George L. Wilcox - US grants

Phencyclidine (PCP) abuse is a major problem among the youth of this country, but we understand little of its mode of action in the CNS. This project will examine the involvement of PCP/Cross Section receptors as possible modulators of the spinal function of excitatory amino acids (EAAs) in the transmission of pain. This goal will be accomplished through electrophysiological, behavioral, receptor binding and autoradiographic studies. Although the spinal cord is likely not involved in the production of psychosis after repeated PCP abuse, understanding its spinal action may assist in analysis of its action in the brain. This project will also improve our understanding of the neurohumoral transmission and modulation of pain information through synapses between primary afferent sensory fibers and the spinal cord neurons that project to the brain. Such an understanding can enhance our ability to design analgesic drugs in the future. The spinal cord is a potentially more fruitful target for analgesics than other CNS locations because subsequent destinations for nociceptive information are diverse both anatomically and pharmacologically. This study will explicitly examine the nociceptive activity of ascending systems and their susceptibility to pharmacological modification. The electrical activity of single neurons with rostrally projecting axons will be identified using standard neurophysiological procedures. The effects of agents acting at EAA and/or PCP/Cross Section receptors on evoked activity will be determined after iontophoretic administration. The current literature suggests that some relation exists between receptors for PCP and the opioid receptors classified as o. A major goal of this study will be to ascertain if PCP/Cross Section binding the spinal cord represents one or more receptors. PCP/Cross Section binding sites in the spinal cord of the rat will be characterized using receptor binding techniques localized using autoradiographic techniques. We expect localization in areas of the spinal cord associated with nociceptive input and/or EAA receptors. Receptor binding experiments involving inhibition of NMDA displaceable 3H-L-glutamate by PCP/Cross Section agonists will address the hypothesis that compounds acting at PCP/Cross Section receptors may competitively inhibit binding to NMDA receptors in the spinal cord. We expect to find anatomical overlap between 3H-TCP, 3H-(+)-NAM and NMDA displaceable 3H-L-glutamate binding sites. Electrophysiological and behavioral studies will test the same compounds with maximum selectivity for each receptor class for excitation or inhibition of activity in ascending nociceptive neurons. These studies may reveal important information concerning the effects of, and treatment for, PCP abuse in humans.

This award provides funds to expand the computing and instrumentation capabilities of the Biomedical Image Processing Laboratory at the University of Minnesota Medical School to meet research needs in the area of spectrofluorometry. The facility is dedicated to the use of digital image processing and three-dimensional graphics in basic biological research. Tasks for which the new equipment will be used include two-dimensional stereology and image classification, two- and three-dimensional digitally enhanced imaging, high speed video motion analysis, and optical sectioning microscopy. The types of image analysis tools funded with this award are key to research at the forefront of modern cell biology. The current revolution in light microscopy and the vastly expanded ability of the electron microscope to provide serial sections are among the most striking spinoffs of the development of digital image reconstruction by astronomers and space scientists.

This application is a request for an ADAMHA RSDA level II award. This award is sought to enable: 1) broadening the range of approaches applied to the study of pain and analgesia; 2) broadening the research skills of the candidate; and 3) promoting interdisciplinary interactions between the candidate and other investigators within and without the home department. Studies throughout the period of support are designed to examine the mechanism of transmission of algesic and analgesic information across synapses in the spinal cord. Past NIDA-supported studies in this laboratory have set the stage for electrophysiological studies which address more directly the cellular mechanisms underlying the behavioral observations. This project will examine the spinal function of excitatory amino acids (EAAs) and substance P (SP) in the transmission of pain, and will elucidate antinociceptive mechanisms and assign cellular sites of action to opioid and adrenergic agonists in the spinal cord. The requested support will encourage formation of new collaborations with several new faculty members in the department whose expertise includes cellular electrophysiological, biochemical and molecular biological approaches. Patch clamp recordings from acutely dissociated dorsal horn projection neurons will examine the nature of ion channels affected by these neurotransmitters. Late in the grant period, measurement of intracellular calcium transients using microspectrofluorimetry will extend these results to include levels of calcium accompanying these changes. Binding experiments will test the hypothesis that the adrenergic-opioid synergism observed in earlier studies of rodent spinal cord results from an interaction at the receptor level. The long term product of this award would be a progression from early experiments well within the PI's expertise to other experiments well beyond his documented training. This substantial broadening of experience is consistent with the PI's current level of research activities, which include electrophysiology, biochemistry, image processing and molecular biology. These current activities together with the rich Minnesota research environment actually make completion of most of the above projects highly likely. It is important, however, that the PI have increased pure research time available to make maximum productive use of this research environment; this award would provide that increased research time.

DESCRIPTION: (Applicant's Abstract) The proposed studies will identify and characterize common mechanisms through which spinally acting analgesic agents produce analgesia and interact with one another. Behavioral and electrophysiological investigations of spinal pain transmission will be conducted in mice and rats. In vitro studies of the cellular effects of these agents and their combinations will be conducted with behavioral and electrophysiological studies in vivo, with rat spinal cord slices in vitro, and with immunohistochemical techniques. The proposed project will examine cellular mechanisms of changes in potency induced by combinations of analgesic agents and by chronic administration of various agents. The agents studied will be drawn from the following list of spinally active analgesic agents: alpha adrenergic, mu opioid, and delta opioid. These receptor systems are thought to share a common transduction system mediated through inhibitory G proteins, Gi or Go. Activation of these G proteins inhibits neuronal activity subserving pain transmission in the spinal cord dorsal horn. The experiments will address questions of potency and efficacy in experimental systems ranging from whole animal to single cells utilizing behavioral and electrophysiological methods. The behavioral studies will employ brief, escapable, noxious, thermal stimuli to determine inhibitory effects of spinally administered analgesics under conditions of heterologous potentiation. The electrophysiological studies will take advantage of technology added to this laboratory with previous support from NIDA; during epochs of electrical or natural stimulation in anesthetized rats, the electrical activity of spinal cord neurons coding pain information will be recorded extracellularly and the effects of iontophoretically applied analgesic drugs and their combinations determined. In slices of spinal cord tissue, similar neurons will be recorded intracellularly to determine the cellular mechanisms of drug action and interaction. Finally, immunohistochemical localization of alpha 2 and delta opioid receptors in spinal cord and dorsal root ganglia will be used to determine co-localization of these receptors in neurons or lack thereof. The results of these studies will improve our knowledge of mechanisms shared by several drugs of abuse. In addition, multi-drug strategies could be developed based on these studies which would maximize efficacy of analgesics while minimizing their morbidity- and abuse-related side effects.

DESCRIPTION: (provided by applicant) This proposal is for an Exploratory/Developmental Grant Application (R21), specifically addressing RFA #PAR-01-047, the Cutting Edge Research Award (CEBRA) program. When opioid and adrenergic receptors are activated, pain transmission is inhibited, certainly in spinal cord dorsal horn by inhibition of release of excitatory transmitters from central afferent terminals and perhaps in skin by inhibition of transmission from epidermal nerve fiber terminals there. The proposed studies will determine whether 1) mu- and delta-opioid receptors (MOR and DOR) and alpha-2 adrenergic receptors (alpha-2A or alpha-2C AR) are colocalized in peripheral terminals of these nociceptive afferent fibers as in their central terminals, 2) their activation similarly results in analgesic effects, and 3) their co-activation yields a supraadditive interaction, synergy. Therapeutic exploitation of synergistic interactions in the periphery presents the opportunity to produce analgesia with minimal adverse effects mediated mostly in CNS (e.g., sedation, respiratory depression, hypotension, cognitive impairment, tolerance, dependence). The proposed research program will identify and characterize mechanisms through which and conditions under which both exogenous and endogenous spinally acting analgesic agents produce synergistic analgesia through the testing of the following hypotheses. HYPOTHESIS 1: alpha-2 AR and MOR or DOR are colocalized in nociceptive peripheral afferent terminals in skin. HYPOTHESIS 2: alpha-2 AR and OR activation inhibits afferent nociceptive activity in peripheral axons or terminals. HYPOTHESIS 3: alpha-2 AR and OR agonists interact synergistically when co-activated in peripheral terminals. These studies will be conducted in normal mice and mice subjected to inflammatory challenge. Electrophysiological studies will complement behavioral studies of peripheral analgesic action and synergy by determining the reduction of afferent activity induced by activation and co-activation of colocalized receptors. Testing the hypotheses will involve conduct of the following studies: 1) immunohistochemical determination of the presence and anatomical relationships between and among receptor subtypes in peripheral afferent terminals in naive vs. inflamed hindpaws; 2) behavioral and electrophysiological determination of analgesic effects and synergism upon co-administration of agonists selective for receptors MOR, DOR and alpha-2 AR; and 3) evaluation of the effect of antagonism of OR or deletion of alpha-2A or alpha-2C AR on the synergistic relationship between their respective agonists. The work is novel because alpha-2 AR localization in epidermal nerve fibers has not been characterized previously, AR-OR colocalization has not yet been studied and the functional consequences of activation or co-activation are unknown. The work is significant because bivalent targeting of analgesia to periphery may bypass centrally mediated side effects like sedation, tolerance, dependence and addiction liability.

DESCRIPTION (provided by applicant): Opioids are an effective treatment for chronic pain, but repeated use can result in tolerance and physical dependence. These changes are thought to require activation of NMDA receptors and nitric oxide synthase (NOS), because blockade of either can prevent induction of tolerance and dependence. Both of these processes are blocked by the small molecule agmatine (decarboxylated arginine), an endogenous NMDA-receptor antagonist and NOS inhibitor. Because the NMDA-R/NOS system is considered a likely mediator of agmatine's protective effect, the proposed study will explicitly compare its action with that of NMDA-R antagonists and NOS inhibitors as well as searching for other target proteins or proteins altered as a consequence of its action. The proposed project will apply mass spectral phosphoproteomic analysis to compare the complement of proteins phosphorylated in morphine tolerance with those phosphorylated when agmatine or other NMDA-R/NOS inhibitors protects subjects from tolerance. The project will test the hypothesis that agmatine blocks the development of spinal morphine tolerance through signal transduction pathways distinct from the NMDA-R/NOS cascade. The first aim will compare agmatine effects with those of other NMDA-R/NOS inhibitors. The second aim will add co-administered morphine to the first aim and identify the phosphorylation cascades specific to morphine tolerance using 2D LC- MS/MS on spinal cord extract. The third aim will validate these changes and determine the neuronal or glial localization of the altered phosphopeptides. The results of these studies should significantly extend our mechanistic understanding of agmatine modulation of neuroplasticity in opioid tolerance. Future studies could then focus on targeting the identified phosphorylation cascades in a broad spectrum of CNS maladaptive phenomena including opioid self-administration and tolerance, chronic pain, and spinal cord injury. The Public Health Relevance: Tolerance to opioid analgesics remains a key factor limiting access of chronic pain sufferers to adequate relief. This project will apply phosphoprotein analysis to identify the signaling processes activated or suppressed as chronic morphine tolerance develops in rodent spinal cord. Compounds that block spinal tolerance and synaptic plasticity by antagonizing NMDA receptors or inhibiting nitric oxide synthase will be the primary focus.