George L. Wilcox - US grants

This project seeks to answer some basic questions concerning the spinal mechanisms of analgesia. The first goal of the studies is to characterize the action of several putative neurotransmitters or nociceptive spinal neurons projecting to the brain. Neurophysiological studies of unit activity of identified spinothalamic projection neurons in anesthetized rats will quantify their responses to thermal stimulation and to a putative primary afferent fiber neurotransmitter, substance P. Subsequent experiments will evaluate the effects of other neurotransmitters (enkephalin analogs and monoamines), opiate analgesics (eg., morphine), heterocyclic antidepressant agents (eg., protryptyline), and their combinations on responses to these stimuli. Behavioral experiments designed to monitor ascending spinal systems in unanesthetized mice using intrathecal injections of these agents will address the behavioral relevance of the neurophysiological data. The second goal of this project is to examine alterations in the spinal pharmacology of the above substances which might accompany opiate tolerance and dependence. Such alterations appear to change the interaction between brain areas activated by opiate and spinal systems affected by opiates. Selected experiments from those described above will be repeated in mice and rats made tolerant to and dependent on opiates. A better understanding of the pharmacological consequences of spinal tolerance/dependence will help us in the design of analgesic combinations with reduced liabilities and of techniques for the moderation of opiate withdrawal. This project is important because it investigates the pharmacology of a system -- the spinothalamic projection system -- which has been clearly identified to be important to human pain perception, but which has not been previously studied pharmacologically.

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.

Current DNA and protein sequencing technology allows rapid acquisition of information concerning the primary structure of proteins. The growth of information concerning three dimensional protein structure, which is critical to understanding protein function is markedly slower, however. Although the primary structure of a protein completely determines its secondary and tertiary structure, no procedure can yet predict the complete structure of a protein form sequence alone. This project will apply neural network simulations to the protein folding problem. A back-propagation neural network architecture, already implemented and optimized on the Minnesota Cray 2 supercomputer, will be configured to form an association between sequence information and three dimensional structure for 100 of the smaller proteins with known structure. After the network has"learned" this training set after perhaps 100-1000 presentations, it will be tested for retention of some of the rules governing protein folding: we will present the network with sequences which are new to it but which have known structures. We will compare its "predictions" with actual structure; successful performance would be 2 A predictions for 95% of the novel proteins. The neural network program we will use was developed here to model arbitrarily large networks. The program includes a Network Description Language (NDL) which allows an experimenter to configure the network for input-output data sets of arbitray structure and dimensionality. Using the Minnesota Supercomputer Center Cray 2, its interactive UNICOS operating system and the University ethernet network, NDL programs can be created and executed interactively and the results of a simulation reviewed graphically at a Sun or Macintosh II workstation. Experiments with the network show that it can learn associations between one dimensional inputs (e.g. sequence) and multi-dimensional outputs (e.g. 3D structure) and has shown recall of some structural elements of small proteins. Support is requested for a year of experimentation with the network using data sets (learning sets) representing relationships between protein sequence and tertiary structure.

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.

Excitatory amino acids (EAAs) likely constitute the most important class of excitatory neurotransmitters in mammalian central nervous system. Their rapid action and ubiquitous presence underlie their pivotal role synaptic communication between neurons at all levels of the neuraxis. The spinal cord is no exception, and primary afferent sensory neurons contain and release glutamate or aspartate in the dorsal horn. The goal of this project is to examine the participation of endogenous EAAs and EAA receptors in the transmission of nociceptive information through the dorsal horn of rats and in synaptic remodeling which may accompany prolonged activation. This project will examine the spinal function of excitatory amino acids (EAAs) and, to a lesser extent substance P (SP), in the transmission of nociceptive signals, and will elucidate antinociceptive mechanisms and assign cellular sites of action to opioid, serotonergic and GABAergic agonists in the spinal cord. We will test several hypotheses concerning the juxtaposition of receptors for these neurotransmitters on common neurons and/or synaptic elements. In addition, we will examine their involvement in development of long term changes in spinal circuitry following chronic changes in afferent input. In conjunction with the first aim above, the experiments will test the participation of EAAs in the relay of information from primary afferent sensory fibers secondary neurons in the spinal cord dorsal horn and of the effects of GABA, opioids, serotonin and cocaine on that transmission. In conjunction with the second aim above, the experiments will evaluate the participation of EAAs in transsynaptic activation of the c-fos protooncogene and in induction of synaptic and neuronal alterations after prolonged activation. The experiments will be carried out at several levels from whole animal electrophysiology, to spinal cord slice intracellular recording, to dissociated neuron recording, to expression of neurotransmitter receptors in oocytes. The results will significantly improve our understanding of spinal nociceptive transmission and analgesic mechanisms, and of spinal mechanisms of adaptation to prolonged stimulation. Understanding analgesic mechanisms at this level of resolution is crucial to the design of new manipulations with enhanced analgesic efficacy and reduced abuse liability.

The studies proposed in this application focus on the mechanisms of excitatory neurotransmission in the nociceptive afferent system in spinal cord dorsal horn of rodents. The goal of these studies is to understand the importance of synaptic transmission and alterations in synaptic efficacy to the overall behavior of the system in the presence of natural nociceptive stimulation and analgesic substances. The hypotheses to be tested address the participation of excitatory amino acids (EAAs), neurokinins and neuronal nitric oxide (NO) in activation of spinal cord neurons and enhancement of synaptic efficacy induced by both acute and chronic sensory and pharmacological stimulation. Some of the excitatory phenomena will be studied in the context of the development of tolerance to acute or chronic analgesics. The proposed experiments are designed to test these hypotheses at several levels of analysis. Some experiments will examine behavior in unanesthetized rodents after intrathecal administration of agents manipulating the transmitters and pathways listed above. Complementary electrophysiological studies will examine the responses of individual neurons in the spinal cord of anesthetized rodents in situ to electrical stimulation of peripheral nerve, natural stimulation of cutaneous receptive fields and iontophoretic application of agents which manipulate the above pathways. In vitro electrophysiological studies of neurons in spinal cord slices will examine cellular, ionic and synaptic mechanisms mediating phenomena seen in the whole animal studies. The experiments will determine if: 1) EAA receptor agonists and antagonists, and NO inhibitors and generators can affect nociceptive neurotransmission in spinal cord; 2) manipulation of NO synthesis can alter SP-induced changes in spinal cord responsiveness; 3) pharmacological removal of inhibitory systems can unmask long term changes in spinal cord synaptic efficacy induced by repeated, intense stimulation; 4) inhibitors of NO prevent or reduce temporary synaptic alterations in spinal sensory circuitry accompanying hyperalgesia and analgesia; 5) manipulation of spinal EAA receptors or NO can alter the development of opioid tolerance. These studies should provide a better understanding at several levels of analysis of the importance of excitatory transmission and synaptic plasticity to spinal nociceptive transmission and opioid action.

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: (Applicant's Abstract) The proposed studies will elucidate temporal characteristics, determine receptor dependencies and characterize mechanisms through which spinally acting analgesic agents induce tolerance. Both behavioral and electrophysiological investigations of spinal pain transmission will be conducted in mice and rats made acutely tolerant to analgesics by single intrathecal injections. In vitro studies of the cellular correlates of tolerance will be conducted in conjunction with the behavioral and in vivo electrophysiological studies and with immunohistochemical studies of receptor localization. The proposed project will examine cellular mechanisms underlying changes in potency and efficacy induced by opioid analgesics given spinally. The experiments will examine the effects on the induction of analgesic tolerance of agents promoting and opposing synaptic plasticity, promoting or inhibiting excitatory transmitter release, mimicking neurotrophins, and inhibiting kineses and protein synthesis. The analgesic agents studied will be selected to act at the following types of spinal analgesic receptors: mu opioid, and delta opioid. The experiments will address questions of analgesic potency and efficacy in experimental systems ranging from whole animals to single cells utilizing both behavioral and electrophysiological methods. The behavioral studies will employ brief, escapable, noxious, thermal or chemical stimuli to determine inhibitory effects of spinally administered analgesics under conditions of homologous and heterologous tolerance. The electrophysiological studies will measure extracellular activity of spinal cord neurons coding pain information during epochs of electrical or natural stimulation in anesthetized rats 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. Most of the studies will induce spinal tolerance using single intrathecal injections of long- and short-acting analgesics alone or combinations of analgesic agents; in addition, systemic injections as well as sustained release preparations of morphine will also be studied, comparing potency and efficacy of agonists between naive and tolerant states. Finally, immunohistochemical localization of excitatory (neurokinin and glutamate) and inhibitory (opioid receptors) in spinal cord will be used to determine changes in localization induced by tolerance. 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 the efficacy of analgesics while minimizing their tolerance-inducing 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.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Opioid and adrenergic agonists produce analgesia synergistically at G protein-coupled receptors (GPCR). Opioid tolerance and neuropathic pain both involve neuronal plasticity that may decrease this synergistic relationship. The proposed research will 1) determine how synergistic interactions occur between opioid receptor (OR) and alpha-2 adrenergic receptor (AR) agonists, 2) compare the mechanisms underlying GPCR-mediated analgesic synergism under normal conditions and in states of neuronal plasticity, and 3) examine the functional relationships between OR and AR transmitters. We will test the following hypotheses: Hypothesis 1A - Synergistically acting receptor pairs sharing localization couple through different intracellular pathways; Hypothesis 1B - Synergistically interacting receptor pairs coupling through the same intracellular pathways reside in different locations; Hypothesis 2 - Persistent alterations in receptor distribution or coupling driven by neural plasticity impact potency or efficacy of analgesic agonists by changing interactions between receptor subtypes. Behavioral studies will examine the mechanism of spinal analgesic synergy under normal, opioid tolerant, and neuropathic conditions. Immunohistochemical localization of OR/AR and quantification of transmitter release from spinal cord slices will define the anatomical substrates for the functional interactions observed. Electrophysiology of dorsal horn neurons will examine the cellular mechanisms of drug action and interaction at the single cell level. The information gained from these studies may identify new therapeutic analgesic combinations and combination receptor targets leading toward reduction of dependence liabilities of analgesic treatments. [unreadable] [unreadable] [unreadable]

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.