Howard L. Fields - US grants

pain; analgesia;

There is a body of evidence implicating the neurons of the rostral ventral medulla (RVM) in modulation of pain and, particularly, in mediating the analgesic effects of systemically administered opiate drugs such as morphine. Endogenous opioid peptide-containing neurons and terminals are present not only within RVM but are also present in other CNS sites that project to and receive projections from the RVM. In addition to enkephalin, there is evidence that serotonin, noradrenalin and other putative neurotransmitters are present in nerve terminals in RVM. Our goal is to elucidate the internal circuitry and function of RVM, particularly the role of opioid peptides. The strategy is to define RVM neuron cell classes on the basis of their receptive field and relationship of their discharge to nocifensive behavior. We will then map the location of each class and determine whether cells of that class project to the spinal cord. The effect on each class of either systemically injected or iontophoresed opiates will then be studied. Using intracellular marking of single RVM cells and double-labelling we plan to determine whether particular physiologically defined classes of cell contain particular transmitters or receive input from cells containing particular transmitters. We will use cross-correlation analysis to study connectivity between simultaneously recorded pairs of cells in RVM. In addition to better understanding of pain-modulating networks in the CNS, these studies, by defining the roles of both opioid and non-opioid neurons may suggest new classes of centrally-acting analgesic drugs.

pain; neural transmission; meeting /conference /symposium; pain threshold;

Opiates are the most powerful analgesic agents presently available. The general goal of the proposed research is to elucidate the circuitry underlying opiate actions that produce analgesia. This proposal is specifically focussed on the mechanisms of opiate analgesia at the level of the brainstem. The rostral ventromedial medulla (RVM) is involved in opioid-sensitive mechanisms that underlie brainstem control of nociceptive transmission. Opiate receptors and endogenous opioid peptides are found in this region, and microinjection of opiate agonists into RVM suppresses behavioral responses to noxious stimulation. Two classes of putative nociceptive modulating neurons have been identified in the RVM. Off-cells, which are thought to suppress nociceptive transmission, are activated by morphine. On-cells, which appear to facilitate nociceptive processing, are depressed by morphine. Other RVM neurons, neutral cells, are unaffected by morphine. We propose to use intracranial microinjection techniques, behavioral tests of nociceptive responsiveness, single unit recording and microiontophoresis to investigate the neurotransmitters involved in opiate control of the activity of physiologically-identified RVM neurons. We plan to investigate the actions of morphine, norepinephrine, serotonin and excitatory amino acids. We are particularly interested in determining how cells containing these transmitters contribute to the antinociceptive action of opiates. Hopefully, by increasing knowledge of how opiates produce analgesia, new drugs or pharmacological strategies can be devised to produce analgesia more selectively.

The Neurobiology of Disease Teaching Workshop has been held for the past seven years in conjunction with the annual meeting of the Society for Neuroscience. The aim of the Workshop is to introduce young neuroscientists to the clinical manifestations and neuroscience of diseases of the nervous system. Enrollment iS limited to 100 students each year; these include graduate students, post-doctoral fellows, M.D.-Ph.D. students, and residents in neurology and psychiatry. A limited number of more senior neuroscientists attend as observers. The teaching program includes patient presentations, lectures by leading investigators, small group discussions, sessions on research approaches and technologies, and a discussion of the personal impact of the diseases on patients and their families. The two-day workshop covers two major diseases or disorders each year. A faculty of 6-10 outstanding investigators and clinician- investigators is gathered for each workshop. The topics for the 1987 Workshop will be The Neurobiology of Hypertension and The Molecular Genetics of Muscular Dystrophy and Huntington's Disease. In prior years the workshop has covered Parkinson's Disease, The AIDS Virus in the Nervous System, Alzheimer's Disease, Regeneration in the Central Nervous System, Multiple Sclerosis, Huntington's Disease, Muscular Dystrophy, Epilepsy, Pain, and Affective Disorders.

This award provides funds to aid in the purchase of optical microscopy equipment. The equipment will be located in a central facility where it will be available to a group of neuroscientists in the Keck Center for Integrative Neuroscience. The scientists are all interested in various aspects of local circuit analysis of the mammalian central nervous system (CNS). The goal of their studies is an improved understanding of how small groups of neurons generate behavior. The instruments requested will be used for combined anatomical and physiological studies of brain function that include the use of double or triple labeling techniques. The development of new instrumentation for optical microscopy and of new methods that permit specific labeling of certain cells or certain cell constituents has been key to progress in our understanding of many aspects of cellular and developmental biology. Neuroscience has benefited greatly from these developments, since they permit the rapid and reliable identification of particular neurons or groups of neurons in the brain or at other sites that typically contain many nerve cells or similar morphology.

The Society for Neuroscience is the major professional organization for scientists who study the nervous system. An important goal of this organization is to encourage scientists in training to undertake research related to diseases of the nervous system. The objective of this grant application is to support teaching workshops that introduce young neuroscientists to current concepts about the etiology and pathogenesis of disorders of the nervous system. For each workshop, about 12 faculty are chosen by the organizing committee. Clinical presentations provide enrollees with an experience of the human dimension of particular diseases. Lectures cover both clinical research and relevant laboratory work. In addition to lectures, enrollees are given a choice of small group workshops that emphasize either specific conceptual or methodological issues. Since its inception, eleven workshops have been held, usually on the day prior to the start of the Society for Neuroscience Meeting. Topics have included: Stroke, AIDS in the nervous system, Epilepsy, Huntington's and Parkinson's disease, Muscular Dystrophy, Multiple Sclerosis, Prion diseases, Drug Addiction, Pain and Affective Disorders, Stroke and Excitotoxicity. Enrollment generally runs between 100 and 200 attendees. Most enrollees are graduate students or postdoctoral fellows. Current plans are to cover the following topics in the near future: Recovery of function after neural injury, Genetic Diseases of the Nervous System and Disorders of the hypothalamus and circadian rhythms. Other topics will be chosen depending on their potential interest to young neuroscientists, their impact on society and the quality of recent research related to that disease area.

Opiates are the most powerful analgesic agents presently available. The general goal of the proposed research is to elucidate the circuitry underlying opiate actions that produce analgesia. This proposal is specifically focussed on the mechanisms of opiate analgesia at the level of the brainstem. The rostral ventromedial medulla (RVM) is involved in opioid-sensitive mechanisms that underlie brainstem control of nociceptive transmission. Opiate receptors and endogenous opioid peptides are found in this region, and microinjection of opiate agonists into RVM suppresses behavioral responses to noxious stimulation. Two classes of putative nociceptive modulating neurons have been identified in the RVM. Off-cells, which are thought to suppress nociceptive transmission, are activated by morphine. On-cells, which appear to facilitate nociceptive processing, are depressed by morphine. Other RVM neurons, neutral cells, are unaffected by morphine. We propose to use intracranial microinjection techniques, behavioral tests of nociceptive responsiveness, single unit recording and microiontophoresis to investigate the neurotransmitters involved in opiate control of the activity of physiologically-identified RVM neurons. We plan to investigate the actions of morphine, norepinephrine, serotonin and excitatory amino acids. We are particularly interested in determining how cells containing these transmitters contribute to the antinociceptive action of opiates. Hopefully, by increasing knowledge of how opiates produce analgesia, new drugs or pharmacological strategies can be devised to produce analgesia more selectively.

In contrast to other sensory modalities such as vision and hearing, pain is, by definition, unpleasant at threshold. This unpleasantness is the subjective correlate of a drive to escape and avoid tissue damaging stimuli. Although the clinical importance of the affective-motivational aspect of pain is generally accepted, the lack of a valid animal model has hampered investigation of its neurobiological basis. The research propose din this project is specifically designed to examine the neural mechanisms of aversion produced by noxious stimuli. We will adapt the place preference apparatus to measure the magnitude of aversion to a context associated with a hindpaw formalin injection (conditioned place aversion or CPA). Rats will receive formalin in a compartment with obvious olfactory, visual and tactile cues. On alternative days they will receive saline in a second compartment. After formalin conditioning, the reduction of time spent in the formalin-associated compartment will be taken as a measure of aversiveness. The effects of formalin elicited CPA of inactivating various CNS structures implicated in nociception will be studied. In the first experiments rats will receive a substance P receptor neurotoxin, the Substance P-saporin conjugate, in the lumbar intrathecal space to selective destroy lamina I spinomesencephalic and spinothalamic neurons. If this reduces CPA, rats will be tested for the effects of lesions or transient inactivation of the parabrachial nucleus and of spinothalamic target nuclei and cortical areas activated by noxious stimuli. Thalamic areas to be studied include the Ventrobasal Complex, the posterior thalamic/posterior intralaminar group, and the medial thalamic/intralaminar region The cortical areas to be studied include the somatosensory, anterior cingulate and dysgranular insular corteces. In complementary studies, cortical areas implicated in aversion by inactivation in aversion by inactivation studies will be stimulated using excitatory chemical agents to determine if local neuronal activity can elicit CPA in the absence of a peripheral noxious stimulus. Finally, we will study the effect on formalin behaviors and CPA of reversible inactivation of descending modulatory systems.

DESCRIPTION (provided by applicant): Stress increases addictive behaviors. Corticotropin-releasing factor (CRF) is released in the ventral tegmental area (VTA) during stressful events, and produces relapse to cocaine seeking. However, the mechanism by which CRF produces stress-dependent relapse to cocaine seeking is poorly understood. The main goal of this proposal is to understand the role of CRF-R1 and CRF-R2, and the CRF-BP in the VTA in modulating dopamine release and stress-induced relapse to cocaine seeking. Over the past four years, my laboratory has collected evidence showing that CRF activates CRF-R2 to increase NMDAR-mediated currents in VTA DA neurons. Furthermore, we have evidence that CRF-R1 activation in VTA DA neurons increases firing activity via activation of Ih. By performing patch-clamp recordings in the VTA, specific aim 1 will elucidate in detail: a) the intracellular pathway responsible for the CRFR1-dependent increase in firing rate, and b) the intracellular pathways responsible for the CRF-R2-dependent increase in NMDAR currents in the VTA. Specific aim 2 will determine the role of CRF-R1 and CRF-R2 in modulating DA release in the ventral striatum. Specific aim 3 will determine the role of CRF-R1- and CRF-R2-dependent pathways in inhibiting footshock- induced relapse to cocaine seeking. Finally, specific aim 4 will take a deep mechanistic look at the CRF-BP. The results from this grant will produced a deep mechanistic and behavioral understanding of the various effects of CRF on VTA neurons. Our results will likely create new therapeutic leads toward agents that disrupt the CRF-R1- and CRF- R2-dependent effects on VTA neurons and thus stress-induced cocaine seeking. PUBLIC HEALTH RELEVANCE: Stress increases addictive behavior. However, the mechanism by which stress-released molecules exert their negative effects on drug-seeking are poorly understood. The main goal of this project is to elucidate the role of CRFR1 and CRFR2 in promoting stress-enhanced relapse to cocaine seeking. Relapse to drug seeking is a major health problem that still has no cure. The results from this proposal could enable us to create new therapeutic targets aimed at inhibiting the ability of stressful event to increase relapse to substance abuse.

DESCRIPTION (provided by applicant): Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) play an important role in the rewarding and reinforcing effects of opioids. Within the VTA, opioids acting at the mu opioid receptor inhibit the release of GABA, which leads to the disinhibition of DA neurons (Johnson and North, 1992a). However, GABA synapses onto VTA DA neurons arise not only from local GABAergic neurons, but also from extrinsic sources, at least some of which are likely to be sensitive to opioids. Two major external sources of inhibition onto VTA neurons arise from the nucleus accumbens (NAc) and the ventral pallidum (VP), both of which have been strongly implicated in opioid reward. These inputs have not been well defined, largely due to the technical challenge of isolating individual connections. Nevertheless, our understanding of the neural circuitry underlying opioid reward requires a thorough investigation of the synaptic properties of these VTA afferents. In this grant, we will make use of recently developed optogenetic tools to independently activate axon terminals arising from either the NAc or the VP in conjunction with whole cell patch-clamp electrophysiology from VTA neurons in midbrain slices. This will allow us to compare and contrast these two inputs and to determine how they are regulated by both acute and chronic exposure to opioids. In addition, by combining this powerful technique with retrograde labeling of VTA neurons from particular projection targets, we will be able to determine the detailed microcircuitry of these afferents within the VTA. The data provided by these studies will provide critical information for the development of circuit based models of limbic system function.