Overview: 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. We have performed over 400 sequencing runs on Illumina HiSeq machines and obtained over 50 billion reads of transcriptome sequence information. and are intensively involved in the analysis of the resulting datasets. We have sequenced the transcriptomes of physiologically or genetically labeled pain-sensing neurons sorted by FACS, neurons in dorsal spinal cord during peripheral inflammation, models of rheumatoid arthritis, inflamed peripheral tissue, axotomized DRG and dorsal and ventral spinal cords and peripheral nerve. We have also begun investigations into transcriptional processes affected by general anesthesia in higher order brain regions. In many cases multiple time points are sampled to follow the evolution and resolution of the intervention with enough samples at each point to permit statistical comparison. Because we sorted for certain neuronal populations we know which genes are in the pain-sensing neurons and which are in mainly non-pain-sensing neurons such as proprioceptive primary afferents, 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. TRPV1 Transcriptome: One important focus of the neuronal sorting experiments 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. Previous experiments demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in dogs and humans. Because of their crucial role in pain transmission, we want to know everything possible about TRPV1-expressing DRG neurons. We isolated TRPV1 neurons by genetic labeling or physiological activation and then performed deep sequencing of the mRNA content using next-gen RNA-Seq. The genetic method expresses a fluorescent marker allowing the TRPV1 DRG neurons to be isolated by FACS. We also killed this population by making them express diphtheria toxin. A second strategy was stimulate TRPV1 neurons with RTX and sort the neurons that display increases in calcium fluorescence. We also killed the cells either genetically or by microinjection of RTX. Our first paper outlines the transcriptome results from the genetically labeled TRPV1 neurons and ganglia in which the TRPV1 neurons had been deleted by expression of diphtheria toxin or microinjection of RTX. This has provided comprehensive, new transcriptomic information on genes expressed by a clinically important population of nociceptive neurons. Analgesia transcriptome: One of the most interesting aspects of the transcriptome analyses is quantitative insight provided by next-gen RNA-Seq. We now know the quantitative relationships between the exact genes that mediate the actions of known analgesic drugs such as morphine, clonidine, lidocaine, ibuprofen, and gabapentin. It has not been clear which paralogs or subunits of drug binding receptors are expressed by different tissues in the pain pathway, yet this becomes clear when expression values for all the relevant genes are obtained quantitatively, at the same time, and with excellent reproducibility between animals and treatments. The transcriptome experiments also point to new targets for potential analgesic drug development. We identified an orphan GPCR that is well expressed in the nociceptive population, and are currently exploring its analgesic properties. In another example, we observe that the Mu opioid receptor is mainly expressed in the DRG and much less so in dorsal spinal cord. This strongly reinforces electrophysiological data that suggest epidural or intrathecal opioid analgesia is mainly mediated by presynaptic actions on primary afferent terminations in spinal cord and provides a framework for therapeutic strategies directed at the peripheral nervous system. Transcriptomics of Peripheral Sensitization: The RNA-Seq data provides a means for amplification of ongoing studies. The RNA-Seq results inform all of our hypothesis-driven studies and those of other groups. An example is our collaborative work with NIAAA. We observe that certain lipids are TRPV1 agonists. Using the transcriptome databases, we extracted the quantitative expression data for all the genes involved in lipid transport, generation, degradation, and the cognate receptors for the relevant lipids from sequencing of skin, DRG and dorsal spinal cord. Differential expression levels therein provided insight into new enzymes that generate particular lipids important for nociceptive sensitization. Canine Ganglionic Transcriptome: This year we completed the extraction and sequencing of canine ganglion and spinal cord tissue from controls and animals with osteosarcoma that were euthanized because of inadequate pain control. Tissues were obtained at autopsy. This study was undertaken to test for genes activated by nociceptive input from naturally occurring bone cancer. This will form a unique dataset that will provide new insight into the transcriptomics of cancer pain in a species that is very similar to humans. Anesthesia Transcriptome: Another project we are in the process of completing is an assessment of the effects of inhalation general anesthesia on cortical and hippocampal transcriptome. This is the initial step to a larger study on the effects of general anesthesia on cognitive function in aged animals. In humans general anesthesia can have a deleterious impact on cognitive function and we hypothesize that we can gain mechanistic insight into the defect state by understanding the molecular level changes in gene expression induced by anesthesia and the capacity for recovery. Our initial results indicate that communication between neuronal synaptic input and nuclear transcriptional control is altered by general anesthesia and that the alterations are more pronounced in cortex than hippocampus. It is quite remarkable that we detect widespread modulation of genes that mediate plasticity of neuronal function and memory formation. Summary: The datasets acquired over the past several years provide unprecedented and extremely fine-grained detail on gene expression in pain-sensing circuits. This may seem complicated but the basic goal is to understand how we sense pain and how we may control it when required. There are a wide variety of painful stimuli that can be encountered in our environment and different neurons exist to sense these different types of pain signals. We are determining exactly what molecules the different types of pain-sensing neurons make and how they work together to do their job. We will use this information to understand pain signaling and how to control it. Taken together these data provide a transformative new resource for the pain research community and will allow a much more precise assessment of experimental manipulations and verification of experimental results.
