Overview: This research program addresses basic molecular and physiological processes of nociceptive transmission in the central and peripheral nervous systems and new ways to effectively control pain. The molecular research is performed using animal and in vitro, cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that innervate the skin and deep tissues and their connections in the dorsal spinal cord, which is the first site of synaptic information processing for pain. Our research has identified the DRG and spinal cord as loci of neuronal plasticity and altered gene expression in persistent pain states. The mechanisms of transduction of physical pain stimuli are also under investigation through examination of events and molecules in damaged or inflamed peripheral tissue and using reductionistic approaches such as cloned thermal and chemo-responsive ion channels expressed in heterologous cell systems or naturally expressed in primary cultures of dorsal root ganglion. Our goals are (1) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (2) to investigate mechanisms underlying human chronic pain disorders, and (3) to use this knowledge to devise new treatments for pain.[unreadable] New Treatments for Pain: We address the new treatment goal by a translational research and human clinical trials program aimed at developing new analgesic treatments for severe pain. The current approach, based on our studies of pain transduction through the vanilloid receptor 1 ion channel, now called TRPV1, has resulted in a clinical trial of resiniferatoxin (RTX) as a new treatment for advanced cancer pain. RTX activates TRPV1, which is a heat-sensitive calcium/sodium ion channel that normally converts painful heat into nerve action potentials by opening the pore of the ion channel. The influx of ions depolarizes pain-sensing nerve endings and triggers an action potential that is conducted to the spinal cord. Capsaicin, a vanilloid chemical and the active ingredient in hot pepper, also stimulates TRPV1 opening, which is why it feels hot. RTX is a very potent vanilloid that will prop open the TRPV1 ion channel, thereby causing calcium cytotoxicity and death of a specific class of pain-sensing neurons. This has proved to be a very effective means of pain control in several pre-clinical models and by several routes of administration. Some routes lead to cell death (e.g. intraganglionic injections) other routes do not, such as application to nerve endings in skin, deep tissue and joints. Through the efforts of an inter-institute working group, established with NIDA's Division of Pharmacotherapies and Medical Consequences of Drug Abuse, we are bringing this novel treatment to human clinical trial. The working group consists of experts on chemistry and manufacture, toxicological, neurobiological, medical, and regulatory affairs as well as anesthesiologists, pain management specialists and pharmacologists from our group. We have (a) established procedures for isolation, purification of RTX and formulation of the drug product, which was tested for stability, (b) conducted preclinical pharmacology, (c) conducted a complete toxicology study, and (d) submitted a clinical protocol to our Institutional Review Board and to the FDA as part the Investigational New Drug Application (IND). The RTX cell deletion treatment will first be tested for its ability to control cancer pain in patients with advanced disease. If it is safe and effective, we shall conduct a second protocol for treatment of head and neck cancer and then work on controlling other acute and chronic pain conditions such as post-surgical pain, joint pain, trigeminal neuralgia, arthritis and neuropathic pain. [unreadable] Basic Pain Mechanisms: Underlying the translational studies are our investigations of molecular regulation of gene expression, neuronal function, behavior, and mechanisms of pain transduction. We are systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord. The goal of this approach is to obtain a comprehensive and informative understanding of nociceptive process. The dorsal root ganglion is quite small but its analysis is made possible by the extensive use of reverse-transcription-PCR, which allows us to make multiple measurements on such small tissue samples. Our studies reveal a complex, dynamic modulation of gene expression at all three steps. We have examined novel molecules as well as neuropeptide, cytokine and chemokine expression and identified prominent roles for new, key molecules with distinct combinatorial patterns of expression among the three tissues. The functional implications of our studies on cytokines suggest a new role for monocyte chemoattractant protein 1 in nociceptive DRG neurons. Our recent work shows that this molecule is quantitatively enriched in the superficial layers of the dorsal spinal cord where it likely functions to affect intercellular communication. Thus, this small protein can affect a specific endpoint in the sensory half of the spinal cord. Further studies demonstrated that this protein was highly enriched in the choroid plexus where it may have a more broad action on the entire nervous system by influencing the epithelial cells that manufacture the cerebrospinal fluid. The above studies were conducted in the laboratory rat, we do not know is whether this molecule is made in the same nociceptive sensory neurons in human DRG and shows the same enrichment in human spinal cord. To answer this question we have established collaboration with the Neuropathology Section of the Clinical Brain Disorders Branch of NIMH to collect human trigeminal ganglia and the spinal trigeminal nucleus when they collect brains for their brain bank. These tissues samples can be used for analysis of specific peptides or proteins and for more expression profiling studies for bridging the gap between rodent and the human spinal circuits and connections with the sensory ganglia. Through this research in animals and humans we hope to obtain a fundamental understanding of the relationships between tissue damage, inflammation and pain sensation. In a broader framework, these studies explore the fundamental molecular basis of synaptic plasticity. We hypothesize modularity in neuronal responses when a new level of synaptic or pharmacological input occurs that will be relevant not only to pain but also to situations such as learning, neurological disorders like epilepsy, and drug abuse. A set of "generic" alterations is combined with circuit-specific genes to meet the demands of new stimulation or activity. Understanding the molecular repertoire and its dynamic interactions will lead to a deeper understanding of mechanisms that trigger and sustain chronic pain and other disorders of the nervous system. [unreadable] Early Translational Investigations: A final set of studies concerns the identification of small chemicals that enhance the action of vanilloid agonists on TRPV1. This ligand-gated ion channel is one of the most important molecular transducers of painful stimuli and understanding how TRPV1 can be blocked, activated or sensitized is of primary importance in understanding pain. We have identified a new action on the TRPV1 molecule via screening of a chemical library. This activity is manifested as an enhancement of calcium influx upon agonist activation of TRPV1. These studies suggest an allosteric modulation of the open state of the TRPV1 ion channel and the presence of a reserve level of activity that can be accessed for pain transmission as well as the existence of a new class of pharmacological agents for pain modulation and pain control.