Hui-Lin Pan, Ph.D., M.D. - US grants

DESCRIPTION: (Adapted from the application) The activation of cardiac sympathetic afferents during ischemia is responsible for conveying cardiac nociception and initiating neural reflexes which lead to hemodynamic alterations and arrhythmias. "Silent" afferents (afferents exhibiting no spontaneous activity and unresponsive to physiological stimuli) are known to play a critical role in nociception, however it remains uncertain whether the heart is innervated by silent cardiac sympathetic afferents. Furthermore, the role of many ischemic metabolites in activation of ischemia-sensitive cardiac sympathetic afferents remains to be established. The following hypotheses will be tested: 1. the heart is innervated by a population of silent sympathetic afferents which function primarily as nociceptors; and 2. myocardial ischemia increases protons and amphipathic lipid metabolites (palmitylcarnitine and lysophosphatidylcholine) concentrations in the myocardial interstitial fluid which contribute to the activation of cardiac sympathetic afferents. The investigator proposes to initially demonstrate the presence of silent cardiac afferents utilizing a stimulating electrode as a search stimulus. The investigator will then determine the responses of silent cardiac sympathetic afferents to cardiac distension, myocardial ischemia, and algesic chemicals. In addition, the investigator plans to use cardiac microdialysis to document the production of protons/lactic acid and amphipathic lipid metabolites in the myocardial interstitial fluid during ischemia. Finally, the investigator will record single-unit activity of afferents to evaluate the role of protons and amphipathic lipid metabolites in the activation of ischemia-sensitive cardiac afferents by buffering the pH of the afferent's receptive field and inhibiting the activity of carnitine acyltransferase-I, respectively. The proposed research will provide insights into the neural encoding mechanism of cardiac nociception and mechanisms of activation of cardiac nociceptors during ischemia. Such information may also suggest alternate interventions designed to treat intractable angina pectoris and to limit potentially dangerous cardiac reflexes.

The applicant's career goal is to conduct independent cardiovascular research in an academic institution. The long- term objective of his research is to explore mechanisms of integrative control of cardiovascular function. This career award will increase substantially his productivity and solidify his establishment as a contributing scientist in this research area. The Wake Forest University School of Medicine provides a supportive environment for the continued development of his research career. The goal of the proposal is to establish an independent research program that will provide insights into the mechanisms governing integrative control of regional blood flows and systemic circulation. Ischemic stimulation of visceral afferents has profound impact on systemic circulation. Sympathetic efferent discharges to the heart and various vascular beds play an important role in cardiovascular reflexes. However, the mechanisms concerning sympathetic discharges during ischemia are not fully known. In this application, he will investigate a complex interaction among vagal afferents, vasopressin, sympathetic efferent nerves and the splanchnic circulation in integrative cardiovascular control during myocardial and mesenteric ischemia. The central hypothesis to be tested in the proposal is that ischemic stimulation of cardiac and abdominal afferents reflexly augments the splanchnic sympathetic outflows through a mechanism of spatiotemporal summation of efferent nerve discharges. This mechanism, in turn, causes a greater reduction of splanchnic blood flow to increase the blood pressure followed by secondary increases in heart rate and myocardial contractility. Increased splanchnic sympathetic outflow is also regulated by circulating Vasopressin due to activation of abdominal vagal afferents during mesenteric ischemia. These studies will yield new information as to how the cardiovascular and sympathetic discharges are influenced by vagal afferents and vasopressin during ischemia. Such information will improve our understanding of the mechanisms of integrative control of cardiovascular function by sensory and autonomic nervous systems in pathophysiological states.

DESCRIPTION (provided by applicant): Diabetic neuropathy is one of the most important complications afflicting diabetic patients. Since chronic pain caused by diabetic neuropathy often is not adequately relieved by traditional analgesics, it represents an important unmet clinical need. The major objectives of this proposal are to study changes in spinal muscarinic receptors and mechanisms of muscarinic analgesia in diabetic neuropathic pain. Preliminary evidence is presented that muscarinic receptors in the spinal cord are up-regulated in diabetes, which may account for the enhanced muscarinic analgesia in diabetic neuropathic pain. Furthermore, the preliminary study suggests that inhibition of the glutamatergic synaptic input to dorsal horn neurons is an important analgesic mechanism of spinally administered cholinergic agents in diabetic neuropathic pain. The following hypotheses will be tested using animal models of diabetes: 1) Muscarinic receptors in the spinal cord dorsal horn are up-regulated in diabetes; Increased spinal muscarinic M2/M4 receptors play a major role in the enhanced analgesic action of spinally administered cholinergic agents in diabetes; 2) Activation of muscarinic receptors causes a more significant reduction in spinal glutamate release from primary afferent terminals in diabetes; Muscarinic receptor agonists elicit GABA release, which activates presynaptic GABAB receptors to inhibit glutamate release onto spinal lamina II neurons in diabetes; and 3) The inhibitory effects of spinally administered cholinergic agents on spinothalamic tract neurons and nociception are mediated, to a greater extent, by spinal GABAB receptors in diabetes. Quantitative measurements of G protein-coupled receptors, single-unit recordings of spinal dorsal horn neurons, whole-cell voltage-clamp recordings of glutamate- and GABA-mediated postsynaptic currents in spinal cord slices, and behavioral assessment of nociception will be used. These integrated studies are important for our understanding of the mechanisms of altered spinal cord pharmacology in diabetic neuropathic pain. This new information also will provide a rationale for development of improved therapies for patients with diabetic neuropathic pain.

DESCRIPTION (adapted from applicant's abstract) The overall objective of this proposal, which is a response to the Program Announcement New Directions in Pain Research, is to study the role of peripheral glutamate receptors in the generation of ectopic discharges from injured nerves as it relates to neuropathic pain. Preliminary evidence is presented that glutamate is accumulated at the site of nerve injury and block of ionotropic glutamate receptors decreases ectopic discharges recorded from injured nerve axons and attenuates pain behaviors in a rat model of neuropathic pain. The following hypotheses will be tested: 1. Blockade of ionotropic glutamate receptors or inhibition of glutamate synthesis at the site of nerve injury attenuates neuropathic pain states; 2. Peripheral nerve injury elicits an augmented expression of ionotropic glutamate receptors at the site of nerve injury and an increased glutaminase activity in the neuroma, dorsal root ganglia and spinal cord; 3. Peripheral nerve injury increases local glutamate accumulation, which is attenuated by inhibition of glutaminase or blockade of ionotropic glutamate receptors; and 4. Glutamate accumulation at the site of nerve injury contributes to the generation of abnormal afferent discharges, which play an important role in the sensitization of spinal dorsal horn neurons. A tight partial sciatic nerve ligation rat model of neuropathic pain will be used. Behavioral assessment of allodynia will be threshold to withdrawal using von Frey filaments. Motor function will also be evaluated. Ectopic nerve activity proximal to the ligation will be measured by extracellular recording from split filaments and assigned to fiber class on the basis of conduction velocity. Single unit recording of dorsal horn neural activity will also be done. Immunocytochemistry and electron microscopy will be used to detect changes in glutamate receptor expression, which will also be measured quantitatively by Western blot. Glutamate and aspartate content of nerve samples will be measured with HPLC. Glutaminase will be measured by HPLC determinations of glutamate over time.

DESCRIPTION (provided by applicant): The sympathetic drive emanating from the brain is increased in animal models of hypertension and in patients with primary hypertension. The paraventricular nucleus (PVN) of the hypothalamus is an important site for the control of sympathetic outflow through its projections to sympathetically related sites in the brainstem and spinal cord. During the previous funding period, we showed that augmented glutamatergic input contributes to increased excitability of PVN presympathetic neurons and elevated sympathetic vasomotor tone in the animal model of hypertension. However, little is known about the molecular mechanisms underlying the sustained increase in glutamatergic input to the PVN in hypertension. Our recent study suggests that group I metabotropic glutamate receptors (mGluRs) in the PVN are critically involved in the support of elevated sympathetic outflow in hypertension. In this competing renewal proposal, we will use spontaneously hypertensive rats and renovascular hypertensive rats as animal models of hypertension to test our central hypothesis that group I mGluRs are upregulated at presynaptic and postsynaptic sites, which leads to increased glutamatergic input and excitability of PVN presympathetic neurons in hypertension. Our specific aims are to determine (1) the changes in the expression and distribution of group I mGluRs in the PVN during the development of hypertension; (2) the contribution of presynaptic group I mGluRs to augmented glutamatergic synaptic input to PVN presympathetic neurons in hypertension; (3)the downstream mechanisms mediating increased excitability of PVN presympathetic neurons by activation of postsynaptic group I mGluRs in hypertension; and (4) the changes in calcineurin activity and their contribution to increased group I mGluR and NMDA channel activity in the PVN in hypertension. The important roles of group I mGluRs and calcineurin in increased glutamatergic input in the PVN have not been recognized previously. Our proposed studies are expected to unravel a cascade of molecular events responsible for the sustained increase in sympathetic vasomotor tone in hypertension. This new information should have a major impact on our understanding of the fundamental neurogenic mechanisms underlying the development of primary and secondary hypertension and on the design of new treatments for hypertension.

Diabetic neuropathy is one of the most important complications that afflict people with diabetes. Because chronic pain caused by diabetic neuropathy is not adequately relieved by existing analgesics, it represents an important unmet clinical need. The cholinergic system in the spinal cord is critically involved in the control of pain transmission. Although distinct M" M3 , and M. subtypes are involved in the regulation of excitatory and inhibitory neurotransmitter release to spinal dorsal horn neurons, little is known about how the function of these mAChR subtypes is altered in diabetic neuropathic pain. The major objectives of this proposal are to study the functional plasticity of spinal muscarinic acetylcholine receptor (mAChR) subtypes in the regulation of nociceptive transmission in painful neuropathy associated with type 1 diabetes. Our preliminary findings suggest that diabetic neuropathy affects primarily the M, and M. mAChR subtypes in the dorsal root ganglion and spinal cord. The specific aims of this project are to determine (1) the functional changes in mAChR subtypes in the spinal dorsal horn and dorsal root ganglion after induction of painful neuropathy in type 1 diabetes and (2) the roles of individual mAChR subtypes in the spinal cord in the control of nociception in diabetic neuropathic pain. Our central hypothesis is that diabetic neuropathy primarily upregulates M, and M. mAChRs on the primary sensory neurons and spinal dorsal horn neurons to inhibit nociceptive transmission. We will use a combination of multidisciplinary approaches including whole-cell patch-clamp recordings of postsynaptic currents in perfused spinal cord slices, real-time RT-PCR, and knockdown of spinal mAChR subtypes with small interfering RNA. These studies will provide substantial novel information about the mechanisms of plasticity in the spinal cholinergic system and mAChR subtypes in diabetic neuropathy. Findings from this project will provide a rationale for the development of new therapies for patients with intractable diabetic neuropathic pain.

DESCRIPTION (provided by applicant): Neuronal Plasticity and Signaling in Neuropathic Pain Project Summary Activity-dependent synaptic plasticity at the spinal cord level is fundamentally important to the development of neuropathic pain caused by traumatic nerve injury and surgery. N-methyl-D-aspartate (NMDA) receptors in the spinal dorsal horn are critically involved in central sensitization and maintenance of neuropathic pain. However, the mechanisms of potentiated NMDA receptor activity in the spinal cord after nerve injury remain poorly understood. Although increased phosphorylation of NMDA receptors after nerve injury is known, the upstream mechanisms of increased NMDA receptor activity in neuropathic pain remain to be determined. In our preliminary studies, we found that inhibition of the protein kinase CK2 in the spinal cord completely reversed increased NMDA receptor activity and produced long-lasting attenuation of allodynia caused by nerve injury. In this application, we will use an animal model of neuropathic pain to test the central hypothesis that nerve injury increases CK2 activity in the spinal cord, which facilitates phosphorylation of the NR1 subunit of NMDA receptors and potentiates NMDA-mediated synaptic transmission in neuropathic pain. The specific aims of this application are to (1) define the role of imbalance between the CK2(phosphorylation) and calcineurin(de-phosphorylation) activities in augmented NMDA-mediated synaptic transmission at the spinal cord level and neuropathic pain;(2) determine the level of CK2 activity and its role in increased phosphorylation of the NR1 subunit of NMDA receptors in the spinal cord after nerve injury;and (3) identify the protein interaction between NR1 and CK2 subunits, the CK2 phosphorylation sites on the NMDA receptor, and their roles in regulation of increased NMDA receptor activity in neuropathic pain. The role of CK2 in the development of synaptic plasticity in the spinal cord induced by nerve injury has not been recognized previously. We expect that new findings from this proposal will be critical not only to the significant improvement of our understanding of the molecular mechanisms of neuropathic pain but also to the development of new strategies to treat neuropathic pain. Because directly blocking NMDA receptors produces intolerable side effects, targeting CK2 and its specific NMDA receptor phosphorylation sites could represent novel strategies for reducing the NMDA receptor activity and neuropathic pain. PUBLIC HEALTH RELEVANCE: Neuropathic pain remains a major clinical problem because available pain medications are less effective and possess serious side effects. This application seeks to define the mechanisms involved in the maintenance of chronic neuropathic pain caused by nerve injury. Findings from this proposal not only are critical to significantly improve our understanding of the cellular and molecular mechanisms of neuropathic pain but also are important to the development of new strategies to treat neuropathic pain.

DESCRIPTION (provided by applicant): The goal of this project is to study the epigenetic basis of chronic pain, with a specific emphasis on understanding the epigenetic mechanisms active during the transition from acute pain to chronic neuropathic pain. Chronic pain leads to prolonged suffering and a reduced quality of life for millions of Americans. Neuropathic pain remains a major clinical problem and therapeutic challenge because existing analgesics are often ineffective and can cause serious side effects. In addition, the mechanisms involved in the sustained alterations in gene expression found in primary sensory neurons and their role in the transition from acute to chronic pain are still poorly understood. One of the characteristic changes in neuropathic pain is the persistent reduction in the expression and function of certain voltage-activated K+ (Kv) and Ca2+-activated K+ channels in dorsal root ganglion (DRG) neurons. Down regulation of Kv1.4, Kv4.2, or large-conductance Ca2+-activated K+ (BK) channels can increase the excitability of DRG neurons, which causes a persistent increase in abnormal nociceptive input and chronic pain. One possibility is that these genes are down regulated by epigenetic changes. Despite the significant advances being made in understanding the role of epigenetics in regulating genes involved in developmental biology and cancer, little is known about the importance of epigenetic changes in the development of chronic pain. In this collaborative and multi-disciplinary project, Dr. Hui-Lin Pan, a neuroscientist with expertise in neural plasticity and neuropathic pain, and Dr. Jean-Pierre Issa, an expert in epigenetics, will collaborate and identify epigenetic changes in the DRG in neuropathic pain. We will test the overall hypothesis that epigenetic mechanisms contribute to the neuroplasticity in primary sensory neurons that is involved in the induction, development, and maintenance of chronic neuropathic pain. The specific aims of this application are to (1) identify changes in DNA methylation and histone modifications of Kv1.4, Kv4.2, and BK genes in the DRG during neuropathic pain development;(2) identify genome-wide patterns of DNA methylation and histone modifications in the DRG during neuropathic pain development;and (3) determine the effects of epigenetic modulation on the development of chronic neuropathic pain and the associated silencing of K+ channel genes in the DRG. Because the critical role of epigenetic mechanisms in the regulation of gene expression and development of neuropathic pain has not been recognized previously, our use of epigenetic approaches to answer these questions could transform our knowledge of how the epigenome defines and contributes to chronic pain development. We expect that new findings from this proposal will provide novel insight into the epigenetic control of chronic pain development and greatly improve our understanding of the molecular pathways that regulate neuroplasticity in chronic pain. In addition, our project may provide novel information, which could lead to the development of new epigenetic therapeutic agents to prevent and treat chronic neuropathic pain. PUBLIC HEALTH RELEVANCE: Chronic neuropathic pain is a major clinical problem because available pain medications are less effective and possess serious side effects. Epigenetic codes include patterns of DNA methylation and histone modifications that regulate the expression of various gene products. This application seeks to define whether epigenetic mechanisms are involved in the development of chronic neuropathic pain. Findings from this proposal not only are critical to significantly improve our understanding of the molecular mechanisms of neuropathic pain but also are important to the development of new strategies to prevent and treat chronic pain.

? DESCRIPTION (provided by applicant): Primary hypertension is the most prevalent type of hypertension, affecting 90-95% of hypertensive patients. However, the underlying etiology of primary hypertension remains poorly understood. The sympathetic drive emanating from the brain is increased in animal models of hypertension and in patients with primary hypertension. The paraventricular nucleus (PVN) of the hypothalamus is an important site in the central nervous system for increased sympathetic outflow through its projections to sympathetically related sites in the brainstem and spinal cord in multiple forms of hypertension. Increased excitability of presympathetic neurons in the PVN are critically involved in the development of hypertension. In this application, we propose to determine the upstream signaling mechanisms responsible for the long-lasting neuronal and synaptic plasticity in the PVN in hypertension. Our preliminary data suggest that Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the PVN is upregulated and contributes to the maintenance of increased sympathetic drive in hypertension. We will use animal models of hypertension to test our overall hypothesis that increased CaMKII activity in the PVN contributes to elevated sympathetic vasomotor tone by augmenting N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory input in hypertension and that reduced calcineurin activity in the PVN in hypertension potentiates CaMKII activity and NMDAR-mediated excitatory input. The important roles of CaMKII and calcineurin in the PVN in synaptic integration and neuronal plasticity associated with development of hypertension have not been previously recognized. Our proposed studies are expected to unravel a cascade of molecular events responsible for the sustained increase in sympathetic vasomotor tone in hypertension. This new information should have a major impact on our understanding of the fundamental mechanisms underlying the development of neurogenic hypertension and on the design of new treatments for resistant and neurogenic hypertension.

Project Summary Neuropathic pain caused by trauma and surgery is a major clinical problem and remains difficult to treat. Neuronal plasticity at the spinal cord level is fundamentally important to the development of neuropathic pain caused by nerve injury. N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated cation channels critically involved in central sensitization and maintenance of neuropathic pain. However, the molecular mechanisms underlying potentiated NMDAR activity in the spinal dorsal horn after nerve injury remain poorly understood. ?2?-1, commonly known as a subunit of voltage-activated calcium channels, is upregulated in the dorsal root ganglion and spinal dorsal horn after nerve injury. Although ?2?-1 upregulation contributes to neuropathic pain, little is known about the mechanisms through which ?2?-1 is involved in neuropathic pain development. In our preliminary studies, we found that ?2?-1 physically interacted with NMDAR subunits and that siRNA knockdown of ?2?-1 profoundly attenuated spinal cord NMDAR activity increased by nerve injury. Furthermore, gabapentin treatment interrupted the ?2?-1 and NMDAR association and normalized nerve injury?induced potentiation of spinal NMDAR activity. In this proposal, we will use a multidisciplinary approach to test our central hypothesis that nerve injury?induced ?2?-1 upregulation critically contributes to potentiation of spinal NMDAR activity and development of neuropathic pain by physically interacting with NMDARs and that gabapentinoids attenuate nerve injury?induced pain hypersensitivity and NMDAR activity at the spinal cord level by disrupting the ?2?-1?NMDAR physical association. Our project is expected to generate substantial new information to unravel a novel function of ?2?-1 as a potent regulatory protein of NMDARs and to identify the important role of ?2?-1?NMDAR complexes in the development of neuropathic pain. Because our studies will provide new molecular insight into mechanisms of neuropathic pain and gabapentinoid actions, our project is highly innovative and underscores a major conceptual advance in understanding the molecular composition, heterogeneity, and function of native NMDARs and synaptic plasticity. We expect that these new findings will significantly advance our understanding of the molecular mechanisms of neuropathic pain and lead to the development of new strategies to treat chronic neuropathic pain.

Project Summary The overall goal of our research program is to elucidate the underlying molecular principles that govern synaptic plasticity associated with chronic pain. Chronic neuropathic pain is a significant and unmet clinical problem. Glutamate ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate the vast majority of fast excitatory synaptic transmission in the mammalian central nervous system. AMPARs are tetrameric cation channels composed of a combinational assembly of four subunits, GluA1 through GluA4. GluA2 is particularly important for the biophysical properties of AMPARs because GluA2-containing AMPARs are impermeable to Ca2+. In contrast, GluA2-lacking AMPARs show inward-rectifying currents and have a high Ca2+ permeability and are thus referred to as Ca2+-permeable AMPARs (CP-AMPARs). The prevalence of synaptic CP-AMPARs of spinal dorsal horn neurons is markedly increased in neuropathic pain. However, the molecular mechanisms underlying the switch of AMPAR subunit composition in neuropathic pain remain little known. The major objective of our proposal is to determine the key molecular mechanism responsible for regulating the assembly and trafficking of CP-AMPARs in neuropathic pain. ?2?-1, often considered a Ca2+ channel subunit, is upregulated in the spinal dorsal horn in neuropathic pain. Our preliminary studies showed that ?2?-1 interacted with AMPAR subunits in vitro and in vivo and that increased ?2?-1 expression promoted synaptic incorporation of CP-AMPARs in the spinal dorsal horn. In this proposal, we will test our overall hypothesis that ?2?-1 potentiates the synaptic CP-AMPAR prevalence in spinal dorsal horn neurons in neuropathic pain through physical interaction with AMPAR subunits to preferentially regulate their subunit composition and synaptic trafficking. We will use a multidisciplinary approach to study ?2?-1?AMPAR coupling and its distinct role in neuropathic pain at molecular, cellular, and behavioral levels. At the completion of our project, we will gain significant mechanistic insight into the poorly defined role of ?2?-1 in synaptic plasticity and neuropathic pain caused by nerve injury and diabetic neuropathy. This new information will redefine the physiological function ?2?-1 and the role of ?2?-1?bound CP-AMPARs in the therapeutic effects of gabapentinoids. Therefore, the findings from the proposed studies will have a sustained positive impact by advancing our understanding of the synaptic mechanism of neuropathic pain, leading to the development of new therapies for chronic neuropathic pain.

Project Summary Opioid analgesics are the mainstay of treatment for severe pain caused by cancer and by tissue and nerve injury. However, clinical use of ?-opioid receptor agonists can cause hyperalgesia and the loss of analgesic efficacy, which lead to opioid dose escalation and remain the major obstacles to adequate pain relief with opioids. Although some evidence suggests a strong link between hyperalgesia and analgesic tolerance induced by opioids, the unifying cellular and molecular mechanisms for these two important phenomena are poorly understood. The major objective in this proposal is to identify the essential signaling mechanisms responsible for opioid-induced hyperalgesia and tolerance (OHT). Opioid administration can cause a persistent increase in glutamate release from TRPV1-expressing primary afferents through stimulation of presynaptic N-methyl-D-aspartate receptors (NMDARs), which represents a key mechanism for OHT. NMDARs are a clinically validated target for treating OHT, but little is known about how opioids lead to increased presynaptic NMDAR activity at the spinal cord level. Also, gabapentinoids can reduce OHT in animal models and in patients through a largely unknown mechanism. Our preliminary data showed that phospholipase C was critically involved in increased spinal NMDAR activity and in OHT. Furthermore, opioid-induced phospholipase C stimulation promoted the interaction between ?2?-1 and NMDARs, and such an interaction was diminished by gabapentin. In this project, we will test our central hypothesis that phospholipase C-??dependent signaling contributes to the development of OHT predominantly by promoting ?2?-1?NMDAR interaction to increase presynaptic NMDAR activity at the spinal cord level and that gabapentin reduces OHT and spinal presynaptic NMDAR activity by interrupting ?2?-1?NMDAR interaction. We will use a multidisciplinary approach, including protein biochemistry, electrophysiological recordings in spinal cord slices, siRNA knockdown, and genetic ?2?-1 and phospholipase C-? isoform knockout mice. The proposed studies are highly significant because it will greatly advance our understanding of the fundamental signaling mechanism involved in OHT. Our findings could also help developing new therapeutic agents targeting PLC-? isozymes and ?2?-1?NMDAR interaction sites for treating OHT and reducing opioid consumption and dependence in patients.

Mechanisms of stress-Induced Persistent Hypertension PROJECT SUMMARY Prolonged and persistent stress is a known risk factor in the development of hypertension. Previous stud- ies have shown that in borderline hypertension, chronic stress-induced increases in arterial blood pressure last for a long period after termination of stress paradigms. However, the underlying mechanisms remain unknown. Chronic stress activates corticotropin-releasing hormone (CRH)-expressing neurons in the hypo- thalamus and central nucleus of the amygdala (CeA), which play a key role in regulating autonomic and cardiovascular functions during psychological stress, fear, and anxiety. The overall objective of our project is to determine the role of CeA-CRH neurons in the development of persistent hypertension and the mech- anisms involved. Our preliminary data showed that selectively inhibiting CeA-CRH neurons through a chemogenetic approach prevented chronic stress-induced hypertension in borderline hypertensive rats (BHRs) and that hyperactivity of CeA-CRH neurons was due to a reduction of K+ channel activity resulting from stress-induced increases in histone deacetylase (HDAC) activity. Our pilot study also showed that in- hibition of HDAC activity in the CeA in chronically stressed rats restored K+ expression and decreased fir- ing activity of CeA-CRH neurons. In this proposal, we will test the central hypothesis that hyperactivity of CeA-CRH neurons is responsible for stress-induced hypertension in BHRs and that a reduction in K+ channel activity caused by stress-induced upregulation of HDAC leads to hyperactivity of CeA-CRH neu- rons in stress-induced hypertension. Because the chronic unpredictable mild stress rat model closely re- sembles precipitation of depression by chronic and low-grade stressors in humans, this model will be used to test the hypothesis. We have 4 specific aims: We will attempt to determine the role of CeA-CRH neurons in the sustained hypertension in chronically stressed BHRs (aim 1), determine the role of CeA-CRH neu- rons in regulating blood pressure and sympathetic outflow in chronically stressed BHRs (aim 2), determine the role of K+ channels in the CeA in hyperactivity of CeA-CRH neurons and heightened sympathetic out- flow in chronically stressed BHRs (aim 3), and identify the epigenetic mechanisms involved in long-lasting downregulation of K+ channels in chronically stressed BHRs (aim 4). Our proposal is innovative because findings from our proposal are expected to provide novel information about the cellular and molecular mechanisms responsible for stress-induced persistent hypertension in borderline hypertension. This new information is significant because it may provide an important rationale for development of new strategies to treat neurogenic hypertension.

Synaptic mechanisms of stress-induced hypertension Project Summary Hypertension is one of the most prevalent health problems and a well-recognized risk factor for many fatal diseases. Thus, it is crucial to identify the mechanisms responsible for the development of hypertension. Prolonged and repeated exposure to stress may cause chronic elevation of sympathetic nerve activity, which contributes to the development of hypertension. Individuals with a genetic predisposition to hypertension may develop a sustained elevation of arterial blood pressure even after removal of the stressor. However, little is known about the neural mechanisms underlying chronic- stress?induced persistent hypertension. Our long-term goal is to determine the synaptic mechanisms involved in stress-induced hypertension. This project will use borderline hypertensive rats (BHRs), the first-generation offspring of cross-breeding of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats, to explore the molecular mechanism responsible for chronic stress-induced hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a key brain site that mediates the stress response and is an important source of the excitatory drive that heightens sympathetic vasomotor tone in the development of hypertension. The preliminary studies showed that chronic stress increased the expression level of ?2?-1 in the PVN, which in turn interacts with the glutamate N-methyl- D-aspartate receptor (NMDAR) to increase its synaptic activity. The central hypothesis is that chronic stress transforms borderline hypertension into persistent hypertension through upregulation of ?2?-1 in the PVN, which interacts with NMDARs and leads to enhanced synaptic NMDAR trafficking and heightened sympathetic outflow. The hypothesis will be tested through pursuit of the following 3 specific aims: 1. Determine the extent to which chronic stress upregulates the expression levels of ?2?-1 and NMDARs in the PVN in BHRs. 2. Determine the role of ?2?-1 in the PVN in chronic stress-induced sustained hypertension in BHRs. 3. Determine whether ?2?-1 interacts with NMDARs and the role of ?2?-1 in enhanced NMDAR activity of PVN presympathetic neurons in BHRs with chronic stress- induced sustained hypertension. The proposed work is innovative because it will be the first study to investigate the interactions between ?2?-1 and NMDARs in the PVN in chronic stress?induced hypertension. The proposed work is highly significant because it will not only identify new molecular mechanisms underlying stress-induced hypertension but also may lead to the development of new treatments for neurogenic hypertension. 1

PROJECT SUMMARY Chronic neuropathic pain is difficult to treat and remains a major clinical problem despite numerous clinical and preclinical studies. Our long-term goal is to identify new targets for treatment/prevention of chronic neuropathic pain. Mounting evidence suggests that epigenetic mechanisms and regulation via microRNAs (miRs) are associated with the transition from acute to chronic pain. The goal of this project is to identify REST- and G9a- related transcriptomic and epigenomic regulatory networks in the dorsal root ganglion (DRG) and their roles in the development of neuropathic pain. The transcriptional repressor REST is a major epigenomic regulator. In nerve injury-induced neuropathic pain, epigenetic silencing by REST overexpression in DRG neurons has been implicated in the repression of targets such as Oprm1, Kcnd3, Kcnq2, and scn10a genes. Histone methyl-transferase G9a is also an epigenomic corepressor of many transcription factors including REST. We recently found that G9a is critical in the epigenetic silencing of almost all K+ channel genes in DRG neurons via increased histone H3K9me2 on the genes' promoters during acute-to-chronic pain transition. Accordingly, mice with G9a ablated from DRG neurons do not develop chronic pain after nerve injury. Thus, both REST and G9a present suitable molecular probes to decipher the genetic and epigenetic bases of chronic pain. However, knowledge is limited about the transcriptomic and epigenomic networks associated with REST and G9a in neuropathic pain development. This multiple-PI grant is a collaboration between two labs with complementary expertise. In addition to the G9a mouse model described above, the two labs have developed an innovative experimental system consisting of complementary Rest conditional knock-out (cKO) and new conditional human REST overexpression (cOE) mouse models. Preliminary results indicate that whereas DRG-specific Rest cKO mice show attenuated pain hypersensitivity after nerve injury, DRG-specific REST cOE mice exhibit pain hypersensitivity. Thus, the two contrasting mouse models recapitulate the chronic pain transition, as anticipated based on work in the field, and provide an in vivo system to study transcriptional mechanisms of chronic pain. Here we propose to the test the central hypothesis that REST and G9a are involved in the development of chronic pain after DRG nerve injury via unique transcriptomic and epigenomic signatures. If successful, our proposed research will identify all REST and G9a targets in DRG neurons including mRNAs and miRs, as well as REST- and G9a-mediated epigenomic changes in the development of chronic pain after nerve injury. We will also functionally identify those REST- and G9a-regulated miRs that could be utilized to block REST- and G9a-mediated chronic pain. We will compare our results to those obtained in chemotherapy- induced chronic pain. In summary, the results from this work are likely to generate new signatures and biomarkers that could potentially be utilized to predict patient susceptibility or resistance to chronic pain development. The results could also be used to identify new actionable drug targets in chronic pain.

Neural Mechanisms of Calcineurin Inhibitor-Induced Hypertension Project Summary The major goal of our project is to determine how the central sympathetic nervous system is involved in calcineurin inhibitor?induced hypertension (CIH). Calcineurin inhibitors, including cyclosporine and tacrolimus (FK506), have revolutionized transplant medicine and substantially prolonged graft survival. However, persistent hypertension remains a major adverse effect associated with long-term use of calcineurin inhibitors. Although calcineurin inhibitors can increase the sympathetic nerve activity, the role of the central sympathetic nervous system in the development of CIH has been largely overlooked. Also, previous work on the neural mechanisms of CIH has focused on the acute effect of a single injection of calcineurin inhibitors. It remains unclear where and how the augmented sympathetic outflow in CIH is generated in the brain. The hypothalamic paraventricular nucleus (PVN) plays an important role in the pathogenesis of hypertension, and calcineurin is abundantly expressed in the PVN. Recent studies indicate that ?2?-1 can directly regulate glutamate NMDA receptor (NMDAR) activity in the central nervous system. Our preliminary studies showed that long-term treatment with FK506 induced a gradual and sustained increase in arterial blood pressure, which persisted for many days even after FK506 was discontinued. Furthermore, blocking NMDARs or inhibiting the ?2?-1?NMDAR complex in the PVN profoundly reduced blood pressure and the sympathetic nerve discharges augmented by FK506 treatment. On the basis of our strong preliminary data, we propose to test the overall hypothesis that prolonged treatment with calcineurin inhibitors increases glutamatergic input to PVN presympathetic neurons by potentiating NMDAR phosphorylation and ?2?-1?mediated synaptic NMDAR activity, leading to a sustained increase in sympathetic outflow and hypertension. We will use several innovative in vitro and in vivo approaches to define the persistent neural plasticity involved in CIH at molecular, cellular, and system levels. Our proposed studies are expected to unravel the cellular and molecular substrates responsible for the sustained increase in sympathetic vasomotor activity in CIH. This new information will greatly increase our understanding of the neural mechanisms of CIH and enable the design of new strategies for treating this condition.

Project Summary Chronic neuropathic pain (CNP) is usually caused by disease or damage involving the somatosensory nervous system, adversely affecting millions of Americans. It is difficult to treat and remains a major clinical problem. Opioids, acting through opioid receptors (ORs: MOR for µ, KOR for ?, DOR for ?, NOP for nociceptin), trigger a complex signaling system and function as powerful analgesics. However, chronic opioid treatment causes hyperalgesia/analgesic tolerance and addiction, which have resulted in an opioid epidemic in the U.S. The opioid-induced hyperalgesia /analgesic tolerance (OIH/AT) and addiction can be modulated by many factors including OR expression levels and heteromer formation with different ORs (for example, MOR-DOR) or with other receptors such as the cannabinoid receptor (CNR1). Our long-term goal is to develop new strategies to enhance opioid analgesic effects and reduce opioid consumption for treatment of CNP. REST is a major epigenetic regulator. We and others have found that overexpression (OE) of REST in the dorsal root ganglion (DRG), causing repression of the MOR gene oprm1, is linked to the onset and maintenance of CNP. Our recent studies indicate that peripheral nerve injury in fact reduces opioid analgesia via the REST corepressor G9a-mediated chromatin repression of oprm1. Further, our preliminary studies suggest that MORs in DRG neurons are essential for OIH/AT. This would suggest that the REST-MOR axis in DRG neurons is a major mechanism regulating both CNP and OIH/AT. However, although the discovery of oprm1 as a REST target using a gene-by-gene approach is useful, it is unclear whether REST regulates the impacts of opioid analgesia in CNP or in OIH/AT by controlling the expression of other ORs or CNR1 or both of these processes. Our preliminary results suggest that REST differentially regulates expression of these receptors in DRG neurons. While it causes a decrease in the expression of MOR and DOR, it causes an increase in the expression of NOP and CNR1, perhaps by repressing the expression of an inhibitor of these genes such as a miRNA. To begin to generate comprehensive insights into the role of REST in CNP, we have now developed an innovative experimental system consisting of Rest conditional knock-out (cKO) mice and REST conditional OE (cOE) mice. Preliminary results indicate that whereas DRG-specific Rest cKO mice show attenuated pain hypersensitivity after nerve injury, DRG-specific REST cOE mice exhibit pain hypersensitivity even without nerve injury. Thus, the two contrasting mouse models recapitulate the chronic pain transition and provide a robust system in which to study mechanisms governing OR expression in primary sensory neurons in CNP and in OIH/AT. Here we propose to test the central hypothesis that REST in DRG neurons is involved in regulating opioid analgesia in CNP and in OIH/AT by governing ORs/CNR1 expression through epigenomic regulation of these genes. Thus, manipulation of REST in DRG neurons could be utilized to increase opioid analgesic efficacy and reduce opioid consumption. The project is responsive to PAR-18-742.