Bradley K. Taylor - US grants

DESCRIPTION: (Applicant's Abstract) Previous studies found that hypertensive SHR rats showed smaller pain responses in acute pain models and suggested that blood pressure control systems can attenuate the processing of brief nociceptive signals. By contrast, preliminary studies found that SHR rats show exaggerated nociceptive responses in a model of inflammatory pain (the formalin test). To test the hypothesis that hypertension decreases nociception in tests of acute pain, and increases nociception in tests of persistent pain, AIM 1A will evaluate nociceptive responses in awake, unrestrained rats in two acute models of nociception and in the formalin test (a short-duration model of persistent inflammatory pain) after: 1) Reduction of blood pressure in SHR rats; and 2) Increases of blood pressure in normotensive rats. Whereas inflammatory pain in the formalin test lasts one hour, inflammatory pain in an arthritic model and neuropathic pain in a spinal nerve ligation model is manifested by hyperalgesia that lasts days to weeks. AIM 1B will evaluate nociception in these models using SHR rats and other experimental models of hypertension. Naloxone eliminates the difference in acute pain sensitivity between normotensive and SHR rats, indicating that tonic endogenous opioid activity is greater in the SHR rat. To test the hypothesis that opioids regulate inflammatory hyperalgesia in the hypertensive rat, AIM 2 will evaluate the effects of selective opioid receptor agonists and antagonists on persistent pain in the SHR rat and other experimental models of hypertension. Sympathetic activity is a crucial determinant of blood pressure, and may also exacerbate the inflammatory hyperalgesia associated with tissue injury. Based on preliminary results obtained with a short-acting opioid agonist, it was concluded that peripheral mechanisms during acute nociception modulate the temporal profile of persistent nociception. To test the hypothesis that hypertension contributes to the magnitude and temporal profile of inflammation, AIM 3A will evaluate the effects of opioids on plasma extravasation and prostaglandin content in multiple models of hypertension. Nociception and cardiovascular systems are also controlled by supraspinal sites, notably the noradrenergic locus coeruleus (LC). Since activity of the LC is depressed in the SHR rat and since activation of the LC is antinociceptive, the exaggerated nociceptive responses in the SHR may reflect abnormal functioning of the LC. To test this hypothesis, AIM 3B will use a novel neurotoxin to selectively lesion the noradrenergic neurons of the LC, followed by evaluation of arterial pressure and nociception. The results of these studies will facilitate our understanding of the mechanisms that contribute to chronic pain in the hypertensive patient.

DESCRIPTION(provided by applicant): Pathological pain often manifested as hypersensitivity to noxious (hyperalgesia) and non-noxious (allodynia) stimuli, is associated with long-term changes in the neurochemical modulators of spinal nociceptive transmission. For example, nerve injury profoundly up-regulates the synthesis of NPY in DRG neurons and the spinal release of neuropeptide Y (NPY), and inflammation increases the number and distribution of dorsal horn neurons containing Y I receptor rnRNA. In studies in our laboratory designed to address the functional consequences of these changes, it was discovered that NPY inhibits nociceptive behavior in well-established animal models of acute inflammation (intraplantar formalin injection), arthritic pain (complete Freund?s adjuvant) and peripheral neuropathic pain (Seltzer model of nerve injury). Based on these data, the long-term objective of this project is to test the overall hypothesis that inflammatory or sensory nerve injury increases presynaptic and postsynaptic elements of NPY signaling at the dorsal horn, thereby inhibiting pronociceptive neurotransmission, and ultimately decreasing allodynia and hyperalgesia. To test the sub-hypothesis that NPY inhibits allodynia and hyperalgesia in animal models of inflammatory and neuropathic pain, Specific Aim #1 will determine the contribution of Yl or Y2 receptors to the actions of NPY using new receptor subtype-selective antagonists and deletion mutant mice lacking the NPY, Yl or Y2 gene. To evaluate the effects of inflammation or nerve injury on NPY release and NPY Y1 receptor expression, and to correlate these with the magnitude of nociceptive behaviors. Specific Aim #2 will use in vivo microdialysis and immunohistochemistry to assess the effects of injury on NPY content in cerebrospinal fluid from the lumbar intrathecal space, Yl-like immunoreactivity in nociceptive regions of the dorsal horn, and nociceptive behavior at multiple time-points. To test the hypothesis that NPY inhibits spinal nociceptive transmission, Specific Aim #3 will evaluate the effects of intrathecal NPY and/or newly-developed NPY receptor antagonists on the activity of spinal nocicresponsive neurons (using c-fos immunocytochemistry) and the spinal release of substance P. The latter will be evaluated with in vivo microdialysis, as well as with quantification of neurokinin 1 receptor internalization, which determines neurokinin release in terms of receptor activation, laminar distribution, and target neuron morphology. An understanding of the endogenous compensatory mechanisms that contribute to the intrinsic decay of aberrant nociceptive signaling may help us to identify new drugs for the treatment of pathological pain.

DESCRIPTION (provided by applicant): The brain exerts a powerful influence on the spinal transmission of pain signals. Recent evidence from our laboratory suggests that the locus coeruleus (LC), an important brainstem noradrenergic center, facilitates the mechanical and thermal hypersensitivity induced by injury to peripheral nerves. These findings led to the hypothesis that in the setting of peripheral nerve injury, descending facilitatory influences from the LC are required for the expression of nerve injury-induced pain (Aim #1), in part by increasing nociceptive processing at the dorsal horn (Aim #2). AIM #1a will test the hypothesis that disruption of synaptic activity in the LC with the microinjection of either a local anesthetic (lidocaine) or a synaptic inhibitor (cobalt) will decrease the tactile and cold hypersensitivity that develops following transection of the tibial and common peroneal branches of the sciatic nerve, leaving the sural nerve intact. Aim #1b will test the hypothesis that irreversible destruction of LC neurons with a noradrenergic neurotoxin (DSP-4) before sham or nerve injury will prevent or reduce the development of injury-induced hypersensitivity. Aim #2 will use similar strategies in combination with measurement of stimulus-evoked expression of c-fos in the dorsal horn to test the hypotheses that disruption of LC function will: (a) prevent or (b) reverse nerve injury-induced spinal nociceptive processing. This two-year R21 study seeks to firmly establish whether the LC exerts facilitatory influences that contribute to the expression of neuropathic pain. The results achieved will provide the basis for an R01 application to further investigate the supraspinal network mediating descending facilitation. Increased understanding of the supraspinal noradrenergic mechanisms underlying chronic neuropathic pain may help to identify entirely new classes of analgesic drugs that may work by directly or indirectly blocking descending facilitation.

PROJECT DESCRIPTION Peroxisome proliferator-activated receptor gamma (PPAR ¿) is well-characterized as a key target of the thiazolinedione (TZD) class of anti-diabetic drugs. Our preliminary results describe the existence of PPAR ¿ mRNA and protein in the dorsal horn. Furthermore, we demonstrate that the mechanical and thermal hypersensitivity associated with inflammation or nerve injury was rapidly reduced by intrathecal administration of rosiglitazone (a TZD) and 15d-PGJ2 (an endogenous PPAR ¿ ligand) in a dose- and PPAR ¿-dependent manner, and by systemic administration of pioglitazone, a BBB-permeant, FDA-approved ligand. The central hypothesis of this proposal is that ligand-dependent activation of PPAR¿ in the dorsal horn decreases injury- induced activation of spinal neurons and glia that then dampens behavioral signs of inflammatory and neuropathic pain. The objective of the present application is to identify the mechanisms underlying PPAR- mediated inhibition of inflammatory or neuropathic pain, with a focus on pioglitazone. The long-term goal of our research program is to harness the therapeutic potential of PPAR signaling to alleviate chronic pain in humans. AIM 1 will test the hypothesis that PPAR ¿ agonists reduce allodynia and hyperalgesia. We will use pharmacological agents and nervous system-specific PPAR ¿ deletion mutants to determine the contribution of PPAR ¿ signaling in the spinal cord to the induction and maintenance of chronic pain. First, we will determine whether single intrathecal or systemic administration of pioglitazone and 15d-PGJ2 reduces behavioral signs of inflammatory and neuropathic pain. We predict that their analgesic actions will be blocked with PPAR ¿ antagonists. Second, we will determine whether chronic intrathecal or oral administration of PPAR ¿ agonists, begun before or after tissue or nerve injury, reduces behavioral signs of inflammatory and neuropathic pain. Third, we predict that anti-allodynic actions will not occur in mice with neuron-specific PPAR ¿ knockdown. AIM 2 will test the hypothesis that PPAR ¿ ligands reduce injury-induced activation of neurons and microglia in the dorsal horn. Somatosensory stimulation of injured rats induces the expression of the immediate early gene, c-fos, in the superficial laminae of the dorsal horn. We predict that intrathecal pioglitazone will reduce inflammation- and nerve injury-induced expression of Fos immunoreactive neurons, as well as the expression of OX-42, a marker of microglia activation. AIM 3 will test the hypothesis that endogenous PPAR ¿ systems tonically inhibit allodynia. First, we will determine if PPAR¿ expression occurs in neurons and/or glia that are activated during pain. Second, in an extension of Aims 1-2, we will determine whether receptor antagonists and genetic deletion increase allodynia and neuronal/glial activation. If affirmative, then we will determine whether the PPAR¿ signaling elements co- vary with allodynia. At various times after nerve injury or persistent inflammation, we will evaluate: behavior and A) PPAR ¿ mRNA and protein; B) phosphorylated PPAR ¿; and C) 15d-PGJ2 levels with LC/MS/MS.

DESCRIPTION (provided by applicant): The long-term objectives of our 12 federal grants (R01-type) are to address important and unique problems in neuroscience and cardiac physiology, including a better understanding how living cells in the nervous system and heart respond to drugs or other changes in the environment. The current application is in response to a significant need of our 8 Major Users to obtain confocal images and electrophysiological recordings, often simultaneously, from live neural, sensory and cardiac cells or tissue slices. Specific needs include the following major functional capabilities: Confocal imaging of Ca2+ transients and fluorescent markers in live tissue slices;ratiometric Fura-2 imaging of Ca2+ transients;high temporal and spatial resolution of Ca2+ imaging during concomitant patch clamp recordings of ion channel activity;and UV Flash photolysis of caged compounds. All of the Major Users have extensive experience with live cell calcium imaging, electrophysiology, and/or confocal microscopy. Collectively, we have published over a hundred papers using one or more of these techniques, including 15 papers that simultaneously used functional confocal imaging and electrophysiological recording in live cells. An inherent problem of laser confocal microscopes is the very intense excitation light, leading to substantial dye decomposition (photobleaching) during live-cell imaging. The proposed Olympus BX- DSU Live-Cell Disk Scanning Imaging System overcomes this problem with optimization of signal detection. It uses spinning disk confocal technology with a new generation, super-cooled EM-CCD camera to generate images with high temporal and spatial resolution. The broad spectrum light source produces substantially less photobleaching than do lasers. Furthermore, this system adds a UV flash illuminator for photolysis of photoactivatable (caged) compounds, complete integration of patch-clamp instrumentation for concomitant electrophysiological recordings, and an upright stand preferred for tissue slice work. The result is a very cost-effective and flexible solution that responds to all described needs of the Major Users. No other instrument at or near the University of Kentucky can provide this constellation of features. The University will provide appropriate space, salary support for experienced technical staff, and maintenance for the system. The system will be set up within a large well-established Imaging Facility at the University of Kentucky by experts in scanning disk technology and electrophysiology. This system will allow the Major Users to continue to achieve new discoveries towards the better understanding and treatment of chronic pain, obesity, auditory disorders, olfactory disorders, pulmonary disease, consequences of brain injury, and cardiovascular disease.

DESCRIPTION (provided by applicant): Severe tissue injury generates central sensitization (increased responsiveness of CNS nociceptive neurons to normal or sub-threshold afferent input) that contributes to hyperalgesia. Latent sensitization (LS) is a silent form of central sensitization that persists after tissue has healed and overt signs of hyperalgesia have resolved. LS can be revealed with opioid receptor antagonists or inverse agonists that rekindle or reinstate hyperalgesia. Thus, pain remission during LS is likely maintained by tonic opioid receptor activity that masks the pronociceptive components of LS. LS is important because it primes nociceptive systems such that, when inhibitory systems fail, a pain episode ensues. A key first step in understanding LS is to demonstrate the translational significance, and we now show that the opioid receptor inverse agonist, naloxone, can reinstate experimental pain when delivered 1 wk after the resolution of secondary hyperalgesia following first degree thermal injury. Specific Aim 1 tests the hypothesis that burn or surgery triggers LS and long-term opioid analgesia in humans. To further study the neurobiological mechanisms of LS, we will also use a mouse model that is long-lasting, powerful, broad range, repeatable, and translates to human studies. We found that mu opioid receptor (MOR) inverse agonists reinstated behavioral and molecular signs of hyperalgesia, even when administered months after tissue injury, and this required NMDA receptor activation of adenylyl cyclase type 1 (AC1). Our results are important because they suggest that any event, such as stress, that interferes with MOR analgesia during LS will lead to relapse of hyperalgesia in chronic pain syndromes in humans. Specific Aim 2 tests the hypothesis that MOR constitutive activity (MORCA) and/or activation of MOR, delta (DOR), or kappa (KOR) receptors by opioid peptides in the DH or rostroventromedial medulla maintains endogenous analgesia and thereby restricts LS to a state of pain remission. Specific Aim 3 determines the extent to which MORs inhibit spatially coordinated neural activity in the DH (using an innovative 64-channel field recording system) and synaptic strength in presynaptic terminals of primary afferent nociceptors or on DH neurons (using patch clamp electrophysiology) during LS. Specific Aim 4 then tests whether MORs specifically inhibit spinal NMDA receptor subunits (GluN2A or GluN2B) and/or Epac1 (exchange protein directly activated by cAMP, recently found to contribute to peripheral pain senstization) to block pain during LS. Completion of this project will bring us closer to our long-term goal of alleviating chronic pain b either: a) facilitating endogenous opioid analgesia, thus restricting LS within a state of remission; or b) extinguishing LS altogether, for example with a selective AC1 or Epac1 inhibitor. Our general model and hypothesis shares similarities with the concept of allostasis: a pathologically-elevated balance between opposing processes (MOR and LS) that facilitate each other by mutual feedback. Our long-term vision is a new conceptual strategy for chronic pain therapy, to restore homeostasis, where there is neither central sensitization nor MOR compensatory responses.

PROJECT SUMMARY Neurons in the central amygdala (CeA) contribute to pain modulation. However, their contribution to the sensory- discriminative and/or emotional-affective dimensions of chronic pain, nor their neurochemical modulation, are understood. Our preliminary data using slice recordings and behavior provide a compelling premise for the idea that drugs targeting the receptors for the bioactive lysophospholipid, sphingosine-1-phosphate (S1P) act within the CeA to inhibit inflammatory and neuropathic pain. This sets the stage for our long-term goal to understand how lipid signaling controls the supraspinal control of acute and chronic pain. The objectives of this proposal are to: determine neurophysiological changes to molecular specific CeA neurons in multiple models of pain (Aim 1), elucidate the effects of S1P signaling on the intrinsic and synaptic excitability of defined subpopulations of CeA neurons (Aim 2), and determine if S1PR agonism in the CeA is analgesic in models of inflammatory and neuropathic pain (Aim 3). In Aim 1, we use transgenic mouse lines, electrophysiology, and optogenetics to test the hypotheses that tissue or nerve injury reduces excitability of specific subclasses of CeA neurons based on their molecular profile. In Aim 2, we test the hypotheses that activation of S1P signaling increases the excitability and synaptic connectivity within a population of molecularly distinct CeA neurons. In Aim 3, we use intracranial drug infusions and chemogenetics to test the hypothesis that activation of S1P receptors in the CeA attenuates inflammatory and neuropathic pain via a specific subtype of CeA neuron. Experimental support of these concepts will facilitate the development of existing (e.g. FDA-approved fingolimod) and novel S1PR compounds for the treatment of chronic pain.