Alvin Beitz - US grants

The research outlined in this proposal will enhance our understanding of the neurotransmitters and circuitry of cerebellar afferents, elucidate the effects of ischemia on cerebellar excitatory amino acids (EAAs) and evaluate the efficacy of a novel class of drugs in attenuating ischemic damage. A growing body of evidence suggests that neuronal cell damage following ischemia may result from the excessive release of EAAs. Cerebellar Purkinje cells receive synaptic input from glutamatergic and possibly aspar- tatergic neurons and have been found to be particularly vulnerable to ischemic insult. Thus the cerebellum provides an excellent model to examine the effect of ischemia on putative amino acid transmitter synthesis, release and metabolism and to determine whether major tranquilizers can reduce ischemic damage by their ability to inhibit amino acid synthesis. This proposal will address these issues as well as outline a series of experiments which are an extension of our work during the previous grant period which will examine the anatomical relationships and neurotransmitters of mossy and climbing fiber collaterals to cerebellar output neurons located in the deep cerebellar nuclei. The proposed research will be accomplished by completing the following four specific aims: 1) To analyze the anatomical relationships of and elucidate the neurotransmitters associated with cerebellar afferent fibers that synapse on deep cerebellar nuclear neurons projecting to the thalamus, red nucleus or inferior olivary nucleus; 2) To determine whether the neurotransmitter associated with cerebellar climbing fibers is aspartate and/or glutamate; 3) To determine the effect of ischemia on amino acid concentrations in cerebellar extracellular fluid and on GDH and glutathione immunostaining and to ascertain if changes occur in the mRNA levels of the amino acid synthesizing enzymes GDH, GAD and glutaminase following an ischemic insult; and 4) To evaluate the effect of the major tranquilizers haloperidol, thiothixene and chlorpromazine on amino acid concentrations in extracellular fluid and to determine their ability to attenuate ischemic effects. These studies will generate new data regarding the circuitry and transmitters of cerebellar input fibers and the effects of hypoxia on cerebellar excitatory amino acids. The final portion of this work could indicate a possible therapeutic use of major tranquilizers in the treatment of ischemic neuropathologies, such as stroke and cardiac arrest.

Recent studies of the trigeminal brain stem nuclear complex have served to reinforce the classic concept of the involvement the spinal trigeminal nucleus (STN), especially its caudal component, subnucleus caudalis, as the essential brain stem release of orofacial nociceptive information to higher levels of the brain. Although a great deal of information is available concerning the anatomy and connections of the STN, no data is available regarding the major neurotransmitters that subserve STN protections. Until such data is obtained little progress can be made toward understanding the pharmacology and biochemistry of this system. The major goals are to investigate the possible presence of several putative neurotransmitters in the STN utilizing novel monoclonal antibodies in conjunction with immunohistochemistry and to evaluate which of these neurotransmitters are associated with the major efferent projections of this nucleus. These goals are defined by the following specific objectives: 1) to establish whether aspartate or cysteic acid containing neurons are present in the STN and to determine whether they comprise neuroral populations which are separate from glutamatergic neurons: 2) to determine if the trigeminothalamic, trigeminospinal, trigeminocollicular or trigeminocerebellar projections arise from glutamate and/or aspartate containing neurons; 3) to evaluate the chemical nature of synaptic inputs onto excitatory amino acid-containing STN neurons which project to the thalamus, spinal cord or other areas utilizing combined electron microscopic-immunohistochemical-immunoautoradiography; and 4) to raise monoclonal antibodies to the putative transmitters taurine, cysteine sulfinic acid, quinolinic acid, aspartyl-glutamate and glutamyl-taurine and if successful to establish whether STN neurons contain any of these substances. The proposed investigation will provide important and novel data concerning the neurotransmitters utilized by STN neurons and will establish a basic foundation for future biochemical and pharmacological studies that may ultimately lead to better therapeutic management of orofacial pain.

Recent studies of the trigeminal brain stem nuclear complex have served to reinforce the classic concept that orofacial nociceptive information is relayed to higher levels of the brain via the spinal trigeminal nucleus (STN). Although a great deal of information is available concerning the anatomy and connections of the STN, very little data is available regarding the neurochemistry or pharmacology of this system. The long term goal of the present proposal is to elucidate the chemical organization of the STN which underlies its pivotal role in nociceptive transmission through the use of anatomical, biochemical, pharmacological and electrophysiological techniques. This goal is defined by the following specific objectives: 1) To identify the neurotransmitters associated with trigeminal ganglion neurons that innervate the tooth pulp by employing double-labeling immunocytochemical methods in combination with retrograde labeling procedures; 2) To analyze the excitatory amino acid receptor subtypes associated with primary afferent synapses and trigeminothalamic neurons using in vitro receptor binding procedures after lesions of the trigeminal nerve or after injections of a retrogradely transported toxin into the thalamus; 3) To identify the transmitters associated with several key inputs to trigeminothalamic neurons by combining tract tracing procedures with immunocytochemistry; 4) To use in vivo microdialysis to analyze the release of substance P, adenosine and amino acid transmitters in the STN following tooth pulp electrical stimulation or veratridine stimulation of the STN. The location of chemically identified neurons contributing to transmitter release into the dialysate will be determined by administering a retrograde tracer directly into the dialysate and performing combined retrograde tracing immunocytochemical procedures. These studies will provide important new data concerning the chemical coded circuitry and pharmacology of the trigeminal system, toward the development of a better therapeutic approach to orofacial pain.

The present shared instrument proposal requests funds to purchase a new state-of-the-art Hitachi transmission electron microscope to be utilized by seven PHS supported investigators to enhance the progress of their current research projects. As indicated in section 2a of the proposal, we have a single antiquated transmission electron microscope in our College and increased usage of this instrument over the past two years has generated heavy scheduling conflicts that have severely limited individual access to a transmission electron microscope. The acquisition of the proposed Hitachi scope would alleviate this problem and provide us with additional essential capabilities that we currently are lacking. Project Summaries: Dr. Beitz's projects involve the combined use of electron microscopic (EM) immunocytochemistry and neuronal tract tracing procedures to define the chemical circuitry of the cerebellar deep nuclei and the spinal trigeminal nucleus. Dr. Johnson's project also utilizes EM immunocytochemistry in combination with morphometric analysis to analyze the localization of a recently discovered islet amyloid polypeptide in beta cells of the pancreas from normal cats, from cats with impaired glucose tolerance and from cats with overt diabetes mellitus. Dr. Louis's work employs EM immunocytochemistry to localize a lens membrane protein to the junctional regions of the lens plasma membrane and to define the structural relationships of the muscle sarcoplasmic reticulum calcium release channel/ryanodine receptor protein. An important component of Dr. Larson's projects involves the use of anatomical double labeling procedures at the ultrastructural level to determine whether anterogradely labeled primary afferent terminals in the spinal cord contain glutamate-like immunoreactivity, whether these terminals are surrounded by astrocytic processes containing glutamate dehydrogenase or glutamine synthestase and whether glutamate immunoreactive terminals co-contain CGRP or substance P. Drs. Madl, O'Grady and Brady also utilize, or plan to utilize, EM immunocytochemistry in their work. Dr. Madl's project requires the immunocytochemical localization of amino acids in axon terminals of neurons subjected to hypoxic conditions while Dr. O'Grady's work involves the ultrastructural localization of a novel cardiac antisecretory peptide in the heart, small intestine and gallbladder. Dr. Brady plans to use novel monoclonal antibodies against carnitine acyltransferases to analyze the location of these enzymes in the mitochondria and peroxisomes of liver cells.

The midbrain periaqueductal gray (PAG) has been implicated in several important functions including pain modulation, defensive behavior, vocalization and reproductive behavior. Despite the fact that a great deal of information is available regarding the anatomical organization of this region, very little data exists concerning the relationship of neuropeptides and neurotransmitters to the anatomical circuitry of this important midbrain area. The long term goal of the proposed research is to elucidate the chemical organization of the PAG which underlies its pivotal role in analgesia through the use of anatomical, biochemical, pharmacological and behavioral techniques. We will reach this goal by completing the following specific objectives: 1) to analyze the possible relationship of the opioid peptides, endorphin, enkephalins and dynorphin, as well as the mu, delta nd kappa opiate receptors to both GABA-containing interneurons and PAG projection neurons using anatomical, biochemical, pharmacological and behavioral techniques; 2) to examine the possible relationship of GABA immunoreactive terminals and GABAA receptor immunoreactivity to PAG projection neurons using light and electron microscopic immunocytochemistry in combination with retrograde transport techniques and intracellular filling of retrogradely labeled cells; 3) to analyze the possible relationship of neurotensin immunoreactive terminals to opioid- and excitatory amino acid-containing neurons in the PAG by utilizing double-labeling immunocytochemical techniques; and 4) to analyze the release of neurochemicals from the nucleus raphe magnus in response to electrical stimulation of the PAG or morphine administration into the PAG by using in vivo microdialysis techniques. These studies will provide novel data that will not only further our understanding of the basic organization of this midbrain region but will also elucidate the neurochemicals involved at the level of the PAG and raphe magnus in PAG mediated antinociception. Such information is essential for developing improved therapeutic approaches to pain management.

The equilibrium receptors of the inner ear detect the position and motion of the body in space. Abnormalities in the receptors or central pathways of the vestibular system can result in oculomotor and postural disturbances. Despite the fact that a great deal of information is available concerning the anatomical and physiological organization, relatively little data exists concerning the neurochemical organization of the vestibular system in general and of the vestibular nuclear complex in particular. The long range goals of the present proposal are to: 1) define the major transmitters and receptors associated with the mammalian vestibular nuclei; 2) determine the major transmitters associated with vestibulocerebellar projections; and 3) ascertain which transmitters are affected by peripheral vestibular lesions or stimulation. These goals will be pursued by the following specific aims: 1) Analyze the presence and distribution of the immunoreactivity for glutamate, aspartate, GABA and their synthesizing enzymes together with glycine, enkephalin and dynorphin and the mRNA expression for these synthesizing enzymes and these opioid peptides In the vestibular complex; 2) Determine which neurotransmitters are associated with vestibulocerebellar projections using immunocytochemistry in combination with retrograde and anterograde tracing procedures; 3) Quantity the location of glycine, GABA-A and excitatory amino acid binding sites in the vestibular nuclei and determine whether they are associated with vestibulocerebellar neurons using in vitro receptor binding procedures alone and in combination with the suicide transport agent, doxorubicin. These studies will be complemented by experiments employing a new combined retrograde transport-in situ hybridization procedure to determine which receptor mRNAs are expressed in vestibulocerebellar neurons; 4) Identify the specific neurotransmitters that interact with vestibulocerebellar neurons by intracellularly injecting Lucifer yellow into retrogradely labeled neurons and then immunochemically localizing certain transmitters The resulting double-labeled sections will then be analyzed using both confocal scanning laser microscopy and transmission electron microscopy; 5) Study the changes in neurotransmitter mRNAs, binding sites and/or release following peripheral vestibular lesions and stimulation. This will be accomplished by using in situ hybridization, receptor binding and in vivo microdialysis techniques. Identification of the major neurotransmitters and receptors associated with the vestibular nuclear complex in combination with data generated concerning which transmitters are affected by vestibular stimulation or damage will provide new information which might facilitate the development of better therapeutic approaches to vestibular disorders.

The midbrain periaqueductal gray (PAG) has been implicated in several important functions including pain modulation, defensive behavior, vocalization and reproductive behavior. Despite the fact that a great deal of information is available regarding the anatomical organization of this region, very little data exists concerning the relationship of neuropeptides and neurotransmitters to the anatomical circuitry of this important midbrain area. The long term goal of the proposed research is to elucidate the chemical organization of the PAG which underlies its pivotal role in analgesia through the use of anatomical, biochemical, pharmacological and behavioral techniques. We will reach this goal by completing the following specific objectives: 1) to analyze the possible relationship of the opioid peptides, endorphin, enkephalins and dynorphin, as well as the mu, delta nd kappa opiate receptors to both GABA-containing interneurons and PAG projection neurons using anatomical, biochemical, pharmacological and behavioral techniques; 2) to examine the possible relationship of GABA immunoreactive terminals and GABAA receptor immunoreactivity to PAG projection neurons using light and electron microscopic immunocytochemistry in combination with retrograde transport techniques and intracellular filling of retrogradely labeled cells; 3) to analyze the possible relationship of neurotensin immunoreactive terminals to opioid- and excitatory amino acid-containing neurons in the PAG by utilizing double-labeling immunocytochemical techniques; and 4) to analyze the release of neurochemicals from the nucleus raphe magnus in response to electrical stimulation of the PAG or morphine administration into the PAG by using in vivo microdialysis techniques. These studies will provide novel data that will not only further our understanding of the basic organization of this midbrain region but will also elucidate the neurochemicals involved at the level of the PAG and raphe magnus in PAG mediated antinociception. Such information is essential for developing improved therapeutic approaches to pain management.

The research outlined in this portion of the program project is designed to define the putative excitatory amino acid transmitters and receptors associated with cerebellar afferent systems in normal rats. Although neurotransmitters in the majority of cerebellar cortical neurons have been identified, information on the transmitters of cerebellar climbing fibers remains controversial and the major transmitters of the mossy fibers have yet to be defined. The experiments proposed will examine several acidic amino acid compounds, nitric oxide and polyamines as possible transmitter candidates for the olivocerebellar pathway and selected mossy fiber pathways. This research will provide basic information relevant to our understanding of the biochemical and pharmacological characteristics of cerebellar afferent systems. Our proposed investigation is described by the following three specific aims: 1. To determine whether glutamate, aspartate, NAAG, nitric oxide synthase or spermidine and putresine immunoreactivity is associated with identified climbing fiber or mossy fiber terminals by employing anterograde tracing with PHA-L and biotinylated dextran amine (BDA) in combination with immunocytochemistry at both light and electron microscopic levels. 2. Based on the results of 1, we will ascertain whether aspartate, glutamate, NAAG, citrulline, spermine or putresine are released in the cerebellar cortex following olivary, vestibular, LRN or middle cerebellar peduncle stimulation by combining electrical or chemical stimulation with in vivo microdialysis and electrophysiological recording or C-fos immunostaining. 3. The types of excitatory amino acid receptors associated with cerebellar afferents will be defined using: A) direct ultrastructural examination of PHA-L or BDA labeled terminals in combination with immunocytochemical localization of glutamate receptor subtypes and B) lesions of selected afferent pathways will be employed in combination with immunoprecipitation techniques and in situ hybridization.

The long term goal of this project is to define the glutamate receptor subtypes associated with synapses in the cerebellar cortex and to elucidate the role of these receptor subtypes in nitric oxide production. Although it is known that multiple glutamate receptor subtypes exist in the cerebellar cortex, the specific subtypes associated with different parallel fiber versus climbing fiber synapses remains to be defined. There is currently no information available as to whether the number or subtypes of glutamate receptors differ between synapses located in zebrin positive parasagittal binds versus zebrin negative bands. The proposed studies will test the hypothesis that: 1) Glutamate receptor subtypes differ between the parallel fiber/Purkinje cell synapse and the parallel fiber/stellate cell synapse and between the parallel fiber/Purkinje cell and climbing fiber/Purkinje cell synapses; and 2) Climbing fiber activation causes nitric oxide (NO) production in the cerebellar cortex via activation of glutamate receptors on stellate cells, while parallel fiber activation produces NO directly. In Specific Aim 1 the first hypothesis will be tested by using immunocytochemistry and electron microscopy to determine the molecular subtypes of glutamate receptors associated with climbing fiber and parallel fiber synapses. In Specific Aim 2 the second hypothesis will be tested by using electrical or chemical stimulation of climbing and parallel fibers in combination with in vivo microdialysis and administration of glutamate receptor antagonists. These studies will provide new data on the synaptic distribution of glutamate receptors in the mouse and rat cerebellar cortex and will determine if differences in receptor subtypes exist between zebrin positive and zebrin negative parasagittal bands. In addition these studies will elucidate the glutamate receptor subtypes involved in cerebellar NO production in vivo. This neurochemical information is important for understanding the basic mechanisms that underlie parallel and climbing fiber neurotransmission and those involved in cerebellar NO release.

DESCRIPTION: The fact that more than 500,000 patients in the United States died from cancer in 1997 indicates that cancer, in its many forms, is still largely incurable and underscores the importance of focusing new treatments and research, at least in pat on optimizing the management of cancer pain. The greatest obstacle to developing new treatments for cancer pain and/or optimally coordinating existing treatments is a paucity of knowledge defining the neurobiology of cancer pain. We have recently developed two animal models of tumor allodynia and hyperalgesia, which we believe will be useful in developing an understanding of the basic mechanisms of cancer pain. In the studies proposed below in vivo microdialysis be utilized to analyze the baseline levels and stimulated release of substance P and putative allogens within a bone tumor and a soft tissue tumor model Weal pursue the following Specific Aims: 1. To measure the basal release of the substance P, PGE.2, endothelin-1, nerve growth factor, and the c$o&ines, TNF-a and IL-1 at tumor sites at several time points as compared to comparable sites in control animals. We will also measure the release of the above substances at the tumor site during peripheral nerve activation. 2. To ascertain whether the tumor cells or accessory cells (such as osteoclasts) at the tumor site are producing the mediators measured by microdialysis in Specific Aim 1. This aim will utilize immunocytochemistry to localize the protein for each algogen at the tumor sites. 3. To determine if the administration of tumor-associated mediators can mimic, and their antagonists reduce, the nociceptive responsiveness in behavioral pain tests. To our knowledge the proposed experiments are the first to utilize the technique of microdialysis to directly measure substances produced and released in vivo by a tumor in awake, freely moving animals aver time. The results should provide important, new data regarding the mediators released in vivo by soft tissue and bone tumors and will dramatically advance knowledge in the area of the neurobiology of cancer pain.

DESCRIPTION (provided by applicant): The interactions between a tumor and its local environment are critical to tumor development and progression, and ultimately to tumor metastasis. Most of our current knowledge of the signaling molecules secreted by tumor cells is based on in vitro studies that have largely ignored the dynamics of the in vivo tumor environment. The overall goal of this proposal is to study the dynamics of signaling peptide and protein secretion by several tumor types (fibrosarcoma, breast, prostate and myeloma) implanted in bone. In the R21 portion of the application we propose to combine a novel in vivo microperfusion procedure, which allows sampling of extracellular fluid from solid tumors over time, with new mass spectrometry methods allowing identification of proteins in mixtures. The R21 phase will be used to further develop MALDI-TOF, ESI-Q-TOF and ESI-MS/MS procedures and to optimize conditions for maximum detection and for identification of proteins and peptides in bone tumor microperfusates. The interactions of various tumor types with the unique micro-environment found in bone will be studied in the proposed R33 phase by analyzing the secretion of selected as well as unidentified proteins produced by breast, prostate and myeloma tumors over time. By permitting the in vivo identification of an array of peptides and proteins present in tumor extracellular fluid over time, this novel microperfusion approach will provide the opportunity to characterize the extracellular peptides and proteins secreted by different bone tumor types which are involved in the growth, progression, latency and metastasis of these tumors. The data obtained from these studies will facilitate cancer detection and diagnosis as well as define new targets or therapeutic and preventative agents.

DESCRIPTION (provided by applicant): Pain is the cancer-related event that is most disruptive to a cancer patient's quality of life. Although bone cancer pain is one of the most severe and common sources of chronic cancer pain, relatively little is known about the mechanisms that generate and maintain this pain. The proposed experiments are designed to study the role of two chemokines, CCL2 and CCL3 in the genesis and maintenance of bone cancer pain using an established mouse bone tumor model and a novel ex vivo model of cancer pain. Four Specific Aims will be pursued: 1) Measure the spontaneous release of CCL2 and CCL3 in vivo and in vitro, from three different tumor types (osteosarcoma, fibrosarcoma and nonpainful melanoma). Based on preliminary data we hypothesize that CCL2 is released only by the fibrosarcoma and osteosarcoma, while CCL3 is released only by the osteosarcoma. 2) Identify the cell types that are producing CCL2 and CCL3 by using immunocytochemistry to localize each mediator to cells at the tumor site. We hypothesize that CCL2 will be localized to fibrosarcoma and osteosarcoma cells and to osteoclasts and macrophages at the tumor site, while CCL3 will be found predominately in the osteosarcoma cells. 3) Investigate the effect of CCL2 and CCL3 on the functional responses of DRG neurons using a novel in vitro tumor/bone-explant/DRG culture system. We hypothesize that CCL2 and CCL3 released from tumor cells or bone produce acute and long-term changes in stimulus- evoked Ca2+ transients that reflect changes in voltage dependent Ca2+ channels and TRPV1 receptors. We further hypothesize that the knockdown of these chemokines will prevent the effect of tumor cells on Ca2+ transients. 4) Determine whether manipulation of expression of CCL2 and CCL3 in tumors cells alters the pain producing phenotype of the cells. We hypothesize that knock-in of CCL2 and CCL3 in nonpainful melanoma cells will result in pain producing tumors, while knock-down of CCL2 and CCL3 in fibrosarcoma or osteosarcoma cells will reduce bone tumor-associated nociceptive responsiveness in behavioral pain tests. The proposed experiments are unique in that they will utilize the technique of microperfusion to directly measure algogens produced in vivo by different tumor types, while an innovative ex vivo tumor/bone-explant/DRG culture system will allow us to measure these substances in the culture media and to analyze their effects on DRG neurons. In addition the use of a new sleeping beauty transposon-shRNA procedure for long-term knockdown or production of these mediators will provide an excellent opportunity to investigate their role in nociception both in vitro and in vivo. Results will extend our knowledge of the mechanisms underlying tumor nociception and will provide a template for developing novel, life-enhancing treatments of cancer pain. For cancer patients, pain relief is central to a high quality of life while they battle the disease, yet current therapies are often distressingly ineffective for some types of cancer pain. To address this critical need and develop new therapies that successfully diminish cancer pain, we need to understand at the cellular level how cancer pain is generated and why it persists. This research focuses on two proteins, CCL2 and CCL3, that are secreted by tumors [and other tissues], and whose absence appears to be linked to decreased cancer pain. Use of an animal model of bone cancer pain and a new ex vivo cancer pain model to test the effects of CCL2 and CCL3 knockdown or antagonism will provide important data that may lead to the development of novel therapies for this dreaded condition.