Summary: The purpose of this research project is to understand the molecular basis of pain sensation and transmission. The goals are to understand human chronic pain disorders, mechanisms of acute pain and to discover new ways to treat pain. The scope comprises comparative transcriptome analysis of nociceptive (pain-sensing) circuits using targeted model systems and, where possible, equivalent studies in human tissue. We aim the identify transcripts that are critical for pain transduction, that are conserved and convergent across species, that display characteristic expression in pain processing tissues relative to other tissues, clear differences in absolute expression levels or state dependent changes indicative of a role in pathological pain states. Several questions we address are: How does the nociceptive circuit meet the challenge of chronic pain? What genes are important to nociceptive processing in humans? What are the effects of analgesic drugs or other manipulations of the pain circuit on gene expression in persistent pain states. These systematic studies will help us understand and manipulate the pain system in a more informed and effective fashion. This project is in the beginning stages. At this point we have established several research methods and protocols, built the supporting infrastructure in terms of equipment, space in the lab and collaborative arrangements, and have hired and begun to train a team of scientists and support personnel to conduct the research project. In terms of infrastructure and personnel I have interviewed and hired a person with a Ph.D. in Bioinformatics and Computational Biology who is skilled at analysis of RNA data from next gen sequencing (RNA-seq) platforms. I have hired a post-BAC IRTA who has experience in RNA extraction, cDNA library construction and RT-PCR analysis and I have a visiting fellow from the Anesthesiology Department of the Catholic University of Rome (supported by his university) working on the project as well. The laboratory has obtained two dedicated computer workstations for high-speed sequence analysis and alignment of the transcriptome libraries with genomic structure. We have also upgraded our Bioanalyzer for RNA quality assessment and our 96 well real-time PCR device for independent verification of alterations seen with the RNA-seq methods and obtained two high-speed PCR devices. We shall use the sequencing services from the National Institutes Sequencing Center (NISC). At NISC we will use the Illumina High-Seq device, generally in multiplex mode to analyze 6 to 8 independent samples/lane. We have met with NISC personnel and will begin evaluation of mRNA library construction using either polyA+ selection or ribosomal RNA depletion to enrich for mRNA. We will also be analyzing microRNAs and long non-coding RNAs during the course of this study. We shall work with the University of Pennsylvania to obtain canine tissue for the RNA studies. These tissues will come from controls and animals with osteosarcoma euthanized because of inadequate pain control or treated with resiniferatoxin. Tissues are obtained at autopsy. The data generated will allow us to test for genes activated by nociceptive input from naturally occurring bone cancer and modulated by treatment. The data will also be used for comparison to parallel studies in rat monkey and human (although the exact models will be different). The transcriptome project will systematically investigate the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord. The equivalent structures for the face and head are the trigeminal ganglia and the medulla. For human studies, it is relatively straightforward to obtain the trigeminal ganglia at autopsy and we have been doing this for nearly two years in conjunction with Dr. Joel Kleinman of NIMH. However, the second order synapse in the medulla is a bit more challenging. The trigeminal nociceptive primaryy sensory neurons synapse with second order neurons in the medulla, which is located at the top of the spinal cord. Thus, by dissecting this region we obtain both neural steps in the pain pathway. For the purpose of dissecting the pain sensing medullary dorsal horn from the surrounding tissue, we have conducted an in-depth neuroanatomical analysis of the human medulla. This region is quite different from the functionally equivalent regions of spinal cord due to the crossing over of the pyramidal tracts, which contain the axons of motor neurons from cerebral cortex. Our studies revealed that the dorsal horn is compressed into a tight circular shape by the fibers crossing over. This arrangement was very favorable for dissection of the medullary dorsal horn from fresh tissue because, due to its compactness, it can be removed in its entirety from a 3 mm slice of medulla. We take several such slices and dissect the dorsal horn from both left and right sides as well as white matter from the dorsal columns and pyramidal tracts for comparison. These same methods will be applied to rat, dog and monkey. Using the Bioanalyzer, we have analyzed total RNA extracted from the human medulla. Analysis yielded moderately good RNA integrity numbers and we are trying to improve the results using a method to remove lipids first. We have begun the initial study on inflammation induced genes in rats. This will include a careful analysis of behavioral hyperalgesia in each cohort using our A-delta and C-fiber selective stimuli and a systematic examination of the time course of gene regulation as it compares to the behavior. Comparisons will be made to several nerve injury models since these are distinct from the inflammation. We are also working with another NIMH laboratory to examine the dorsal root, trigeminal ganglia and spinal cord/medulla from non-human primates and have filed and taken the appropriate biosafety registrations and training courses, respectively.