Lawrence Toll - US grants

The NOP receptor, the fourth member of the opioid receptor family, has been the target of investigation since its discovery in 1993, with respect to pharmacology, anatomy, and behaviors elicited by agonists and antagonists. Despite considerable research efforts, the lack of suitable receptor antibodies has prevented the appropriate interpretation of many studies pertaining to the details of receptor location, internalization, and dimerization. In order to test hypotheses pertaining to NOP receptor function, Dr. Brigitte Kieffer and colleagues have generated knock-in mice that carry green fluorescent protein (eGFP) coupled to the NOP receptor. Similar GFP-tagged delta receptor and mCherry-tagged mu receptor knock-in mice have proven very useful in understanding the relationship among anatomy, cellular localization, and function of delta and mu opioid receptors. Like the mu receptor, NOP receptors are found in very high numbers in all of the pain-related brain regions, including PAG, RVM, MHb, thalamus, etc. However, NOP receptors are unlike the other members of the opiate receptor family in that NOP receptor agonists block opiate analgesia when administered i.c.v. while having antinociceptive activity when administered intrathecally. In addition, NOP receptor agonists appear to be more effective rather than less effective in chronic pain states. This is surprising since NOP receptor mRNA decreases in DRG and anterior cingulate cortex (ACC) and NOP receptors decrease in certain spinal cord laminae in spinal nerve ligated mice. Using NOP-eGFP(+/+) mice, immunohistochemical experiments will be carried out to better understand the circuitry and the cellular localization in brain, spinal cord, and DRG that leads to these unusual properties of N/OFQ and other NOP receptor agonists. These will be correlated with behavioral experiments subsequent to microinjections into specific brain regions to understand how NOP receptor activation modulates the sensory as well as the affective component of pain. Specific Aim 1 will carefully examine the location of NOP-eGFP receptors in DRG as well as determine the pain modalities (heat, cold, touch) attenuated by systemic administration of NOP agonists and antagonists and determine how these parameters change during chronic neuropathic and inflammatory pain. Specific Aim 2 will examine spinal cord NOP-eGFP expression as well as characterize spinal projections both to the brain and to the periphery. These results will be compared with the effects of NOP agonists on different pain modalities after intrathecal administration in sham and neuropathic mice. Specific Aim 3 will examine neuropathic pain-induced changes in NOP-eGFP receptor levels in brain with particular emphasis on regions involved in the sensory (PAG) and affective (ACC) components of pain. Direct injections of NOP receptor agonists and antagonists into these brain regions will be used to better understand the NOP receptor-related circuitry that modulates thermal, tactile, and emotional pain through these brain regions. These experiments will clearly identify the role of NOP receptors in acute and chronic thermal and tactile pain, allodynia and hyperalgesia.

Abstract Inflammatory and anti-inflammatory cytokines contribute to neuronal hyperexcitability in pain transmission pathways. One of the inflammatory cytokines, Interleukin-1 (IL-1), is involved in many neuroimmune responses. In particular, it has been demonstrated that IL-1 induces acute pain, reduces morphine analgesic activity, is involved in tolerance development, and is necessary for chronic neuropathic and inflammatory pain. This has been studied in many ways, including use of an endogenous IL-1 receptor antagonist, and the use of IL-1 and IL-1R knockout mice. The problem with these experiments from a mechanistic standpoint is that IL-1 receptors are found endogenously on a variety of cell types in the brain, including astrocytes, microglia, endothelial cells, and neurons, and global knockout experiments can't define the cell types that mediate the actions of IL-1. The development of novel genetic models, in which the IL-1 receptor, IL-1R1, can be reciprocally knocked out and restored (IL-1R1r/r) from a global knockout, into individual cell types in the brain, spinal cord, and dorsal root ganglion permits a much more specific identification of the actions of IL-1 with respect to pain. Specific Aim 1 will study morphine analgesia and tolerance development in 5 mouse genotypes, wild type, global IL-1R1 KO, and mice in which the IL-1R1 receptor is restored selectively into neurons, endothelial cells, and astrocytes. We expect to find that the global KO animals and two of the restored mouse genotypes will have more potent and prolonged morphine analgesia and reduced tolerance development, as described in the literature. Furthermore, one of the restored mouse lines will act like the wild type animals with reduced morphine activity and will exhibit tolerance development. Aim 2 will examine two chronic pain models, spinal nerve ligation and Complete Freund's Adjuvant, using the same mouse genotypes, to determine which cell type mediates the development of chronic pain. Aim 3 will develop a new mouse model by crossing the IL-1R1r/r mice with c-Fos-Cre/ERT2 (TRAP2) mice. With this new genetic model, IL-1R1 will be restored only in mice that have been subjected to some pain stimulus. The use of these novel genetic models will pinpoint the actions of IL-1 with respect to opioid analgesia and pain to specific cell types and develop a collaboration that will be able to determine the mechanisms of IL-1 actions, in future R01 applications.