Alan R. Light, PhD - US grants

Acute and chronic pain continue to be the most common complaints which physicians are asked to treat. While acute pain can be managed in most cases, physicians are constantly searching for new methods with fewer side effects and less risk of addiction to treat it. Chronic pain is a different matter all-together. It is estimated that over 60 billion dollars are spent annually on this disorder not including loss of productivity. At present, our understanding and ability to treat chronic pain are limited. the present proposal will investigate the brain's own capacity to modulate pain. A thorough understanding of these mechanisms is essential for the rational development of new therapies for the treatment of acute and chronic pain. The continuing long term objective of this proposal is to determine the cellular and synaptic mechanisms by which sensory input to the dorsal horn and in particular the marginal zone (lamina I) and substantia gelatinosa (lamina II) is modulated by higher brain centers. The specific aims of this five year proposal are to: 1) continue to define the functional effects of focal brain stem stimulation on physiologically and anatomically identified marginal zone and substantia gelatinosa interneurons as well as marginal zone neurons identified as projecting to the midbrain; 2) determine the putative neurotransmitter utilized by identified descending fibers by using immuno-cytochemical double labeling techniques both at the light and electron microscopic level; 3) determine the physiological chacteristics of inhibitory interneurons in the marginal zone and substantia gelatinosa by using intracellular recording and labeling combined with immunocytochemical labeling both at the light and electron microscopic level;l 4) determine the effects of stimulating descending systems on the expression of the proto-oncogene, c-fos, in spinal cord neurons; 5) determine the characteristics of neurons in the parabrachial region of the midbrain/pontine junction which receive nociceptive inputs from the spinal cord. These specific aims will be accomplished by using techniques which combine intracellular recording of neurons with intracellular labeling so that the neuron and its axonal arbor can be examined with the light and electron microscopes. Putative neurotransmitters in these neurons will be assessed by using immunocytochemical techniques at both light and electron microscopic levels. Immunocytochemical techniques will also be used to demonstrate the expression of c-fos which will be used as a marker of cellular modulation by descending pathways.

Subcortical brain regions have been shown to have direct projections to the marginal zone (lamina 1) and substantia gelatinosa (lamina 2). These projections presumably play a role in the modulation of nociceptive input, which is known to relay in these laminae. The current proposal is to study the extent and influence of newly discovered direction projections from the cerebral cortex to spinal cord laminae 1 and 2. These initial studies will use single axon recording and intraaxonal injection of HRP to label physiologically identified corticospinal axons and their spinal terminations for both light and EM analyses. The results will provide information on the function of direct projections from the cerebral cortex to the superficial dorsal horn.

Opioid peptides cause relatively specific inhibition of pain when applied to midline brain structures or when applied directly to the spinal cord. The neural mechanisms which mediate this specific inhibition are only partially understood. While "pain" can be studied only in humans, "nociception" (neural responses activated by tissue damaging or potentially tissue damaging stimuli) can be studied in animal models. Nociceptive neural responses are inhibited by opioids paralleling the inhibition of pain. We, therefore, propose to determine whether the specific inhibition of nociception is due to direct, postsynaptic effects on nociceptive neurons in laminae I and II of the spinal cord. To test this, we will record extra- and intracellularly from both nociceptive and non-nociceptive neurons in laminae I and II. We will then apply opioids via micro-iontophoresis and micro-pressure ejection to determine whether they cause conductance changes specifically in the nociceptive neurons. The neurons will be labeled with horseradish peroxidase via the recording micropipette to determine whether cellular morphology contributes to the opioid effects. In other experiments we will determine whether nociceptive neurons in laminae I and II are specifically inhibited when opioid peptides are injected into midline brain structures such as periaqueductal gray. Further experiments are designed to determine the mechanisms of this descending inhibition by using various specific antagonists to block the inhibition at the spinal level. These experiments are designed to determine whether opioid peptides act by producing postsynaptic effects specifically on nociceptive neurons. Future studies could determine which of the various opiate receptor subtypes mediate specific postsynaptic effects, if they are found. The ultimate goal is a better understanding of the mechanism of the opioid blockade of pain with the hope that this understanding will lead to the production of opioids which are more selective, less addicting, and lead to fewer side effects.

The long term objective of this project is to determine the neural mechanisms of opioid inhibition of nociception at the spinal cord level and compare this effect with the opioid inhibition of nociception at the brainstem level. The ultimate goal is a better understanding of the mechanisms of the opioid blockade of pain with the hope that this understanding will lead to the rational development of analgesic drugs and other treatments which are more selective, less addicting and lead to fewer side effects. The development of epidural analgesics is an example of the treatments which a more thorough understanding of the opioid mechanisms has led to. The specific aims of the next grant period are to: l) determine the effects of activating and inactivating specific opioid receptor subtypes on the responses of extra- and intracellularly recorded lamina I and II neurons. 2) determine the site of action of different opioid receptor subtypes in lamina I and II (pre- vs. postsynaptic). 3) determine the relationship of activating specific opioid receptor subtypes on nociceptive behavior in the rat and correlate these effects with the production of FOS onco-protein at both the spinal and brainstem level. 4) determine at which site, spinal cord or brainstem, specific opioid agonists are most effective when applied systemically. A series of in vivo extra and intracellular recording studies is proposed in anesthetized cats combined with microiontophoresis and intrathecal and systemic application of opioids to address the first specific aim. In vitro whole cell patch clamping experiments in spinal cord slices are proposed to address specific aim 2. Whole animal behavioral studies in rats combined with immunocytochemistry of FOS protein are proposed to address specific aims 3 and 4.

The Histology Core is responsible for overseeing all histological procedures used in this Program Project Grant, including trouble-shooting immunohistochemical and in situ hybridization procedures in conjunction with personnel from the individual projects. A full-time Research Technician and a supervising Research Analyst (whose salary is provided by the Department of Cell and Molecular Physiology) will conduct many of the procedures listed in this proposal; they will be assisted by research staff from several projects within this application with other protocols. Alan R. Light will be as an advisor to the core. The Histology Facility has been responsible for a number of techniques that have allowed more efficient use of histological methods on Principal Investigators' past projects in a Program Project Grant, previously led by Edward R. Perl. The Histology core occupies 600 ft/2 in an area on the ground floor of the Department of Cell & Molecular Physiology, and it is easily accessible for Projects 1 and 3. This facility houses all the major equipment necessary for the histological procedures described in the projects.

The enhancement of pain following injury occurs acutely within a few minutes, and can last for several days or weeks. In some cases, the enhance pain can last indefinitely, either because the injured cannot be repaired, or because of some unknown mechanism that may not be related to peripheral injury. In these cases, the pain is defined as "chronic pain." It has been proposed that long-term exposure to painful stimuli can cause profound changes in the central nervous system that are very difficult to alter and are the cause of chronic pain. Chronic pain has proven to be extremely debilitating for affected individuals, and has been refractory to most treatments. We hypothesize that one of the CNS locations in which pain transmission is altered by persistent nociceptive inputs is the spinal cord. Just as a number of peripheral events contribute to short-term sensitization of peripheral nociceptors (see Projects 2 and 3), it is likely that long-term spinal cord sensitization of peripheral nociceptors (see Projects 2 and 3), it is likely that long-term spinal cord sensitization requires a combination of factors involving damaged peripheral tissue, afferent traffic from that tissue, and spinal responses to the peripheral injury. We propose that understanding, long-term spinal sensitization requires systematic manipulation and evaluation of combinations of peripheral, afferent and central effects that have been implicated individually as contributors to persistent pain. Once these factors are understood, rational treatments can be developed. The specific aims for the next 5 years are to determine: 1. the contribution of primary afferent firing in our present model of long- term hyperalgesia. 2. whether blockade of afferent activity, axonal transport of peripheral nerve ending destruction a) affects the glial activation and allodynia/hyperalgesia produced by peripheral injection of formalin, and/or b) produces glial activation and allodynia/hyperalgesia without peripheral tissue damage. 3 whether activation of spinal cord glial cells (astrocytes and microglial cells) is involved in short- and long-term hyperalgesia. 4. some of the potential mediators that activate astrocytes and microglia and induce long-term hyperalgesia in our formalin model. Combinations of single afferent recording, behavior, and immunohistochemistry will be used on rats and transgenic mice to accomplish these aims.

"Pain has a profound impact on the quality of life and health-associated costs for all Americans...costing the American public more than $100 billion each year in health care, compensation and litigation"(from NIH Guide New directions in Pain Research). Long-term exposure to painful stimuli can elicit changes in the nervous system that are the cause of chronic pain. These changes can be very difficult to reverse. Chronic pain is debilitating for those individuals it affects, and is refractory to most treatments. The goal of our group is to further determine the cellular mechanisms that mediate the persistent alterations in pain observed following injury, by studying both the peripheral nerve and the spinal cord mechanisms initiated by persistent nociception. This is a complex process involving interactions of supporting tissues, the immune system, the somatic and autonomic nervous systems, and all of the extracellular and intracellular messenger systems. The interaction of so many diverse systems makes understanding this process complex and requires a multi-disciplinary team approach for optimal progress. Using the Program Project Mechanism, we propose to form such a team. Project 1 (PI, Alan Light) will study the mechanisms by which astrocytes and microglia become activated by noxious peripheral inputs and how these cells interact with neurons to mediate long-term behavioral changes in pain responses. Project 2 (PI, Lorne Mendell) will make use of in vitro recording methods to examine the mechanisms by which nerve growth factor sensitizes the responses of nociceptive neurons in rat DRG and to determine how this is modified by inflammation. These studied will also address the specificity of the response of nociceptors to different neurotrophins, the time course of their action and the interaction of neurotrophins with other substances (e.g., serotonin) known to activate or sensitive nociceptive afferents. Project 3 (PI, Gerry Oxford) will study the molecular mechanisms by which growth factors, intracellular modulators, and the newly cloned vanilloid receptor interact to produce sensitization in rat dorsal root ganglion cells.

DESCRIPTION (provided by applicant): Molecular receptors on Group III-IV sensory neurons detecting muscle metabolites Fatigue due to chronic heart failure, chronic obstructive pulmonary disorder (COPD), and other chronic fatiguing disorders is serious, debilitating, and creates poor prognoses for long-term outcomes in these patients. Many more patients are affected by idiopathic, injury, or disease-caused short-term fatigue and myalgia that sometimes remits with treatment or for unknown reasons becomes chronic. Considerable evidence indicates that peripheral sensory dysregulation of group III/IV muscle afferents, and autonomic dysregulation may cause or contribute to the excessive fatigue of chronic heart failure. Our long-term goal is to determine the fundamental mechanisms that signal fatigue to sensory and motor systems, and determine the mechanisms that cause enhanced fatigue in diseases such as heart failure and COPD. In the next three years, we propose a comprehensive evaluation of the molecular receptors on group III/IV muscle afferent neurons that endow these specialized endings with the ability to detect and signal the increases in muscle metabolites that occur with muscle contraction and exercise. Experiments proposed here will use: 1) innovative neuron harvesting and quantitative real-time, PCR (qPCR) to determine which molecular receptors are expressed selectively in group III/IV afferent neurons. 2) calcium imaging, cell harvesting and qPCR to determine how fatigue-selective neurons selectively encode non-painful levels of metabolites. 3) immunohistochemistry to determine if mRNA expressed is translated protein inserted into membrane. 4) whole-cell recording of metabolite activated currents to determine the function of the molecular receptor proteins; and 5) single unit electrophysiological recording in a nerve muscle preparation to determine which metabolites activate these sensory neurons in situ. The results of these experiments will provide the basic science background for the concept of fatigue as an integrated system with powerful influence on the cardiovascular/autonomic system, the sensory-perceptual experience of fatigue, and motor system inhibition. This concept, and the molecular receptors discovered will lead to rational, targeted effective treatments for the excessive fatigue experienced by heart failure patients, patients with COPD, and other patients suffering from prolonged, unexplained fatigue.

? DESCRIPTION (provided by applicant): GCAMP6 mice for determination of mechanisms of chronic muscle ache, pain and fatigue. Muscle pain and muscle fatigue are symptoms in many chronic diseases including Fibromyalgia, Chronic Fatigue Syndrome, Myofacial Pain, Chronic Tension Headache, and Temporomandibular Disorder. Many more patients have degraded quality of life because of short term myalgia and fatigue that sometimes remits with treatment, or for unknown reasons, becomes chronic. Our long term goal is to determine the fundamental mechanisms that signal intense muscle pain, ache and fatigue to sensory and motor systems. We have previously used discoveries in mouse models to prove that combinations of protons, lactate, and ATP are necessary and sufficient to activate muscle sensory neurons. In translational studies in human subjects we showed that combination of these three metabolites activated the sensations of muscle ache and fatigue in human subjects. Here we propose to create a transgenic mouse that will make it possible, for the first time, to image the activity of sensory neurons that signal pain and fatigue in functioning skeletal muscle. This will allow us to establish the molecular and cellular mechanisms of the sensory signaling pathways for cognitive sensations of muscle pain and muscle fatigue. It will also make it possible to directly observe the mechanisms of several controversial phenomena in muscle pain including trigger points, the pulsating nature of muscle ache and chronic and migraine headache, lymphatic disease association with muscle fatigue and muscle pain, and sympathetic activation enhancement of muscle pain. The specific aims for this proposal are: 1) Create mice that have endogenous calcium sensors with promoters that allow expression in Group III/IV muscle innervating sensory neurons. 2) Use these mice to record III/IV afferents responses to low and high metabolites using anesthetized in vivo preparations. This will allow comparisons with the digit muscle recordings collected from teased muscle nerve fibers previously, and dorsal root ganglion neurons recorded with calcium imaging. With this method we will also determine the location and structures that physiologically identified Group III/IV receptor endings innervate (both nociceptors and ergoreceptors of all types e.g., mechanoreceptor and metaboreceptor).

Myalgic encephalomyelitis/Chronic fatigue syndrome (ME/CFS) and Fibromyalgia syndrome (FM) are chronic multi-symptom disorders, which are frequently comorbid. The primary goal of this study is to identify unique genetic mutations seen in ME/CFS and/or FM but not in depression or other functional disorders like migraine. Identifying such variants would provide specific biomarkers for diagnosis of ME/CFS and FM and for identifying causes and targets for treatment. Both ME/CFS and FM have been proposed to involve mitochondrial dysfunction. Recently, we employed RNA-Seq and VarScan2 to examine mutations in leukocytes (not whole blood) for the full human transcriptome. In a pilot sample, 94% of the 17 ME/CFS patients had High or Moderate Impact variants in mitochondrial genes affecting NADH dehdrogenase 4, and the 17th had mitochondrial variants affecting cytochrome B. Both are proteins in the respiratory chain complexes that have critical roles in production of ATP, the cell's major energy source. Known mitochondrial disorders are associated with other mutations affecting these same complexes. Like ME/CFS, these mitochondrial disorders involve symptoms of muscle weakness, fatigue, neuropathies and exercise intolerance. Most of our 17 patients also had a second mitochondrial variant affecting ATP production and also a chromosomal DNA variant affecting either mitochondrial replication or autoimmune function. Thus, we hypothesize that mutations in mitochondrial genes (especially in combination with other mutations) increase risk or contribute causally to the development of ME/CFS and/or FM, and may be specific biomarkers of these disorders. Specific aims of the study are: First, to use RNA-Seq and VarScan2 to compare the frequency of leukocyte mitochondrial mutations of 150 patients with ME/CFS and/or FM versus 150 healthy controls, patients with migraines or depression (total sample= 300). Second, to examine the relationship between greater heteroplasmy of these mutations and severity of physical fatigue, mental fog and widespread pain in all 150 CFS/ME and FM patients. Third, in mothers of 40 ME/CFS or FM patients with mitochondrial mutations, to assess whether they share their child's mutation, indicating what proportion of patients have maternally inherited vs. acquired somatic mutations.

Muscle pain and fatigue affect nearly all people at some time in their lives. At present, effective treatments for short term muscle pain are clearly inadequate and adequate treatment for chronic muscle pain is even worse. Considerable evidence indicates that peripheral sensory dysregulation of group III/IV muscle afferents, and autonomic dysregulation may cause or contribute to both short-term and chronic muscle pain and fatigue.Our long term goal is to determine the fundamental mechanisms that signal intense muscle pain, ache and fatigue to sensory and motor systems. We have previously used discoveries in mouse models to prove that combinations of protons, lactate, and ATP are necessary and sufficient to activate muscle sensory neurons. In translational studies in human subjects we showed that combination of these three metabolites activated the sensations of muscle ache and fatigue in human subjects. We propose here to use several transgenic mice that will make it possible, for the first time, to determine the molecular, cellular, and structural mechanisms of the sensory signaling pathways for the cognitive sensations of muscle pain and muscle fatigue. These mice will also make it possible for us to determine the many different types of afferents that selectively signal the many different aspects of autonomic function that allow us to function over a wide range of muscle blood flow. Finally, these mice will make it possible for us to directly image not only the cell bodies of the muscle innervating neurons, but to directly observe the activation of the nerve endings in skeletal muscles by both metabolites and mechanical stimulation. These images may allow us to determine the mechanisms for ?trigger points?, the pulsating nature of muscle ache, lymphatic disease associated with muscle fatigue and pain, and sympathetic activation of enhanced muscle pain. The specific aims of this proposal are: Aim 1: Define the different types of mechanosensitive, and metabosensitive sensory neurons innervating skeletal muscle based on their responses to muscle contraction. Aim 2: Determine if chemical mediators produced during muscle contraction are responsible for the potentiation seen among a subset of mechanosensitive muscle sensory neurons during muscle contraction. Aim 3: Determine if metaboreceptors and metabo-nociceptors also respond to mechanical forces generated during muscle contraction. Aim 4: Determine the location and structure of skeletal muscle sensory neurons signaling pain and fatigue via direct imaging of neuron terminals.