modulate the physiology of primary sensory neurons involved in pain and temperature sensation. We have developed a new approach to study the role of NGF in neuronal development that utilizes a transgenic mouse model system. Three lines of transgenic mice have been isolated that express increased levels of NGF in the skin, a peripheral target tissue that normally produces NGF. These mice express a transgene containing tissue-specific regulatory sequences from a human epidermal keratin gene (K14) linked to the mouse NGF cDNA. The K14-NGF transgene targets NGF expression to the basal cells of the epidermis and other stratified, squamous epithelium. Expression in the skin begins at approximately E14, the time that endogenous NGF levels decline and neuronal cell death is initiated. We have found that the forced upregulation of NGF in the skin during this critical period has profound effects on the development of the peripheral nervous system. The goal of the proposed research is to elucidate the effect of NGF over- expression on trigeminal and dorsal root ganglia, focusing on changes in cell number, neuronal phenotype, and axon growth. The specific aims of these studies are to: 1) Determine if the amount of NGF affects the number and size of primary sensory neurons in trigeminal and dorsal root ganglia. NGF mRNA will be measured using reverse PCR and in situ hybridization analysis. Relative levels of the NGF polypeptide will be measured using western blot analysis. 2) Determine if increased NGF expression induces changes in primary sensory projections to peripheral and central targets. Immunocytochemistry and western blotting for neurofilaments and NGF will be used to examine the density of innervation of the transgenic skin. In addition, tract tracing will be used to determine if central projections of sensory afferents are affected. 3) Determine if over-expression of NGF differentially affects subpopulations of sensory neurons. In situ hybridization will be used to neurochemically identify primary sensory neurons in the trigeminal and dorsal root ganglia in normal and transgenic mice. Once identified the number and somal size of these subpopulations will be determined. Initial studies will use cRNA and oligonucleotide probes for mRNAs known to be regulated by NGF, including preprotachykinin, calcitonin gene- related peptide, cholecystokinin, somatostatin and tyrosine hydroxylase mRNAs. This model provides a unique means to test the major tenet of the neurotrophic hypothesis in an in vivo system in which changes in the level of NGF are restricted to a single target tissue of peripheral neurons.
|
0.961 |
1995 — 2000 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Overexpression of Nerve Growth Factor
DESCRIPTION: Neurotrophins are known to control not only neuronal survival, but may modulate phenotypic differentiation and adult neuropathic pain. In transgenic mice overexpressing NT-3 or NGF, changes are noted in sensory neuron number, projections and pain threshold. The collaborating lab has the ability to determine the "Comprehensive phenotype" of adult sensory neurons, using intracellular recording and labeling. These techniques will be used to examine individual sensory neurons of control and transgenic mice to assess if neurotrophin overexpression alters the phenotype (aims 1 and 2). It is also hypothesized that neuropathic pain recapitulates neurotrophin dependent processes. In aim 3, the phenotype of neurons responsible for neuropathic pain will be characterized, and antineurotrophin antisera will be used to block formation of the sympathetic/sensory neuron connections thought to mediate neuropathic pain.
|
0.961 |
2001 — 2004 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Overexpression of Nerve Growth Factors @ University of Pittsburgh At Pittsburgh
Sensory neurons responsive to painful stimuli, called nociceptors, are crucial for the health of an organism as they are responsible for initiating behavioral responses that prevent or limit tissue damage. Under normal circumstances pain is an important sensation. However, following injury or disease, pain can become persistent, seriously disrupting the lives of those affected. Development and maintenance of nociceptors is known to be dependent on two growth factors, nerve growth factor (NGF) and glial-line derived neurotrophic factor (GDNF). Furthermore, it has been proposed that NGF and GDNF support two different populations of nociceptors and that these two populations are responsible for different types of persistent pain. In addition, changes in nociceptor function induced by NGF and GDNF also modulate nociceptive processing in the adult spinal cord, suggesting a multi-level, growth factor-driven plasticity in the somatosensory system. In our previous studies we determined that NGF plays a dramatic role in determining the "comprehensive phenotype" of nociceptors, influencing physiological, anatomical and functional properties of NGF-dependent nociceptors. The next step is to determine how GDNF affects nociceptors, how changes in NGF and GDNF affect spinal cord neurons receiving nociceptive input, and what affects these changes have on persistent pain. This proposal consists of three Specific Aims employing a "genes to behavior" approach. Specific Aim 1 examines how increases in the level of GDNF affects the comprehensive phenotype of nociceptors. This data will be compared with the results from the NGF studies to provide a complete analysis of the role of growth factors in nociceptor development. Specific Aim 2 examines how NGF- and GDNF- induced changes in nociceptors affects dorsal horn anatomy and neurochemistry. Analysis of spinal cord is crucial because alteration in spinal cord homeostasis can exacerbate or compensate for persistent pain. Specific Aim 3 examines how mice with hypertrophied NGF and GDNF phenotypes respond in two models of persistent pain. Whole animal behavior, nociceptor physiology and changes in dorsal horn neurochemistry will be examined. Upon completion of these specific aims, we anticipate that the differences between NGF- dependent and GDNF-dependent nociceptors will be understood with respect to their anatomical, physiological and neurochemical traits. In addition, it should be possible to identify how these two classes of nociceptors affect dorsal horn development and function. Finally, we will gain insight into how these two classes of nociceptors and their spinal cord targets, respond to induction of persistent pain.
|
1 |
2005 — 2012 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization and Plasticity of Visceral Nociceptors @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Pathological pain arising from visceral sensory neurons is integral to organ dysfunction and can occur in the absence of any obvious structural abnormalities (e.g. irritable bowel syndrome). Visceral pain also contributes significantly to the debilitating nature of many chronic diseases (e.g. pain associated with Crohns's disease). The long-term goal in visceral sensory biology is to identify the causes of these persistent pain states and to develop therapies that specifically target these conditions. Recently, significant advances have been made in understating somatic pain. In large part, this is due to the development of molecular and genetic models that allow a greater level of resolution than has been previously possible. It is well established that visceral afferents are very different from somatic afferents [26]. However, if the techniques used to examine somatic nociception could be adapted for visceral sensory systems, equally significant progress could be made to elucidate basic mechanisms underlying visceral pain. To that end, the present experiments combine behavioral, anatomical, physiological and genetic approaches to study visceral afferents in the mouse. A novel "ex vivo" physiology paradigm that preserves the entire sensory neuron, including its peripheral connection to the organ (in this case the colon) and its central connection to the spinal cord, will be used. This application has 4 specific aims: The first is to determine the heterogeneity of visceral afferents projecting to different abdominal and pelvic organs. The second goal is to use the ex vivo preparation to determine the comprehensive phenotype (CP) of individual visceral sensory neurons. The CP includes an anatomical description of central and peripheral processes, neurochemical characterization, and analysis of action potential shape, conduction velocity and response properties (e.g. threshold, adequate stimulus typing). This information is crucial for the third goal, which is to determine how different populations of visceral afferents (identified on the basis of the CP) respond to insult. For the fourth aim we will repeat these studies in mice lacking the transient receptor potential vaniiloid subfamily receptor 1 (TRPV1, previously known as VR1) to determine if, similar to somatic afferents, this channel is required for development of hypersensitivity.
|
1 |
2007 — 2011 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Growth Factors in Persistent Pain @ University of Pittsburgh At Pittsburgh
The neurotrophin family (NGF, NT-3, BDNF and NT-4) and the GDNF family that includes, artemin and neurturin have been shown to be crucial for sensory neuron development. Nociceptors have been shown to be particularly dependent on NGF and GDNF. Preliminary studies from our laboratories show that artemin and neurturin can, like NGF, affect nociceptor survival and responsiveness. These observations have led us to propose the general hypothesis that NGF is required for nociceptor embryogenesis but that during late development, NGF and the GDNF family combine to regulate nociceptor plasticity and phenotype. The goal of this proposal is to test three hypotheses addressing the roles of artemin and neurturin. SA1 tests the hypothesis that neurturin modulates the comprehensive phenotype (CP) of nociceptors. Specificially, we predict that neurturin is important for the development and differentiation of IB4-positive polymodal nociceptors and that it directly affects the heat responsiveness of these neurons. SA2 tests the hypothesis that like NGF, neurturin and artemin can regulate postnatal sensory neuron phenotype and plasticity. We have preliminary data that neurturin and artemin may be even more potent than NGF for sensitizing primary afferents. This is an exciting result as this is the first time that this family of growth factors, that like NGF is made in the targets of sensory neurons, has been shown to alter sensory neuron response properties. SA3 tests the hypothesis that the alterations of primary afferents induced by inflammation are dependent on artemin and neurturin. NGF has been thought to play an important role in inflammatory pain because of its ability to sensitize primary afferents and because it increases during inflammation. Our preliminary data shows that artemin mRNA increases 10-fold more than NGF mRNA 24 hrs after induction of inflammation. In SA3 we will extend these studies and attempt to block inflammatory pain using siRNAs for artemin, neurturin and NGF and their receptors.
|
1 |
2011 — 2012 |
Davis, Brian M |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Phenotyping Pain in a Mouse Model of Pancreatic Cancer @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Pancreatic cancer, and specifically pancreatic ductal adenocarcinoma (PDAC), is among the five leading causes of cancer death in Western countries including the United States. Despite advances in diagnostic imaging and surgical techniques, mortality still approaches incidence as non-specific and often late symptoms complicate early detection and the frequent spread limit chances of cure. One of the important clinical manifestations of pancreatic cancer is pain. The often intense pain is thought to arise from invasion of tumor cells into the pancreatic nerves. This nerve-cancer interaction may also contribute to the aggressive behavior of pancreatic neoplasms. Together these findings suggest an intimate relationship between the peripheral nervous system and pancreatic cancer, wherein tumor-produced growth factors induce nerve sprouting and hypertrophy, increasing nerve-cancer interactions, which in turn promote cancer growth and invasion. We have begun to breed a transgenic mouse model that expresses the most common genetic lesions seen in human pancreatic cancer (a gain-of-function Kras mutations and loss of the p53 tumor suppressor gene) and develop tumors with virtually all of the features seen in the human disease. But before we develop a comprehensive program to exploit these mice for the study of cancer pain we need to determine if in fact they exhibit quantifiable pain behaviors, and whether these behaviors are correlated with changes in the peripheral sensory nervous system as has been seen in humans. Our hypothesis is that pancreatic cancer in Kras/p53 mice will produce pain behaviors that are correlated with tumor growth and the production of neurotrophic factors that have been shown to regulate anatomy and function of adult nociceptors (e.g., NGF and artemin). To test this hypothesis we will complete the following aims: SA1: Phenotype pain behaviors in Kras/p53 mice during progression of PDAC. a) Using Kras/p53 and control mice, we will conduct longitudinal studies of behaviors that should be affected by ongoing pain, including open field behavior, gait analysis, and hunching, as well as testing for the development of referred hypersensitivity (e.g., to abdominal skin). SA2: Test the hypothesis that PDAC increases neurotrophic factors (e.g. NGF, artemin, GDNF) and induces hypertrophy of visceral afferents . This hypothesis will be examined by: a) Determining changes in growth factor and growth factor receptor expression in pancreatic tumors (via real- time PCR, Western blots). b) Documenting changes in pancreatic innervation for growth factor dependent afferent fibers during the course of PDAC. PUBLIC HEALTH RELEVANCE: Pancreatic cancer remains a common and extremely painful disease, affecting about 40,000 individuals in the United States each year. This proposal will determine if a mouse model of pancreatic cancer, one that has many of the genetic, anatomical and biochemical features of the human disease, also experiences ongoing pain. These studies have to potential to lay the foundation for future experiments that will elucidate the mechanisms that cause pancreatic cancer pain, with the goal of developing novel therapies for cancer pain.
|
1 |
2013 — 2017 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Neurogenic Inflammation in Pancreatic Cancer @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC), like many cancers, has a major inflammatory component that is integral to disease progression. In humans, patients with familial chronic pancreatitis (CP) have a 53-fold increase in their risk for PDAC [92] and 40% go on to develop pancreatic cancer [63]. The link between CP and PDAC is so strong that physicians often recommend prophylactic pancreatectomy for patients with severe CP. The mechanistic link between pancreatic inflammation and cancer is not known. However, studies by our laboratories and others indicate that inflammation accelerates the disease, driving maturation of precancerous lesions into frank cancer [40, 41]. Furthermore, in a genetically engineered mouse model (GEM) of PDAC, pancreatic inflammation increased dissemination of pancreatic cells into the blood and liver [78]. Experiments in our labs show that pancreatic inflammation can be regulated by the nervous system. The importance of neurogenic inflammation is supported by studies that show ablation or silencing of pancreatic afferents attenuates/prevents acute and chronic pancreatitis [45, 67, 81, 82]. PDAC converts the pancreas into a tissue that produces pathological levels of neurotrophic factors that cause hypersensitivity and sprouting of sensory neuron terminals and this likely underlie disease-related neurogenic inflammation. Importantly, preliminary data from our lab indicate that growth factor upregulation begins early in the disease process, even before the appearance of frank cancer (Preliminary Data). These observations in combination with the role that inflammation appears to play in progression of PDAC leads to the central hypothesis of this application: PDAC-generated neurotrophic factors induce neurogenic inflammation that drives PDAC progression. This hypothesis will be tested in the following specific aims: Aim 1: Determine the effect of PDAC-produced neurotrophic factors on pancreatic afferents (dorsal root and nodose ganglion neurons) and whether blocking these growth factors attenuates the release of neurogenic inflammatory molecules. Aim 2: Determine the contribution of neurogenic inflammation to PDAC progression and tumor growth. These studies will directly test the role of the nervous system in PDAC. Three different methodologies will be tested for their ability to block neurogenic inflammation and slow/halt disease progression. Each of these methods is either currently available or in development for use in the clinical setting. If successful, any of these approaches could be used for patients with high-risk for PDAC or those in which the disease is already diagnosed.
|
1 |
2014 |
Davis, Brian M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Peripheral Contributions to Bladder Hypersensitivity @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Disorders of urinary bladder sensation (discomfort, pain and increased sensitivity) characterize painful bladder syndrome/interstitial cystitis (PBS/IC). PBS/IC is a chronic and debilitating disorder in which urinary frequency and urgency are associated with small bladder volume. Pain, however, is the most common and most troubling symptom in PBS/IC patients and is typical of visceral pain in general - 94% of registrants in a PBS/IC database report pain referred to another pelvic area. Urodynamic studies in PBS/IC subjects confirm that bladder sensations and fullness are perceived at lower intravesical volumes, resulting in a leftward shift of the normal psychophysical function (i.e., bladder hypersensitivity). Because the visceral innervation is unique (organs are innervated by two nerves) and noxious visceral stimuli are unlike noxious tissue-damaging somatic stimuli, peripheral mechanisms of bladder hypersensitivity differ from those of somatic hyperalgesia. Mechanical and/or chemical hypersensitivity of the bladder largely underlies the pain and discomfort experienced by PBS/IC patients, yet the molecules and mediators that confer mechanosensitivity and/or sensitization of bladder sensory neurons and their contribution to urinary bladder pain are poorly understood. The ion channels TRPV1 and P2X3 have been implicated in bladder hypersensitivity. We hypothesize that TRPV1 and P2X3 contribute to mechanotransduction and/or sensitization of the bladder innervation and we will study their contributions: 1) using cystometry to functionally evaluate bladder hypersensitivity, 2) to mechano- and chemo-sensitivity of pelvic nerve and lumbar splanchnic nerve receptive endings in the bladder using an in vitro bladder-nerve preparation, and 3) to whole cell currents and excitability of bladder sensory neurons, identified by content of retrograde tracer.
|
1 |
2015 — 2016 |
Davis, Brian M Glorioso, Joseph C. (co-PI) [⬀] |
U18Activity Code Description: To provide support for testing, by means of a research design, the effectiveness of the transfer and application of techniques or interventions derived from a research base for the control of diseases or disorders or for the promotion of health. The project should be capable of making conclusions which are generalizable to other sites. These are usually cooperative programs between participating principal investigators, institutions, and the sponsoring Institute(s). |
Novel Viral Tools For Control of Bladder Function and Pain @ University of Pittsburgh At Pittsburgh
? DESCRIPTION (provided by applicant): There are a number of diseases that affect the bladder that do not have adequate treatments. For example, interstitial cystitis (IC) and overactive active bladder (OAB) are chronic urological disorders characterized by increased micturition frequency and urgency; IC is distinct from OAB in that patients additionally suffer from pelvic/suprapubic pain. Likewise, inability to empty the bladder is a common debilitating problem (often associated with severe pain) among spinal cord injury patients and for some, is the major contributor to decreased quality of life. In all of these examples, it is not completely clear how distinct subpopulations of bladder afferents differentially drive these symptoms and thus, there are basic science issues that need to be addressed before mechanism-based approaches can be explored. Our laboratory (the Davis lab) has been using genetic mouse lines that express channelrhodopsin (ChR2) or halorhodopsin (NpHR) in sensory neurons. Preliminary data presented in this application demonstrate that these light sensitive channels can be used to regulate the visceromotor reflex (a surrogate for bladder pain) as well as control the micturition reflex. Unfortunately, our previous studies rely on transgenic mouse models that have significant limitations inherent in a genetic technology that produces permanent gene changes that are activated during embyogenesis and lack temporal and spatial control. New tools are needed to determine if our observations can be extended to other relevant animal models, as well as developed into effective treatments for human. This application is in response to RFA-RM-15-002 that strives to develop tools that will be tailored to the specific use case/mechanism under study and whose end deliverable is a tool or technology, NOT a biological discovery (from Common Fund pdf provided to potential applicants). The co-PI on this application (Dr. Glorioso) is a pioneer in the use of sensory neuron-specific viruses that can be used to activate or silence sensory neurons. In particular, he has developed a novel, druggable chloride channel (ivermectin) construct that is effective in blocking somatic pain. In addition, the Glorioso lab has produced other novel viral constructs that have passed phase I human trials, allowing the proposed studies to meet the criteria of translatability that is require by this RFA. This proof of concept data while exciting, will only be applicable to human bladder (as well as other animal models) if appropriate strategies are developed that will allow expression of these molecules in humans. The proposed research program will combine the efforts of these two laboratories; one studying pain and regulation of visceral organ function and a second that has a long track-record in designing sensory neuron-specific viruses that can be used to target expression of novel genes to primary afferents. We will produce and test 9 different viral constructs (expressing three novel genes, under three different promoters), targeted to different sensory neuron populations and determine their effectiveness for control micturition and the visceromotor reflex (a surrogate for bladder pain).
|
1 |
2016 — 2020 |
Albers, Kathryn Marie [⬀] Davis, Brian M Koerber, H Richard (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of Epithelial-Neural Communication @ University of Pittsburgh At Pittsburgh
? DESCRIPTION (provided by applicant): The transduction of cutaneous stimuli has been previously thought to be solely a function of sensory fibers. It is now recognized that production of growth factors and neuroactivators (e.g., NGF, ATP, ACh, glutamate) by epidermal keratinocytes can have a profound effect on this process. To unravel these complex interactions and advance our understanding of the mechanisms regulating neural-keratinocyte communication, we developed optogenetic mouse models in which light activated channelrhodopsin (ChR2) is targeted to cutaneous sensory neurons. Light stimulation of the skin of these mice was found to elicit a robust nocifensive behavioral response. Electrophysiological analysis of this activation using a skin-nerve-ganglia and spinal cord ex vivo preparation showed preferential activation of C-fiber nociceptors. Thus, blue-laser light penetrates the epidermis and activates ChR2 at levels that depolarize peripheral nerve terminals. Interestingly, light activation of some neurons did not elicit response properties identical to those obtained using direct mechanical or thermal stimulation of the skin. We hypothesized this lack of a full response reflected a missing stimulus from the skin. We therefore isolated mice in which ChR2 was targeted exclusively to K14 keratin expressing keratinocytes. Remarkably, light stimulation of keratinocytes expressing ChR2 evoked changes in behavioral and electrophysiologic response properties of cutaneous sensory neurons. We also found that different subtypes of cutaneous afferents are activated at different levels suggesting heterogeneity in skin-neural communication. Using these new genetic models we propose three specific aims to advance these findings: Aim 1 experiments will examine how light-induced release of neuroactivators (e.g., ATP) from ChR2- expressing keratinocytes activates subtypes of primary sensory afferents. Aim 2 will determine how light activation of ChR2 or halorhodopsin expressed by subtypes of sensory afferents or keratinocytes affects afferent response properties. We will also determine how this activation compares to mechanical and/or thermal stimulation of the skin. Aim 3 experiments will determine the contribution of changes in keratinocytes and sensory neurons to thermal and mechanical hyperalgesia in a model of inflammatory pain. These studies will determine if hyperalgesia is caused by changes in primary afferents, skin keratinocytes or both. The ability to control activation of either keratinocytes or sensory afferents will provide new insights into how the skin and sensory nervous system communicate under normal and inflamed conditions.
|
1 |
2020 — 2021 |
Albers, Kathryn Marie (co-PI) [⬀] Davis, Brian M Gold, Michael S (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cross-Talk Between the Colon Epithelium, Colon Afferents and Sympathetic Neurons Regulate Pain in the Normal and Inflamed Colon @ University of Pittsburgh At Pittsburgh
SUMMARY Visceral pain is notoriously difficult to treat, often persisting long after the precipitating injury/disease is no longer evident. In this application we will explore a novel, multicellular peripheral circuit that we hypothesize explains many of the intractable features of chronic, visceral pain. We now know that epithelial-neuronal communication is widespread, with numerous epithelial cell types releasing neuroactive substances (e.g., ATP, ACh, 5HT, glutamate). This is particularly apparent in the colon where we have found that channelrhodopsin (ChR2) -induced activation of colon epithelial cells produces high frequency bursting of colon extrinsic primary afferent neurons (ExPAN?s), phenocopying physiologic stimuli and inducing robust behavioral responses (visceromotor responses (VMR), a validated assay of hypersensitivity). Building on these findings, new surprising data indicate colon epithelium also receives functional input from sympathetic neurons; activation of sympathetic projections to the colon induces large, phase-locked calcium signals in the epithelium. Closing the loop, we found that activation of ExPAN?s via colorectal distension (CRD) induces calcium signals in the post-ganglionic sympathetic neurons projecting to the colon, and that ChR2- induced activation of ExPAN?s induces cFos expression in these same neurons. That this multicellular circuit plays a role in visceral pain is supported further by preliminary data that shows that inflammation (acute and/or chronic) is correlated with increased signaling in all portions of this circuit. Thus, the goal of the proposed experiments is to test the hypothesis that persistent visceral hypersensitivity is due, at least in part, to amplification in an epithelial-ExPAN-sympathetic circuit such that it is possible to treat pain by breaking any limb of this feed-forward circuit (Fig.1). This hypothesis will be tested in 3 aims: Aim 1: Determine if persistent hypersensitivity induced in a model of IBD (DSS (dextran sulfate sodium)) is due to increased epithelial signaling and/or ExPAN excitability, Aim 2: Determine if DSS-induced inflammation increases the ability of ExPANs to activate sympathetic neurons in prevertebral sympathetic ganglion (PrSG) directly (via synapses in PrSG) or indirectly (via a spinal cord circuit) and, Aim 3 Determine the ability of sympathetic neurons to drive activity in epithelial cells in naïve mice and in the DSS model of IBD.
|
1 |
2020 — 2021 |
Albers, Kathryn Marie (co-PI) [⬀] Davis, Brian M Heuckeroth, Robert O (co-PI) [⬀] Howard, Marthe J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular and Functional Analysis of Hirschsprung Defects in Humans and Mouse @ University of Pittsburgh At Pittsburgh
Hirschsprung?s disease (HSCR) is a birth defect where the enteric nervous system (ENS) is absent from the distal bowel. Most HSCR patients have reduced activity of RET and many have reduced EDNRB (endothelin receptor 3) activity. Although only the distal colon is aganglionic (missing neurons and glia) in 80% of children with HSCR, proximal bowel regions (and the rest of the GI tract) innervated by enteric neurons harbor the same mutated genes. Since RET and EDNRB are needed for many aspects of ENS development, it seems likely enteric neurons in ganglionic regions of the colon and their connections (hereafter referred to as the ENS connectome) are abnormal. Consistent with this hypothesis, HSCR surgery to remove aganglionic bowel and reconnect ?normal bowel? to the anal verge does not alleviate all HSCR symptoms; up to 50% of children have ongoing issues post-surgery including bowel distension, inflammation, explosive diarrhea, blood in the stool, lethargy and poor feeding. The long-term goal of this research project is to determine how mutations in HSCR-related genes affect the entire ENS and contribute to GI tract dysfunction. The short-term goals are: 1) to identify the gene-expression changes downstream of mutations in RET and EDNRB that occur in HSCR patients and in mice in that contain HSCR-relevant mutations, and 2) Perform anatomical and functional studies in HSCR mouse models to determine how these gene defects negatively impact the enteric nervous system. The overall hypothesis guiding these experiments is that for the innervated portion of the colon, different HSCR mutations produce defects in motility resulting from specific changes in communication between unique subsets of neurons (in the myenteric plexus and autonomic nervous system (ANS)), ICC, and glia. Aim 1: Conduct pooled and single cell RNA-Seq analysis on enteric neurons of wild type and HSCR mice models; compare murine data with RNA-Seq analysis from HSCR patients and controls. Aim 2: Determine the effect of HSCR-associated mutations on ENS/ICC/glia communication. Aim 3: Examine extrinsic parasympathetic and sympathetic drive of myenteric neurons, ICC, glia and associated smooth muscle contractions in HSCR mouse models. Impact: Transcriptomics indicate that the different HSCR mutations will negatively affect the function of different cell types in the ENS and ANS (neurons, glia and ICC). The consequence of the involvement of multiple cell types is that patients may appear similar symptomatically, but the underlying cause, and hence appropriate treatment, may be very different. This research program is designed to identify mutation-specific mechanisms of disease as a basis for development of patient-specific treatments.
|
1 |