Overview: The objectives of this project are to understand the molecular biology of pain-sensing neurons and peripheral tissues at the transcriptome level and modulation of transcriptomic parameters in acute and chronic pain models and in human patients or post-mortem samples. The laboratory has established research methodology and protocols, built an infrastructure of hardware and software, formed collaborative arrangements, trained a team of scientists and support personnel to utilize the methodology of RNA-Seq, in situ hybridization and tissue procurement. We have performed hundreds of deep sequencing runs in various species and models resulting in many billions of reads of transcriptome sequence information. We are intensively involved in the analysis of the resulting datasets that encompass physiologically or genetically labeled pain-sensing neurons, neurons in dorsal spinal cord during peripheral inflammation, models of inflamed or surgical incision peripheral tissue, axotomized dorsal root ganglion (DRG) neurons, dorsal and ventral spinal cords, peripheral nerve, and peripheral tissue as well as human tissues comprising the nociceptive circuit. We are also investigating transcriptional processes affected by general anesthesia in higher order brain regions. Use of the newer high-throughput sequencing devices allows sampling of multiple time points to follow the evolution and resolution of the intervention with enough read depth and number of samples at each point to permit thorough assessment and statistical comparison, respectively. Because we isolated certain neuronal and non-neuronal cell populations, we know which genes are in pain-sensing neurons and which are in mainly non-pain-sensing neurons such as proprioceptive primary afferents, and supporting cells or Schwann cells. The ability to form incisive hypotheses regarding pain physiology is greatly advanced by this type of tissue and neuron-specific information. We now have quantitative information on all the genes that mediate DRG and spinal cord sensory and motor functions and formation of the myelin sheath which, in turn, permits us to build new levels of understanding of how pain is generated, transmitted, processed and modulated in the peripheral and central nervous systems in animal models and humans. TRPV1 Transcriptome: One important focus for our group is the subpopulation of DRG neurons that express the thermo-, chemo-, pH-, and lipid-responsive ion channel called TRPV1. This ion channel is also gated by capsaicin, the active ingredient in hot pepper. We have demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in dogs and humans indicating a crucial role for TRPV1+ neurons in transmission of clinical pain. Because of the efficacy of manipulations aimed at the TRPV1-expressing DRG neurons, we performed deep RNA sequencing (RNA-Seq) on mouse, rat, canine, and human ganglionic preparations targeting TRPV1 neurons. We published initial reports on the comprehensive transcriptomic profile of this clinically important population of nociceptive neurons, followed by a second investigation that distinguished the contribution of Schwann cells versus neurons to the DRG transcriptome and extended the analysis to TRPV1+ neurons functionally identified by agonist-activated calcium fluorescence and DRGs obtained at autopsy from one of our human cancer pain patients who had been treated with RTX, a cohort of canines with cancer pain that were also treated for pain with RTX and controlled treatments in the rat. In combination, these data demonstrate that the most sensitive neuronal component is the centrally projecting axons that contain TRPV1 whereas the cell bodies in DRG are comparatively resistant to RTX. This important mechanistic insight was gained from transcriptomic analyses and is being used to fine-tune the administration protocol in our human clinical trial. Analgesia transcriptome: One of the most interesting aspects of the transcriptome analyses is quantitative insight provided by next-gen RNA-Seq. This is a high-resolution, transformative technology that provides sequence-based counting of transcripts to categorize, for example, genes that are well-expressed versus those expressed at an inconsequential level. Additionally, we can make qualitative assignments as to which molecular paralogs are in the nociceptive populations allowing a more informative mechanistic framework to emerge. In this cycle we examined the endogenous opioid peptide precursors and peptides preproenkephalin and pre prodynorphin in dorsal spinal cord after an experimental inflammation through the combined use of radioimmunoassay and HPLC and gel filtration chromatography for peptide levels, RNA-Seq for the mRNA levels, and in situ hybridization for combined cellular localization as well as behavioral characterization of the animals alteration in nociceptive behaviors. The focus was on the dynorphin family of endogenous opioids, and this opioid peptide and mRNA was strongly up-regulated in dorsal spinal cord neurons. We also performed a direct comparison of transcriptome level alterations in dorsal horn after inflammation compared to surgical incision. Dynorphin was a strong neuronal signature in both models. Transcriptome analysis also showed upregulation of anaplastic lymphoma kinase (ALK) in the spinal cord sample. In situ hybridization showed that ALK and dynorphin were in the same subpopulation of neurons and that these constituted a population of glutamatergic dorsal horn neurons. Analyses of the kappa opioid receptor system using a selective antagonist to block the action of endogenous dynorphin The results suggest that endogenous dynorphin acts to aid in resolution of the hyperalgesic state engendered by peripheral inflammation. Thus, we identify a critical component, dynorphin, and a critical cell population, dynorphinergic-glutamatergic excitatory dorsal horn neurons that participate in the regulation of spinal cord hyperexcitability. We hypothesize that these neurons are of adaptive significance, such that they reduce tonic hyperexcitability and allow an injured organism to continue to forage for food while protecting the injured limb from further damage. Anesthesia Transcriptome: We are in the process of completing a transcriptomic assessment of the effects of inhalation general anesthesia and ketamine infusion on cortical and hippocampal transcriptomes and associated proteins identified from the gene analysis. These are initial steps to a larger investigation of the general anesthesia on cognitive function. In humans, general anesthesia can be deleterious to cognitive function. We hypothesize that mechanistic insight into the defect state can be obtained by understanding the molecular-level changes induced by anesthesia and the capacity for recovery. Our results indicate that communication between synaptic input and nuclear transcriptional control is strongly inhibited by general anesthesia and that the alterations are more pronounced in cortex than hippocampus. We detect widespread modulation of genes that mediate functional plasticity and memory formation. Corresponding decreases in several of the proteins are also observed. The data suggest that general anesthesia can transiently uncouple synaptic activity from neuronal transcriptional control. The situation turned out to be quite different for ketamine where we saw activation of a subset of immediate early transcription factors rather than a suppression of their basal expression as well as activation of the Nrf2 pathway.
