Zhizhong Z. Pan - US grants

DESCRIPTION: Opioids are the most powerful analgesics currently used for the treatment of pain, but repeated administration induces tolerance which significantly decreases the analgesic effect of opioids. The general goal of this project is to elucidate the contribution of different types of opioid receptors to the mechanisms of opioid tolerance. The nucleus raphe magnus (NRM) in the medulla is critically implicated in opioid analgesia through its direct projections to the spinal dorsal horn, the first relay for pain transmission. Our previous studies have shown how agonists of the mu-opioid receptor act in the NRM to produce analgesia. Recently we have demonstrated the neural mechanism by which kappa-opioid receptor agonists in the NRM oppose the mu-receptor-mediated analgesia. The hypothesis for the current project is that opioid tolerance results from a functional reduction of the mu-receptor mediated actions and an up-regulation of other endogenous anti-mu systems such as the kappa-receptor system and the GABA transmission system, a major target of inhibitory actions of analgesic opioids. Using the NRM as a model system, the proposed studies are designed to determine the functional changes both in the synaptic properties of NRM neurons and in the actions mediated by mu- and kappa-receptors in opioid-tolerant rats versus opioid-naive rats. Both intracellular/whole-cell patch clamp recording in NRM slices in vitro and NRM microinjection in freely-moving rats in vivo will be used to characterize the cellular mechanisms of the adaptive changes and their behavioral significance in animals. This research will significantly expand our knowledge of the neural mechanisms for opioid tolerance and contribute to our efforts to improve the treatment for chronic pain and other chronic opioid-related problems.

[unreadable] DESCRIPTION (provided by applicant): Opioid actions are mediated primarily by three classic opioid receptors: mu, delta and kappa. Currently, opioid analgesics, mainly mu-opioid receptor (MOR) agonists such as morphine and fentanyl, are still the most effective pain relievers available and are widely used for the treatment of many forms of chronic pain. However, repeated use of opioids causes analgesic tolerance and physical dependence as a result of prolonged stimulation of MOR, which significantly reduces the analgesic efficacy of MOR agonists and limits their long-term clinical use for chronic pain management. In contrast to the well-characterized MOR actions, the function of delta-opioid receptors (DOR) is much less clear. DOR agonists produce little to weak analgesic effect both in animals and in humans under normal conditions. Recent studies including ours have demonstrated that the weak DOR effect is primarily due to the predominant intracellular localization of non- functional DOR in normal conditions, and more interestingly, the intracellular DOR is trafficked to plasma membrane and becomes functional after several forms of long-term neurochemical stimulation, including chronic morphine exposure. However, the molecular signal and mechanisms mediating the exocytotic DOR trafficking are completely unknown at present. In our preliminary studies exploring the issue recently, we have discovered that nerve growth factor (NGF), a neurotrophin with diverse functions in neuronal growth and differentiation, can trigger and maintain similar DOR trafficking and induce functional DOR in pain-modulating brainstem neurons under normal conditions. This represents a novel NGF function that has not been reported to date. Therefore, this proposal is aimed to identify the molecular mechanisms mediating the NGF- and chronic morphine-induced DOR trafficking and analgesic function, and to determine the synaptic and behavioral interactions of the emergent DOR with MOR in regulation of both central synaptic activity and pain inhibition in the context of chronic morphine. Findings from this project will provide the first evidence for NGF as a critical molecular mediator of membrane trafficking of G-protein-coupled receptors including DOR in central neurons, and will demonstrate the potential for DOR agonists as effective analgesics for chronic pain under chronic opioid conditions with decreased MOR efficacy. PUBLIC HEALTH RELEVANCE Repeated use of opioid analgesics, the current pain reliever of choice, causes analgesic tolerance and physical dependence, which significantly reduce their analgesic efficacy and limit their clinical use for adequate control of moderate to severe pain. This research proposal investigates the cellular and molecular mechanisms underlying a new analgesic function of different opioid compounds in brain cells after repeated opioid administration. These studies may provide neuropharmacological evidence for using these opioid compounds as more effective analgesics under an opioid-tolerant and dependent state. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chronic pain due to various health conditions is a common clinical problem in patients. Currently, opioid-based analgesics remain the most effective pain reliever and have been increasingly used for pain control in recent years. However, repeated use of opioids causes aversive side effects, including those causing opioid misuse and addiction due to strong rewarding properties of opioids. In fact, as medical use of opioids is increased, so is misuse of prescription opioids and opioid addiction. Concerns over non-medical misuse of opioids and risk of developing opioid addiction have significantly limited optimal use of opioid therapies in non-malignant pain patients. Nevertheless, there are few preclinical or clinical studies concerning the effects of repeated opioids for pain control on the risk of developing compulsive behaviors of opioid misuse and addition. One of the key issues in question is how repeated opioid treatment for pain control alters individual's response or sensitivity to opioid's rewarding effect, the pharmacological driving force for compulsive behaviors in opioid misuse and addiction. Recent studies including ours have shown that prolonged opioid exposure and chronic pain both induce new functional delta-opioid receptors (DOR) in pain-modulating brain areas, leading to enhanced opioid analgesia. However, the role of DOR in opioid reward is largely unknown at present. In our pilot study in animal models, we have found that prior exposure to repeated opioids increases the sensitivity to the rewarding effect of subsequent opioids. This intriguing and provoking finding prompted us to explore this important issue in this R21 proposal. Based on our preliminary findings, this research proposes two specific aims to focus on two foremost issues: 1) how do presence of pain and repeated opioid exposure affect reward sensitivity to subsequent opioids? And 2) does DOR play a critical role in the changed reward sensitivity? Aim 1 studies will use rat models of persistent pain and behavioral measures of opioid reward to determine whether pain exacerbates repeated opioid-induced increase in reward sensitivity. Aim 2 studies will determine whether new functional DOR is recruited by repeated opioids and by persistent pain in a reward-related brain area, resulting in DOR-mediated increase in reward sensitivity. Combining synaptic analysis and behavioral measures, these proposed studies may identify a novel function of DOR in the adaptive properties of brain's reward function and provide a fundamental platform for future studies on the cellular and molecular mechanisms underlying DOR-mediated changes in reward sensitivity. Understanding the impact of chronic pain and prior opioid exposure on the rewarding property of opioids will provide insights necessary for the development of improved opioid therapies that produce optimal pain control while minimizing the risk of opioid misuse and addiction. PUBLIC HEALTH RELEVANCE: Repeated use of opioid analgesics for control of chronic pain has the potential to cause compulsive behaviors of opioid misuse and addiction due to the strong rewarding effect of opioids. This research proposal investigates how the presence of pain and repeated opioid treatment may change individual response and sensitivity to the rewarding effect of subsequent opioids, the pharmacological driving force for the compulsive behaviors. Information obtained from these studies will provide necessary insight for the development of effective opioid therapies that provide optimal pain control while minimizing opioid misuse and addiction.

DESCRIPTION (provided by applicant): Chronic pain due to various health conditions remains a common clinical problem affecting millions of people worldwide. Opioid therapies are still most effective for control of chronic pain and have been increasingly used clinically for desired and sustained pain relief. However, beside analgesic effects, opioids also have strong rewarding effects and highly addicting properties after repeated use, leading to opioid dependence and addition. In fact, there have been increased case reports of non-medical opioid misuse, abuse and incidences of opioid addiction among chronic pain patients under a long-term opioid therapy. This trend is significantly weighing on physicians'decisions on prescribing opioids of long-term, thus limiting the optimal use of opioid analgesics for maximum pain control. Nevertheless, little is known currently about how an existing pain would change the reinforcing and addicting properties and consequently abuse potential of opioids through their functional interactions. Focusing on neurons in the central amygdala, a brain area increasingly recognized as crucial for emotional modulation of both chronic pain and drug addiction, this project is aimed to identify synaptic adaptations specifically induced by chronic pain and by a rewarding opioid, and to determine how chronic pain- induced changes affect the rewarding properties and resultant abuse potential of opioid analgesics used for pain control through interacting adaptations on the key glutamate neurotransmission. Two common rat models of chronic pain will be used: rats with complete Freund's adjuvant-induced persistent inflammatory pain and rats with spinal nerve ligation-induced prolonged neuropathic pain. The behavioral measure of opioid reward and addiction will be achieved by using two commonly used rat models: opioid-induced conditioned place preference and opioid self-administration. Our preliminary studies in these rat models have revealed important and interesting data, which indicate that chronic pain may induce similar synaptic adaptations to those induced by repeated opioids in amygdala neurons and facilitate the reinforcing effects of opioids and related behaviors. Based on these preliminary findings, our central hypothesis is that chronic pain facilitates synaptic adaptations induced by rewarding opioids through glutamate neurotransmission, exacerbating opioid-seeking and intake behaviors. To test this hypothesis, this project will employ multidisciplinary approaches to determine the impact of chronic pain on opioid addiction and underlying synaptic mechanisms in the amygdala at cellular, molecular and behavioral levels. It is traditionally believed that pain would serve as a negative emotional stimulus against the rewarding properties of opioids, but supporting experimental evidence is lacking. Therefore, this project is expected to provide conceptually new information regarding the functional interactions between chronic pain and opioid reward. Findings of this project will help to identify pharmacological targets for the development of new opioid therapies that minimize abuse potential of opioid analgesics while maximizing their analgesic effects. PUBLIC HEALTH RELEVANCE: Chronic pain, a common health condition affecting millions of people, requires long-term use of opioids, the most effective analgesics currently available, for efficient and sustained pain control. However, repeated use of opioids causes drug dependence and sometimes addiction, characterized by compulsive behaviors of drug misuse and abuse, due to the strong reinforcing effects of opioids. This project investigates how an existing pain condition changes the reinforcing effects of opioids and consequently the risk of developing behaviors of opioid misuse and addiction. The goal of this project is to identify pharmacological targets to reduce the abuse potential of opioids taken for pain control while maintaining their analgesic effects.

? DESCRIPTION (provided by applicant): It is well documented in clinical reports that some individuals are more vulnerable than others to developing chronic pain with considerable variability in response to treatment with pain drugs. However, this individual pain vulnerability and differential drug response are rarely addressed in preclinical research on animal pain models. The individual pain vulnerability is thought to be largely mediated by epigenetic changes that are induced by prior stressful events and pain experience, and lead to differential responses to subsequent pain stimuli and analgesic drugs. This proposal is designed to investigate the role of DNA methylation, a long- lasting form of chromatin modification that mediates gene repression, in individual variance and vulnerability to developing chronic pain. In preliminary studies, we found that animals display considerable variance in developing negative emotions of affective pain under chronic pain and stress conditions; and the vulnerability to developing the negative emotions is closely associated with downregulation of DNA methyltransferases 3a (Dnmt3a) that catalyzes de novo DNA methylation in pain- and emotion-regulating central amygdala (CeA). We propose multidisciplinary studies to test our central hypothesis that pain- and stress-induced Dnmt3a downregulation and associated DNA hypomethylation in central amygdala is a key process that leads to increased individual vulnerability to developing chronic pain. Aim 1 will systematically characterize the individual variance and vulnerability in rat and mouse models of orofacial pain and establish a functional link between pain and stress by determining whether animals vulnerable to chronic pain are also vulnerable to stress in developing the negative emotions of affective pain. Aim 2 will determine the functional role of CeA Dnmt3a in the individual vulnerability by associating the downregulation of CeA Dnmt3a with the individual vulnerability and by mimicking the effect of pain and stress with functional knockdown of CeA Dnmt3a. Aim 3 will determine molecular manipulations that reduce the individual vulnerability by reversing pain-induced downregulation of CeA Dnmt3a and examine the function of pain inputs onto CeA neurons in induction of the pain vulnerability. These proposed studies are expected to provide key knowledge on the role of DNA methylation in the individual vulnerability and to move the field forward with potential molecular targets for the future development of individually targeted pain medicine.

PROJECT SUMMARY More than 100 millions of people suffer from daily chronic pain worldwide. Opioids remain the mostly prescribed medications to treat chronic pain. However, long-term use of opioids causes paradoxical hyperalgesia and in addition, opioids produce strong rewarding effect and can cause opioid abuse and addiction, which may have contributed to the current opioid epidemic. Due to these severe side effects of opioids, the analgesic efficiency and clinical benefits of long-term opioids for chronic pain have been increasingly questioned in recent years. Therefore, there is an urgent need for new drugs or therapeutic options to treat chronic pain, which requires better understanding of chronic pain-induced changes in the brain without opioids and in the context of long-term opioids. However, it is still unclear what changes in the brain that mediate the development of chronic pain from acute pain and how chronic pain may change responses to opioid reward for the altered liability of opioid abuse under chronic pain. Our preliminary studies have found that Dnmt3a, a DNA methyltransferase that catalyzes DNA methylation for gene repression, is significantly downregulated in the brain in a time-dependent manner during the development of chronic pain. Moreover, repeated opioid treatment also downregulates Dnmt3a. Therefore, this proposal will investigate whether Dnmt3a acts as a key protein in the brain for the development of chronic pain and whether Dnmt3a could be a novel epigenetic target for the development of new drugs and therapeutic options for the treatment of chronic pain. We propose three specific aims to test our central hypothesis that persistent pain induces adaptive Dnmt3a decrease in key pain-modulating brain regions, which results in DNA hypomethylation and aberrant neuronal hyperactivity, leading to the development of chronic pain and increased sensitivity to opioid reward. Aim 1 will identify Dnmt3a as a key epigenetic protein in three representative pain-modulating brain regions for the development of chronic pain. Aim 2 will manipulate Dnmt3a levels in the brain regions to validate Dnmt3a as a potential drug target for the treatment of chronic pain. Aim 3 will identify any abuse liability of Dnmt3a upregulation as a treatment option and how it affects the rewarding effect of opioids for opioid abuse. These studies are expected to reveal critical roles of brain Dnmt3a in the development of chronic pain and provide fundamental insight for Dnmt3a as the target of novel therapeutic options that inhibit chronic pain development while decreasing abuse liability of opioids.