Jianguo Gu - US grants

Description: (Modified from Applicant's Abstract) Sensory information including pain is conveyed to the CNS through primary afferent fibers and transmitted synaptically by the release of glutamate and neuropeptides from the central terminals of primary afferent fibers. The ATP P2x receptor is a new family of ligand-gated cation channels for which extracellular ATP is the endogenous ligand. We have found that P2x receptors are localized at presynaptic terminals on cultured DRG neurons. Activation of these receptors not only enhances spontaneous glutamate release, but surprisingly, it directly elicits action potentials at DRG presynaptic terminals, triggering synchronous multiquantal glutamate release. Studies proposed here will test two central hypotheses: 1) Presynaptic P2x receptors act as a 'central sensory signal generator' to directly initiate sensory signals at the central terminals of afferent fibers. 2) P2x receptor activation enhances glutamate release evoked by conventional sensory impulses. In addition, we will also examine whether endogenous ATP is release from DRG presynaptic terminals. Electrophysiological, pharmacological and immunocytochemical approaches will be applied and experiments will be conducted on a spinal cord slice preparation and a DRG-dorsal horn culture system. The proposed P2x receptor functions will be examined on the primary afferents that are potentially nociceptive types. The long-term objective of our studies are to explore the roles of P2x receptors in both centrally generated pathological pain and in sensory hypersensitivity such as hyperalgesia.

Sensory inputs to the brain are modulated by excitatory and inhibitory activity even at the first central synapses where peripheral inputs contact central nerve cells. In the somatosensory system of vertebrates, for touch, heat and pain detection, spinal afferent nerves carrying incoming sensory information terminate in the spinal cord structure called the dorsal horn, where they synapse on central spinal neurons. One of the neurotransmitter compounds, a peptide called Substance P ('SP'), has traditionally been considered to have only excitatory effects in the dorsal horn, though there have been some inferential suggestions of inhibitory effects. This project presents a novel challenge to the traditional view, with direct tests based on recent evidence that SP may increase inhibitory synaptic activity in the dorsal horn. Electrophysiological recordings using the powerful technique of patch-clamping, coupled with new cellular labeling techniques, allow studying cellular and synaptic mechanisms by which SP produces inhibitory activity in the dorsal horn.
Results will have a substantial impact in challenging a traditional dogma; the importance extends beyond somatosensory neuroscience to modulation in other senses, and to modeling neuronal circuits. This project also has teaching and interdisciplinary training components for graduate students, including international collaborations with Japan.

ABSTRACT: Trigeminal neuropathic pain is the most debilitating pain disorder but current treatments including opiates are not effective for it in most patients. The pain can often be triggered by a breath of cooling air blowing on the face. Mechanisms of trigeminal neuropathic pain exacerbation at cooling temperatures are not well understood, which prevents us from designing mechanistically based therapy to effectively treat this devastating pain. Our recent studies in rat models have suggested that thermally sensitive two-pore domain K+ channels (thermal K2Ps) may play a key role in trigeminal neuropathic pain exacerbation at cooling temperatures. Our central hypotheses here are: 1) Thermal K2Ps provide a brake to prevent nociceptor hyperexcitability under physiological conditions, 2) Trigeminal nerve injury causes thermal K2P down- regulation to impair the brake leading to trigeminal nociceptor hyperexcitability and neuropathic pain, and 3) Cooling temperatures further suppress thermal K2Ps to lead to trigeminal neuropathic pain exacerbation. We will test these hypotheses with the following specific aims. ?Aim 1. Elucidate the role of thermal K2Ps in controlling trigeminal nociceptor excitability and determine the effects of cooling temperatures on K2P functions. We will use immunohistochemistry to characterize thermal K2P expression in trigeminal nociceptors of normal rats. We will use our newly developed in situ patch-clamp recordings to characterize thermal K2P- mediated leak K+ currents in trigeminal nociceptors and their role in controlling trigeminal nociceptor excitability of normal rats. We will study effects of cooling temperatures on thermal K2P functions in trigeminal nociceptors of normal rats. ?Aim 2. Elucidate that trigeminal nerve injury down-regulates thermal K2P expression leading to trigeminal nociceptor hyperexcitability. We will use two established rat models of trigeminal neuropathic pain, the infraorbital nerve chronic constrictive injury (ION-CCI) and oxaliplatin-induced trigeminal neuropathy models. We will study changes of thermal K2P expression and thermal K2P-mediated leak K+ currents in trigeminal nociceptors of these models. We will use pharmacological and genetic approaches to elucidate that down-regulation of thermal K2Ps leads to trigeminal nociceptor hyperexcitability. We will determine how cooling temperatures further exacerbate hyperexcitability of trigeminal nociceptors of these rat models. ?Aim 3. Elucidate that thermal K2P down-regulation underlies trigeminal neuropathic pain, and establish thermal K2Ps as therapeutic targets for trigeminal neuropathic pain in rat models. We will use orofacial operant tests to study whether thermal K2P down-regulation leads to trigeminal neuropathic pain manifested with cold allodynia and hyperalgesia. We will use ION-CCI and oxaliplatin models to determine whether trigeminal neuropathic pain can be alleviated by pharmacological and genetic enhancements of thermal K2P functions in trigeminal afferent nerves. ? Completion of these Aims will fill the scientific gap of trigeminal nociception, elucidate a new mechanism of trigeminal neuropathic pain, and establish new therapeutic targets for this debilitating pain.

DESCRIPTION (provided by applicant): The sense of light touch is critically important for daily life but this important sense can be altered to result in sensory dysfunctions such as tactile anesthesia and mechanical allodynia under pathological conditions. How mammals can sense light touch has been one of the biggest mysteries in science. This lack of knowledge prevents development of potentially effective approaches for preventing or treating mechanical sensory dysfunctions. Our long-term-goal is to uncover the cellular and molecular mechanisms underlying the sense of light touch in mammals. As the first stage of our long-term goal, the overall objective of this application is to study mechanisms underlying mechanical transduction of Merkel cell-neurite complex, a sensory structure essential for sensing light touch in mammals. Our central hypothesis is that Merkel cells are mechanical transducer cells that express mechanically activated ion channels (MA) and that activation of these channels triggers Merkel cells to fire action potentials and release excitatory transmitters. This hypothesis is based on ou preliminary results obtained by using our recently developed patch-clamp recordings from Merkel cells situated in whisker hair follicles (Merkel cell in situ patch-clamp technique). This innovative technique has, for the first time, led us to successfully record MA currents from Merkel cells. We have further discovered that Merkel cells in situ fire action potentials in response to mechanical stimulation. Our unique expertise of Merkel cell in situ patch-clamp recording technique places us at an advanced position to test the hypothesis with the following specific aims: 1) Elucidate ionic mechanisms of MA currents that excite Merkel cells in situ and characterize Merkel cell MA channel properties; 2) Identity ion channels that encode mechanical activity in Merkel cells; and 3) Delineate the mechanisms underlying the transmission of mechanical activity by Merkel cells. The outcomes of the above investigations will provide scientific knowledge about the sense of light touch at a cellular and molecular level. The study may have clinical implications ranging from sensory dysfunctions seen in diabetes and other disease conditions to Merkel cell malfunctions such as Merkel cell carcinoma.

Nodes of Ranvier are highly specialized axonal regions on myelinated nerve fibers of sensory, motor and central nervous systems where action potentials are propagated by saltatory (leap in Latin word) conduction. Saltatory conduction through nodes of Ranvier ensures timely sensory and motor responses and precise signal processing in the CNS. A number of neurological diseases affect nodes of Ranvier to impair saltatory conduction leading to motor disorders, such as paralysis and sensory dysfunctions, such as pain, numbness, and other abnormal sensations. Knowledge of ion channels and their functions at mammalian nodes of Ranvier is a key to fully understanding saltatory conduction under both physiological and pathological conditions, and for potential treatments of those sensory and motor disorders. The overall goal of this project is to study ion channel mechanisms for securing saltatory conduction of action potentials at mammalian nodes of Ranvier. We have recently developed the in situ patch-clamp recording technique for nodes of Ranvier in somatosensory afferent fibers of rats. In the preliminary studies we have found that nodes of Ranvier express surprisingly high levels of the two-pore domain potassium channels (K2P channels), a unique family of ion channels that constitutively open, and the function of which, in action potentials, as well as in nerve conduction was previously unknown. Functionally, our preliminary studies strongly suggest that K2P channels are key molecules for securing saltatory conduction in myelinated somatosensory afferent fibers of mammals. In this application, we will use the in situ patch-clamp recording technique in conjunction with pharmacology, gene knockdown, and immunochemistry approaches to achieve the following specific aims. Aim 1. Characterize K2P channels and elucidate their molecular identities at the node of Ranvier of rat somatosensory afferent fibers. In this aim we will pin down K2P channel subtypes at the node of Ranvier and profile their pharmacological and single channel properties. Aim 2. Study specific roles of K2P channels in securing saltatory conduction at the node of Ranvier of rat somatosensory afferent fibers. This aim will elucidate that the K2P channels at the node of Ranvier play a key role in rapid action potential repolarization and in securing high speed and high frequency saltatory conduction. Aim 3. Elucidate that K2P channels at the node of Ranvier play a key role in temperature-dependent saltatory conduction on rat somatosensory afferent fibers. This aim will test the idea that K2P channels at the node of Ranvier are highly thermal sensitive, which is a determinant factor controlling the velocity and fidelity of saltatory conduction at different temperatures. This aim exemplifies that biological factors affecting K2P channel activity will highly impact saltatory conductions in myelinated nerve fibers. Completion of the 3 Aims will elucidate a novel ion channel mechanism that secures saltatory conduction, which may have implications in sensory and motor disorders with impaired saltatory conduction at the node of Ranvier.