Li-Yen M. Huang - US grants

The long-term objective of the proposed research is to understand the mechanisms of action of antiarrhythmic drugs on ionic channels in cardiac cells. The specific aim of these studies is to determine the modes of action of a number of antiarrhythmic drugs on ionic channels in developing heart. The drugs include Dilantin, lidocaine, digoxin and amiodarone. The experiments are designed to study the effects of these drugs on ionic channels in different ages of cultured cardiac cells using voltage clamp and patch clamp techniques. The objectives of these experiments are (a) to determine the properties of ionic channels in fetal and neonatal ventricular myocytes, (b) to characterize the actions of these drugs on ionic channels in cardiac cells which undergo developmental changes, and (c) to study the effects of lidocaine and Dilantin on digoxin or toxin-treated cardiac cells. The ionic current will be measured in different ages of cultured rat aggregate under voltage clamp conditions. Patch pipette voltage clamp technique will be used. The fundamental knowledge obtained from these experiments will be essential in defining the mechanisms of action of antiarrhythmic agents in developing heart.

The long-term objective of the proposed research is to understand the physiology and pharmacology of the neural systems that transmit and modulate pain. The specific aim of these studies is to determine the electrical properties of isolated spinothalamic tract cells and the mode of action of a number of transmitter substances on these cells. The objectives of these experiments are (1) to determine the voltage dependent properties of identified spinothalamic cells and (2) to characterize the actions and interactions of peptides and biogenic amines on these cells. The ionic current of identified single STT cells will be measured under voltage clamp conditions. Patch pipette voltage clamp technique will be used. The fundamental knowledge obtained from these experiments will be essential in defining the mechanisms of action of the putative transmitter in spinothalamic cells.

The long term objective is to understand the physiology and pharmacology of the neural system that transmit and modulate pain. The specific aim is to determine the elecrical properties of isolated spinothalamic and trigeminothalamic cells (dorsal horn projection cells), and the mode of actions of a number of transmitter substances on these cells. The objectives of these experiments are (1) to determine the voltage dependent properties of identified projection neurons and (2) to characterize the actions and interactions of amino acids, peptides and biogenic amines on these cells. The ionic currents of identified single projection cells will be measured under voltage clamp conditions. Patch pipette voltage clamp technique will be used. The fundamental knowledge obtained from these experiments will be essential in defining the mechanisms of action of the putative transmitters in spinothalamic cells.

This research protect is concerned with the physiology and pharmacology of the neural systems that transmit and modulate pain. The long term goals are to understand the conductance mechanism and the regulation of chemosensitivities of identified dorsal horn projection neurons. The specific projects involve 1) the studies of gating behavior of single calcium channels. 2) the modulation of Ca channels by guanine nucleocides, and 3) the characterization of actions of amino acids, peptides and biogenic amines on these cells. The ionic currents and single channel activities of isolated dorsal horn projection neurons will be measured under voltage clamp conditions. Whole-cell and single channel recording techniques will be used. The knowledge obtained from these experience will greatly increase our understanding of the functional properties of projection neurons and facilitate the designing of better pain therapy.

This research project is concerned with the functional role of the trigeminal (V) spinal subnucleus caudalis in the processing of pain information. Our long term goal is to determine how pain information is transmitted and regulated in the central nervous system. The specific goal of this proposal is to examine primary afferent-evoked synaptic responses and the action potential in trigeminothalamic neurons in thin medulla slices. The ionic currents and single channel activities of trigeminothalamic neurons will be measured using patch clamp recording techniques. The information obtained will enhance our understanding of the role that trigeminal system has in processing pain-related messages and will facilitate the designing of the therapy for the treatment of orofacial pain.

The long term goal of this project is to determine the mechanism by which nerve dysfunction results in chronic neuropathic pain. The objective of this proposal is: (1) to understand the modulatory actions of opioids on excitatory amino acid-evoked (EAA) responses in trigeminal neurons; and (2) to define the role of opioids in generating and maintaining the sustained responses to EAA. We hypothesize that binding of an opioid activates G proteins which in turn raise the level of second messengers inside the cell and result in a change in the EAA-evoked responses. The experiments are designed to test this hypothesis. The EAA-activated currents will be measured using the patch clamp technique. Experiments will be performed on trigeminal neurons, labeled trigeminothalamic cells isolated from the spinal trigeminal subnucleus caudalis and the same type of neurons in thin medullary slices. The information obtained from this project will advance our understanding of the pain mechanism and help us design a better therapy for treatment of neuropathic pain.

DESCRIPTION (Investigator's Abstract): The long term goal of this research is the determination of cellular mechanisms of opioid actions on nociceptive (pain) neurons. Because opioids are among the most effective agents for relieving pain, they are thought of as inhibitory agents. Yet, there are many examples in which opioids exert direct excitatory actions on nociceptive neurons. One of the clinically important features of opioid antinociception is that opioids are very effective in relieving inflammatory pain, but are ineffective for neuropathic pain. The focus of this grant is to determine the role(s) of the opioid-induced excitatory responses in inflammatory and neuropathic pain states. The applicants hypothesize that opioid actions are a balance between excitatory and inhibitory effects and a major difference between the actions of opioids on inflammatory as opposed to neuropathic pain is that the balance is tipped towards the excitatory side in the neuropathic state. To test this hypothesis, they will (1) compare the mechanisms by which opioids affect excitatory and inhibitory amino acid responses in dorsal horn neurons isolated from normal, inflamed (i.e., arthritic) and neuropathic animals, and (2) compare the effects of opioids on Ca2+ mobilization in dorsal horn neurons and in primary afferent terminals of these animals. The experiments will be performed on single dorsal horn neurons and spinal cord slices. The membrane currents will be monitored with perforated patch electrodes; the intracellular Ca2+ concentrations will be measured using fluorescence Ca2+ indications. These experiments will provide information about the mechanisms underlying excitatory actions of opioids in the normal, inflammatory and neuropathic states. The knowledge will aid us in developing new drugs and better strategies for the treatment of pain. In addition, the study will facilitate a better understanding of mechanisms underlying the plasticity in nociceptive systems in general.

The long-term goal of this research is to develop gene therapy for chronic pain. Opiates have remained to be the drugs of choice for treating patients with severe pain. The side effects of opiates, including respiratory depression, tolerance and dependence, have often limited their use. The focus of this grant is to design viral vectors that allow for efficient and stable delivery of wild type or mutant mu opioid receptor (muOR) genes to sensory neurons. We hypothesize that appropriate muOR gene delivery and expression in dorsal root ganglion (DRG) neurons will lower the doses of mu-opioids required for analgesia and reduce tolerance, thus decreasing the side effects of opioids. To test this hypothesis, we will (1) deliver the wild type or mutant mu OR gene into sensory neurons using an adeno- associated virus (AAV) vector, (2) evaluate the effects of mu- opioids on pain behaviors in inflamed rats injected with recombinant AAV-mediated muOR transgene (rAAV-muOR), (3) evaluate the effects of mu-opioid in tolerant rats that receive injections of rAAV-muOR and (4) evaluate the effects of mu-opioids on membrane conductance and receptor trafficking in rAAV-muOR- transduced DRG neurons isolated from non tolerant and tolerant rats. The experiments will be performed on rats treated with complete Freund's adjuvant (CFA) and on DRG neurons isolated from those rats. Pain behaviors will be monitored by paw withdrawal latencies, membrane currents will be measured with perforated patch electrodes and receptor trafficking will be examined with immunofluorescence staining and receptor binding assays. These experiments will establish the feasibility of a genetic approach to pain treatment. If successful, the study will provide clinicians with a new tool to treat patients with severe pain and provide researchers a model for studying the role of opioid receptor regulation in the development of opioid tolerance.

DESCRIPTION (provided by applicant): Inhibitory synaptic transmission in nociceptive neurons is largely mediated by glycine receptors (GlyRs). It has been shown recently that intracellular Ca2+ causes a large potentiation GlyR-mediated responses. There is good evidence suggesting that the potentiation is mediated by a diffusible cytoplasmic protein (CytoP). The goal of this project is to develop a new technology to identify the CytoP and study its interactions with GlyRs in trigeminal neurons. We hypothesize that a diffusible CytoP binds to GlyRs at low intracellular concentrations ([Ca2+]i), keeping GlyRs in a low activity state. When [Ca2+]i rises, the protein is rapidly dissociated from GlyRs, resulting in an enhancement of channel activity. To test this hypothesis, we will combine molecular biology, patch-clamp and imaging techniques to determine these interactions in molecular details. The CytoP will be isolated by the two-hybrid method. The interaction of the CytoP with GlyRs will be determine by simultaneously monitor (i) GlyR-mediated currents using the patch clamp technique, (2) intracellular Ca2+ transients using fluorescent Ca2+indicators and (iii) dynamic interactions between GlyRs and cytoplasmic proteins using the frequency resonance energy transfer (FRET) imaging technique. We will then extend this technology to identify CytoP and study the GlyR-CytoP interaction in trigeminal neurons. A better understanding of the mechanism of GlyR modulation will provide important information about the neuronal signaling and may lead to new therapy for orofacial pain.

DESCRIPTION (provided by applicant): ATP is one of the substances released from damaged tissues. It activates P2X receptors in primary sensory neurons and contributes to pain and discomfort. The long-term goal of this research is to understand the role of P2X receptors in nociception. In this grant our focus will be to understand the plasticity in P2X receptors following inflammation and nerve injury. We hypothesize that activation of P2X receptors is greatly enhanced under injurious conditions as results of an increase in the activity of protein kinases, interactions with other receptors and changes in expression of P2X receptors. In vivo and in vitro approaches will be used to test this hypothesis. We will (1) determine the effects of ATP on nociceptive behaviors, (2) identify the functional properties of sensory afferents responsive to ATP and quantify the responses of these afferents to ATP, (3) determine the properties of ATP-induced currents and intracellullar Ca 2+ mobiliTation and (4) determine the mechanisms underlying the potentiation of ATP responses by examining the effects of protein kinases and nociceptive mediators, i.e., bradykinin and prostaglandin E2, on ATP-evoked current and Ca 2+ responses and by determining the expression of P2X receptors. Behavioral experiments will be performed on normal, inflamed and nerve injured rats in vivo. Electrophysiological experiments will be performed on skin and DRG-nerve preparations and single DRG neurons isolated from these rats. Membrane currents will be measured with patch electrodes. Single-unit activity will be monitored with extracellular electrodes, intracellular Ca2+ concentrations will be studied using Ca2+ dyes and P2X receptor expression will be examined with Western analyses. These studies should provide a better understanding of the mechanisms underlying the plasticity of P2X receptors under injurious conditions. The information will be essential for developing new strategies for the treatment of chronic pain.

DESCRIPTION (provided by applicant): Recent studies of cytokine actions in the spinal cord and dorsal root ganglia suggest that neuron-glia interactions are important in initiating and maintaining chronic pain states. How this is accomplished remains unclear. Disorders of the orofacial complex often produce intense chronic pain in patients and results in devastating consequences in their well being. The long-term goal of this research is to understand neuron-glia interactions in the trigeminal ganglion (TG), the primary afferent structure for the orofacial region. Our focus will be to understand the critical role that transmitters released from neuronal somata (cell bodies) of TGs play in neuron- satellite glial cell communication. We hypothesize that excessive firing in TG neurons elicits somatic release of ATP from neuronal somata. ATP activates satellite cells and evokes cytokine release from these cells. Cytokines in turn sensitize the neuronal somata and further increase their activity. In vitro and in vivo approaches will be used to test this hypothesis of positive feedback loop. We will (1) identify the transmitters released from neuronal somata of TGs (2) examine the communication between neuron and satellite cells (3) determine the effects of cytokines on P2X and TRPV receptor-mediated responses, (4) examine the action of cytokines on nociceptive responses in rats and determine the possibility of using siRNA to down-regulate receptors involved in neuron-glia communication and thus to relieve orofacial nociception. Normal, inflamed and nerve-ligated rats will be used. Transmitter release and membrane currents will be measured with patch pipettes;intracellular Ca2+ concentrations will be monitored with Ca2+ dyes;expression of P2X and TRPV receptors will be examined with immunocytochemistry and Western analyses. These studies should provide a better understanding of the mechanisms underlying neuron-glia interactions in TGs. The knowledge will be critical for designing better therapies for the treatment of orofacial pain.

DESCRIPTION (provided by applicant): Our recent studies of the communication between neuronal soma and satellite glial cells (SGCs) in dorsal root ganglia (DRGs) suggest that somatic release of ATP in response to afferent nerve stimulation activates purinergic P2X7 receptors (P2X7Rs) in SGCs. Under normal conditions, tonic activation of P2X7Rs in SGCs reduces P2X3R expression in neuronal somata, thus exerting inhibitory control of neuronal activity. Following high frequency nerve stimulation, activation of P2X7Rs evokes cytokine release causing an enhancement of P2X3R activity in somata and an increase in the excitability of neurons. The P2X7R-mediated SGC-neuronal soma feedback controls under inflammation and nerve injury conditions have not been well established. This knowledge is essential for understanding the generation and maintenance of abnormal chronic pain. The goal of this application is to understand changes in neuronal soma-SGC communication during different phases of chronic pain development. We hypothesize that following injury, an increase in cytokine release switches P2Rmediated SGC-soma communication from inhibitory to excitatory feedback control, thus initiating the development of abnormal pain states. A subsequent large increase in ATP release, which activates P2X7Rs and pannexin-1, produces a large enhancement of SGC-soma communication and thus leads to the transition from the development to the maintenance phase of chronic pain. We will (1) determine if P2R-mediated SGC-soma communication changes during the development and maintenance phase of chronic pain, (2) determine the mechanisms underlying the change in SGC-soma communication and (3) determine if modulation of SGC-soma communication is a valid strategy for the management of chronic pain.