Virginia S. Seybold - US grants

Considerable biochemical and pharmacological evidence has accumulated to support the role of histamine as a neurotransmitter in the nervous system, however, the lack of a histological marker for histaminergic neurons has impeded the precise localization of histaminergic neurons in mammalian brain. With an antiserum against histamine, we have the ability to visualize histaminergic neurons at the levels of the light and electron microscopes, and thus begin to acquire a morphological perspective for histamine-containing neurons. In addition to mapping the distribution of histaminergic fibers, terminals and cell bodies by immunohistochemistry, we propose to examine connections of the group of histamine-immunoreactive cell bodies in the hypothalamus by double-labeling cells via histamine-immunoreactivity and the retrograde transport of horse-radish peroxidase. Because of the general involvement of the hypothalamus in the regulation of endocrine and autonomic function, we believe projections of these cells to the median eminence and autonomic nuclei in the brainstem may form the neuronal circuitry whose activation is mimicked in neuropharmacological studies of histamine. Studies of hypothalamic histamine-containing fibers at the ultrastructural level will add insights into the extent to which histamines effects on endocrine function are mediated via neuronal synapses. Finally, we propose to isolate liver and brain histidine decarboylase and raise monoclonal antibodies against the enzymes. The antibodies will be used in immunohistochemistry as an alternate approach to the visualization of histaminergic neurons.

The general direction of my research is to determine the organization and function of chemically-coded neuronal circuits in the dorsal horn of the spinal cord. The specific aims of this proposal are to characterize the distribution, projections and synaptology of neurotensin and met-enkephalin containing neurons in the dorsal horn. Because these peptidergic neurons occur in regions of termination of primary afferent neurons, they may modulate or mediate the transmission of sensory information through the spinal cord. Both peptides are reported to have analgetic properties, so determining the circuitry of neurons containing neurotensin and met-enkephalin will add insights into the morphologic basis for their physiological effects. The distribition of met-enkephalin neurons in the dorsal horn will be determined immunohistoochemically. Of particular interest is whether met-enkephalin perikarya exhibit periodicity in a longitudinal dimension similar to that observed for neurotensin neurons. The studies of neurotensin perikarya have indicated that a neurochemical organization exists in the rostrocaudal dimension of dorsal horn laminae that has heretofore been unappreciated. A comprehensive comparison of the distribution of neurotensin and met-enkephalin in rat and cat is also proposed. Preliminary studies indicate differences occur in the distribution of these peptides in the 2 species. Since both animals are widely used in studies of nociception and analgesia, confirmation and documentation of these differences is important. The retrograde transport of horseradish peroxidase and changes in the distribution of peptide after transection of the spinal cord will be used to determine supra-spinal, intra-segmental and intersegmental projections of spinal neurotensin and met-enkephalin neurons. Ascending projections of spinal enkephalin neurons may be important in the activation of descending systems which modulate nociception in the spinal cord. In addition, the synaptology of neurotensin-containing neurons in the superficial laminae will be characterized at the ultrastructural level. The ultrastructural studies may add insights into the morphological basis for why neurotensin is excitatory on the dorsal horn neurons and opiates are inhibitory, yet both are analgetic.

While the physiological characterization of primary afferent neurons has been extensive, the relevant morphological criteria that predict functional categories of these neurons have been established. The first hypothesis posed is that peptide content, in conjunction with cell diameter, peripheral target and projection of the central process in the spinal cord, can be used to classify populations of primary afferent neuron perikarya. Ultimately, this classification of neurons may be used to predict their functional properties. To examine this hypothesis, the mean diameter for each population of primary afferent perikarya that is immunoreactive for a given peptide will be determined. Co- existence of peptides in these neurons will also be characterized in order to establish the peptide signatures that discriminate the smallest populations of neurons. The nodose, trigeminal, and one lumbar ganglion believed to contain a minimum of visceral afferent neurons will be analyzed in order to determine whether specific peptide signatures are selective markers for visceral versus somatic primary afferent neurons. Whether co-existence of peptides in primary afferent neurons is a plastic phenomenon, and therefore a reliable criterion, will be examined by comparing the frequency of co-existence in ganglia from normal animals to ganglia from an animal model for chronic pain. Preliminary correlations of peptide signatures with primary afferent neurons associated with A-delta or C fibers will be based on the diameter of the neuron, the pattern of distribution of peptide immunoreactive axons of primary afferent origin in the spinal cord, and whether the axons are capsaicin sensitive. The second hypothesis to be tested is that axons of primary afferent neurons may contain presynaptic receptors for some of the peptides that occur in the highest frequency in these neurons. In vitro receptor autoradiography will be used to determine a correlation of selected peptide binding sites with primary afferent fibers. To determine whether these binding sites reflect functional receptors, changes in the density of peptide binding sites will be determined in the spinal cord as a function of chronic pain, a physiological stimulus for the small diameter primary afferent fibers known to contain the peptides. Data from these studies will provide a meaningful classification of primary afferent perikarya and provide insights into the functional significance of some peptides contained within these neurons, particularly with respect to nociceptive systems.

The aim of this project is to determine whether painful peripheral neuropathies in humans are associated with abnormalities of spinal cord neuropeptide synthesis and neuropeptide receptor regulation. Such abnormalities have been detected in rats with an experimental version of painful peripheral neuropathy; the question addressed in this project is whether or not similar abnormalities occur in humans. Autopsy specimens (donated by patients or the family of the deceased) from spinal cord will be obtained as soon as possible after death, with particular care taken to correctly identify the spinal segments associated with the peripheral location of the pain symptoms. The specimens will be divided at the time of autopsy into two samples. One sample will be processed with immunohistochemical staining for the presence of 8 neuropeptides; the other sample will be processed for the autoradiographic demonstration of receptor binding for 5 peptide ligands. Tissue donors with pre-morbid history of reflex sympathetic dystrophy or causalgia will be the primary focus of this work because of the similarity between the symptoms and antecedents of these conditions and those of the experimental model. However, the study will also accept material from patients with other kinds of painful peripheral neuropathy (e.g., post- herpetic neuralgia and diabetic neuralgia). This is justified because of evidence indicating that neuropathies with different etiologies may nevertheless share fundamentally similar pathogenic mechanisms. In all cases, material will be accepted only if the abnormal pain symptoms were unilateral; this will allow side-to-side comparisons for the identification of abnormalities.

It is well known that an inflamed area is hyperalgesic. However, the mechanism underlying sensitization is not understood. Our overall goal is to understand how the process of sensitization occurs, how it can be interrupted, and to define the chemical identity of the neurons that become sensitized. The mechanism of sensitization is likely to involve an interplay between primary afferent neurons and substances generated by the immune system since sensitization is generally associated with tissue damage. In these exploratory studies we will focus on whether eicosinoids and cytokines act directly on primary afferent neurons to enhance the response of these neurons to the algogens bradykinin and serotonin. Since different populations of primary afferent neurons may respond differently to sensitizing substances, we will also define some of the neurochemical characteristics of the sensory neurons that are assayed for their responsiveness. The most relevant portion of the primary afferent neuron for studies of sensitization is its peripheral process, and specifically, the peripheral terminal. The technical evolution of activity dependent fluorescent dyes, and microfluorimetric detection systems for these dyes, makes it possible to study biochemical changes in individual processes of primary afferent neurons. We propose to use Indo-1 to measure changes in intracellular Ca2+ ([Ca2+]i) as a bioassay. Since depolarization of primary afferent neurons opens voltage-sensitive calcium channels in the neuronal membrane, changes in [Ca2+]i will provide an indirect measure of neuronal depolarization. Our experiments will address the following questions: 1. Do substances associated with inflammation increase [Ca2+]i in primary afferent neurons? The activity of two prostaglandins (PGE2 and PGI2) and two cytokines (interleukin 1-beta and tumor necrosis factor-alpha) will be explored. 2. Do prostaglandins and cytokines potentiate the action of known algogens in causing changes in [Ca2+]i? Bradykinin and serotonin depolarize sensory neurons and cause overt pain. These algogens also increase [Ca2+]i. Since prostaglandins and cytokines may not have a direct effect on [Ca2,]i, we will determine whether these substances potentiate the [Ca2+]i that occurs in response to bradykinin and serotonin. 3. What populations of primary afferent neurons, based on their peptide content, exhibit sensitization? We will determine whether the neuronal processes that were assayed contained calcitonin gene-related peptide and/or substance P immunoreactivity. The occurrence of RT97 immunoreactivity, which is a predictor of axon myelination, will also be determined. The above studies will initially be conducted in primary cultures of neonatal rat dorsal root ganglion neurons. We will also explore whether these studies can be conducted in vitro on peripheral processes of sensory neurons in tooth pulp obtained from adult rats.

DESCRIPTION: (Provided by Applicant): Purpose: This institutional training program that combines a broadly based curricular training in Neuroscience with research training focused upon neuroscience oriented approaches to drug abuse. Our program provides this training in the context of the interdepartmental Neuroscience Program of our Graduate School, related graduate programs in the basic health sciences and a research environment which is characterized by a critical mass of NIDA-supported investigators who have a productive history of collaborative, interlaboratory research. Program: The training program capitalizes on 4 general arenas of trainer-trainee interaction: curriculum, research training, multiple venues for discussion of research (seminars, retreat, travel to scientific meetings) and structured programs that promote trainee planning for success. The curriculum is based on the graduate program in Neuroscience. The curriculum emphasizes cellular and molecular, systems, and behavioral components of neuroscience, and includes a course in Neuroscience Principles of Drug Abuse. The research encompassed by trainers involves cellular neuroscience integrated with molecular and/or behavioral approaches to problems associated with drug abuse; research training is enriched by the tradition of collaboration among trainers of this program. Training in research will be under the direction of 13 faculty members, all of which are members of the graduate faculty in Neuroscience. Trainees: The proposed program provides training for 7 predoctoral. and 5 postdoctoral trainees. Predoctoral trainees pursuing a Ph.D. in Neuroscience, or students in the departmentally-based graduate programs of Pharmacology or Molecular Veterinary Biosciences who elect to minor in Neuroscience, will be eligible for training under the auspices of the proposed training program. Postdoctoral training by its nature is more customized and varied according to trainer and trainee. The principle to be followed in the proposed program is that the research conducted should include collaborative efforts between laboratories so as to insure an experimental neuroscience perspective. Facilities: Resources available to the trainees are state-of-the-art facilities in the various faculty laboratories. There are well-equipped facilities for electrophysiology, biochemistry, molecular biology, histochemistry, autoradiography, microscopy (including confocal), image analysis, psychophysical testing of nociception, smooth muscle drug and transmitter assays, behavioral analgesia assays, ion and fluid transport assays, HPLC of amines and peptides, radioirnmunoassays, and the synthesis and characterization of novel opioid ligands. Two highly specialized facilities include the Biomedical Image Processing Laboratory and the Minnesota Microchemical Facility.

DESCRIPTION (provided by applicant): Maintenance of hyperalgesia requires changes in the expression of proteins that lead to an increase in synaptic strength. Data generated in this proposal will test the hypothesis that peptides released in the spinal cord in response to tissue damaging stimuli activate receptors on spinal neurons that increase gene expression, resulting in increased expression of proteins that underlie hyperalgesia. Data will be generated to address the following questions: 1) Does SP activate NFAT-dependent gene transcription in spinal neurons? 2) Does SP facilitate effects of CGRP on CREB-dependent gene transcription in spinal neurons? 3) Do SP and CGRP increase expression of proteins relevant to hyperalgesia in spinal neurons in vitro? 4) Do SP and CGRP increase expression of these proteins in spinal neurons in vivo? 5) Does treatment with SP or CGRP increase secondary hyperalgesia? 6) Do endogenous SP and CGRP contribute to the maintenance of secondary hyperalgesia during peripheral inflammation? Immunoblot analysis and real time PCR will be used to quantify the effects of SP and CGRP on the expression of four proteins that are known to be increased in the spinal cord during peripheral inflammation and that contribute to hyperalgesia: neurokinin-1 receptor, cyclo-oxygenase 2, type 1 nitric oxide synthase and inositol triphosphate receptor. Behavioral assays of nociception will be used to test whether treatment with SP and CGRP is sufficient to increase hyperalgesia, antagonists of SP and CGRP receptors will be used to determine whether endogenous SP and CGRP are necessary to support the increase in nociceptive responses that occurs in a model of peripheral inflammation. The experiments will generate new information concerning the effects of SP and CGRP on protein expression in spinal neurons in general as well as 4 proteins that contribute to hyperalgesia. If the hypothesis is proven to be true, the data will provide evidence for a new therapeutic strategy that may be effective in treating peripheral inflammatory diseases.

DESCRIPTION (provided by applicant): As cancer progresses, pain is increasingly associated with destruction of tissue, and severe pain occurs with bone destruction. Derivatives of Cannabis sativa (cannabinoids) are potent analgesics, and endogenous cannabinoids share this property. Anandamide (AEA) and 2-arachidonoylglycerol (2AG) are two endocannabinoids that are synthesized on demand from membrane phospholipids, and they may play fundamental roles in modulating our sensitivity to noxious stimuli. Indeed, we have generated evidence that increased degradation of AEA in skin is associated with mechanical hyperalgesia in a murine model of bone cancer pain. Cannabinoid-1 receptors mediate the inhibitory effects of AEA on somatosensory neurons, and expression of these receptors is increased in dorsal root ganglion neurons ipsilateral to tumors in tumor bearing mice. Activation of the increased pool of receptors by endogenous AEA can be manipulated by decreasing AEA degradation, resulting in prolonged anti-hyperalgesia in tumor-bearing mice. These data provide evidence to support the hypothesis that the local inhibition of endocannabinoid degradation in the periphery reduces mechanical hypersensitivity in a murine model of bone cancer pain through CB1 and CB2 receptor-dependent mechanisms. Studies outlined in this proposal will test this hypothesis further and address specific aims that explore 2AG metabolism in the same model of bone cancer pain. Specific aim 1 addresses whether inhibition of 2AG degradation is more effective than inhibition of AEA degradation in attenuating mechanical hypersensitivity in tumor-bearing mice. Monoglyceride lipase (MGL) degrades 2AG in vivo. Specific aim 2 examines the sources of MGL and cannabinoid receptors that underlie the anti- hyperalgesic effect of MGL inhibition in the periphery and whether expression of these proteins changes in tumor-bearing mice. Specific aim 3 examines the capacities of cells relevant to cutaneous mechanosensation to synthesize AEA and 2AG and whether the capacity for synthesis is changed in the cancer condition. Specific aim 4 addresses whether an MGL inhibitor acts directly on sensory neurons to reduce a depolarization-evoked calcium signal. Together with parallel data generated for AEA signaling, the data generated under specific aims 1-4 will provide extensive information on the cellular mechanisms through which inhibitors of AEA and 2AG degradation reduce mechanical hyperalgesia in cancer pain. The data will provide a rationale for the manipulation of endocannabinoids as a novel therapeutic strategy in the treatment of cancer pain.