Volker Neugebauer - US grants

DESCRIPTION: (adapted from applicant's abstract) Persistent pain has a strong emotional component. As part of the limbic system the amygdala plays a key role in the affective and autonomic aspects of behavior, the evaluation of the emotional significance of sensory stimuli, emotional learning and memory, and stress responses. A novel pathway from the pontine parabrachial region to the amygdala has been described and may provide an important link between spinal and brainstem regions that receive pain input with higher brain centers including the central nucleus of the amygdala (CeA). The role of the amygdala and CeA neurons, in particular, in persistent pain is not clear and is the subject of this proposal. The effects of persistent pain on the activity of CeA neurons will be studied using the kaolin/carrageenan inflamed knee joint model in the rat. Extracellular single unit recording in vivo and whole cell voltage- and current-clamp recordings from brain slices in vitro are used to examine functional properties of CeA neurons and to assess the influence of glutamate receptor subtypes on cellular responses. Specific Aim 1 compares the nociceptive and non-nociceptive responses of CeA neurons before and during persistent inflammatory pain in vivo. In parallel experiments in vitro, synaptic transmission and membrane properties at two sources of input to the CeA, the pontine parabrachial complex-CeA (pPB-CeA) input and the basolateral amygdala-CeA (BLA-CeA) are analyzed. Specific Aim 2 determines the involvement of glutamate receptors (ionotropic and metabotropic) in the inflammation-enhanced nociceptive processing in vivo. In parallel experiments in vitro, the influence of glutamate receptor subtypes is analyzed for effects on synaptic transmission including pre- versus postsynaptic influences and membrane properties after inflammation.

DESCRIPTION (provided by applicant): The multidimensional character of pain presents a therapeutic challenge that calls for better understanding of higher brain functions that regulate its complex emotional-affective and cognitive aspects (NIH PA-10-006). This project will continue to provide valuable insight into these higher brain mechanisms. Neuroplasticity in the amygdala, an emotional brain center, is now recognized as a key factor in the emotional-affective dimension of pain. Amygdala dysfunction in pain also causes cognitive deficits by impairing medial prefrontal cortex (mPFC) function. For that reason, control of amygdala activity is a desirable therapeutic goal in pain management. Here we advance the novel concept that cognitive deficits and impaired cortical output result in the persistence of pain and its emotional affective component. Based on our previous studies and preliminary data we propose the novel hypothesis that the cognitive control system for negative emotions consists of mPFC-driven inhibition of excessive amygdala activity, which is impaired in pain but can be restored for pain relief. Three Specific Aims (SAs) will determine synaptic and cellular mechanisms and behavioral consequences of a vicious cycle in which abnormal deactivation of the mPFC in a rat model of arthritis pain causes failure of mPFC-driven inhibition of amygdala output. Cortical control of output neurons in the central nucleus of the amygdala (CeA) requires activation of inhibitory neurons in the intercalated cell mass (ITC) of the amygdala. The goal is to identify pharmacological targets that can restore cortical control of amygdala dysfunction in pain. These include metabotropic glutamate receptor mGluR5 to activate mPFC neurons, cannabinoid receptor CB1 to release excessive synaptic inhibition of mPFC neurons, and novel neuropeptide S (NPS) to activate selectively ITC cells that inhibit CeA neurons (feedforward inhibition). Behavioral experiments (SA1) will test the hypothesis that restoring mPFC-amygdala control pharmacologically will decrease pain and shorten its duration. Nocifensive, emotional-affective and cognitive behaviors will be measured. Electrophysiology in vivo (SA2) will examine dysfunction of the mPFC-ITC-CeA pathway in pain, measured as mPFC and ITC deactivation and CeA hyperactivity. Pharmacological rescue strategies will be tested. SA1 and SA2 will use stereotaxic and systemic drug applications. Patch-clamp studies in brain slices (SA3) will determine pain-related changes of synaptic and cellular modulation of mPFC output by mGluR5 and CB1 and of feedforward inhibition of CeA neurons by NPS. Patch-clamp analysis will clarify the usefulness of pharmacological targets to restore normal transmission at individual synapses of the mPFC-ITC-CeA circuitry. These conceptually novel studies will identify cortico-amygdala control deficits as an important mechanism of persistent pain. They will provide novel targets to restore cognitive control functions for pain relief. The mechanistic analysis of higher brain functions and drug targets in pain will boost basic science knowledge required for evidence-based medicine and provide new and improved strategies for pain management.

1,1,1-Trifluoro-2-Chloro-2-Bromoethane; 1-(3,4-dihydroxyphenyl)ethane-1,2-diol; 2'-Nor-2'-deoxyguanosine; 2'NDG; 2-Amino-1,9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-6H-purin-6-one; 21+ years old; 3,4-dihydroxyphenylglycol; 6H-Purin-6-one, 2-amino-1,9-dihydro-9-((2-hydroxy-1-(hydroxymethyl)ethoxy)methyl)-; 7-(hydroxyimino)cyclopropan(b)chromen-1a-carbxoylic acid ethyl ester; 9-[(1,3-Dihydroxy-2-propoxy)methyl]guanine; Abdominal Pain; Action Potentials; Active Oxygen; Adjuvant Therapy; Adult; Affective; Affective Disorders; Agonist; Amygdala; Amygdaloid Body; Amygdaloid Nucleus; Amygdaloid structure; Animals; Area; Arthritis; Autonomic pain; Behavior; Behavioral; Biochemistry; Biochemistry and Pharmacology; Biochemistry and Pharmacology Cancer Activity; Blood Coagulation Factor IV; Blotting, Western; Body Tissues; Brain; Brain Stem; Brainstem; CPCCOEt; Ca Release Channel-Ryanodine Receptor; Ca++ element; Calcium; Calcium-Ryanodine Receptor Complex; Cell Communication and Signaling; Cell Nucleus; 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[unreadable] DESCRIPTION (provided by applicant): Cognitive impairment such as the inability to make advantageous decisions is one of the consequences of persistent pain but the underlying neural mechanisms are not known (NIH PA-06-544). The role of the prefrontal cortex in cognitive function, including decision-making and avoidance of emotion-based risky choices, is well established. Impaired prefrontal cortical function was recently shown in pain patients with cognitive deficits. A major source of input to the mPFC is the basolateral amygdala (BLA), a key element in the emotional-affective amygdala circuitry. Our previous studies showed enhanced synaptic transmission from the BLA to the central nucleus of the amygdala (CeA) in an arthritis pain model. We hypothesize that the BLA is an important structure underlying pain-related emotional-affective behavior (through projections to the CeA) and cognitive deficits (through connections with the mPFC). To determine the role of the BLA-mPFC interaction in cognitive effects of pain, we will use a multidisciplinary approach that combines behavior, systems and cellular electrophysiology and pharmacology. We will continue to use our well-established pain model, the kaolin/carrageenan-induced knee joint arthritis. The following specific hypotheses will be tested: 1. Restoring normal function in the BLA and mPFC improves pain-related decision-making deficits. 2. Pain-related sensitization of BLA projection neurons inhibits mPFC neurons. 3. Pain leads to synaptic plasticity in the BLA and increases inhibitory transmission from the BLA to mPFC neurons. The Specific Aims are: 1. Determine if restoring normal function in the BLA (deactivation with APS, an NMDA receptor antagonist) and in the mPFC (removing inhibition with bicuculline, a GABAA receptor antagonist) improves pain-related cognitive impairment in a novel behavioral test modeled after a decision-making gambling task in humans. Arthritic and control animals decide between disadvantageous high-risk and advantageous low-risk strategies based on food reward. 2. Analyze the effect of arthritis on BLA and mPFC neurons and determine if inhibiting BLA sensitization (with APS) or disinhibiting the mPFC (with bicuculline) reverse pain-related inhibition of mPFC neurons in anesthetized rats in vivo. 3. Determine the effect of arthritis on excitatory and inhibitory synaptic transmission in the BLA and at the BLA-mPFC synapse in vitro, using whole-cell patch-clamp recordings in brain slices from arthritic and control animals. This translational research project will determine the neurobiological mechanism by which pain produces clinically documented cognitive deficits. If our hypotheses are correct, the proposed studies will be the first to demonstrate that the amygdala impairs mPFC function resulting in pain-related decision-making deficits. The long-term goal of this project is the better understanding of higher brain functions involved in the different pain components to improve pain management strategies and decision making. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The multidimensional character of pain presents a therapeutic challenge that would benefit greatly from a better understanding of higher brain functions that regulate its complex emotional-affective aspects. Neuropathic pain is generally believed to result from maladaptive neuroplasticity but underlying mechanisms, particularly those in higher brain centers, are not well understood. This project will focus on abnormal function of the amygdala, a brain area that is recognized as a key player in the emotional-affective dimension of pain. Our goal is to mitigate maladaptive amygdala plasticity and block the development of chronic neuropathic pain. A critical determinant, we believe, is pain-related plasticity of serotonin 5-HT2C receptor (5-HT2CR) control of corticotropin-releasing factor (CRF) signaling in the amygdala because CRF is associated with 5-HT2CR- mediated negative affective states and CRF1 receptors mediate amygdala plasticity in inflammatory pain. Here we advance the novel concept that abnormal function of 5-HT2CR in the amygdala is a critical mechanism of chronic neuropathic pain and its emotional-affective component, and is also the likely cause of the limited efficacy of selective serotonin reuptake inhibitors (SSRIs) to treat neuropathic pain. Specifically, we propose the novel hypothesis that 5-HT2CR in the basolateral amygdala (BLA, amygdala input region), drives a vicious cycle involving CRF1 receptors that results in abnormal activity in the central nucleus (CeA, output region). 5- HT2CR-driven maladaptive plasticity in the BLA-CeA circuitry plays a critical role in chronic neuropathic pain. Three Specific Aims (SAs) will determine synaptic and cellular mechanisms and behavioral consequences of manipulation of 5-HT2CR function in the amygdala in the spinal nerve ligation (SNL) rat model of neuropathic pain. Complementary pharmacological and novel viral vector knockdown strategies will be utilized in all aims for local inactivation or elimination of 5-HT2CR in the amygdala. Behavioral experiments (SA1) will determine the role of 5-HT2CR and CRF1 in the BLA in the emotional-affective component of neuropathic pain. Electrophysiology in vivo (SA2) will examine the hypothesis that 5-HT2CR in the BLA drives CRF1 activation and central sensitization of CeA output neurons. Patch-clamp studies in brain slices (SA3) will determine excitatory and (dis-)inhibitory synaptic and cellular mechanisms of plasticity in the BLA-CeA network that results from abnormal 5-HT2CR function driving persistent CRF1 signaling. Systemic application of a 5-HT2CR antagonist and SSRI in SA1 and SA2 will validate their clinical utility and viability. These conceptually novel studies will characterize the 5-HT2CR/CRF1 interaction in the amygdala as an important mechanism of chronic neuropathic pain. We will also identify strategies to eliminate or disrupt this signaling mechanism to block maladaptive amygdala plasticity and thus neuropathic pain. The mechanistic analysis of higher brain functions and drug targets in pain will boost basic science knowledge required for evidence-based medicine and provide translational strategies for pharmacotherapeutics and/or gene therapy.

Project Summary Chronic pain remains a major health care issue and presents a therapeutic challenge. A complex disorder with different dimensions, pain involves and affects neural function at various levels. And so it is not surprising that chronic pain mechanisms are still not well understood. To address this important knowledge gap we will build on our NIH-funded work (since 1999) that impacted the field by identifying neuroplasticity in the amygdala, a brain center for emotions, as a key mechanism for emotional-affective aspects of pain and pain modulation (Neugebauer, 2015). Control of amygdala activity has emerged as a desirable therapeutic goal (see Simons et al., 2014;Baliki and Apkarian, 2015), but mechanisms of abnormal excitability and amygdala output in chronic pain remain poorly understood. The proposed project will test the novel hypothesis that loss of function of small conductance calcium-activated potassium (SK) channels is a critical mechanism of uncontrolled amygdala output in neuropathic pain, which allows the abnormal persistence of pain behaviors but can be mitigated with a gene transfer based rescue strategy (AAV-mediated SK2 expression). SK channels are important regulators of neuronal excitability and hold promise as targets for neuropsychiatric and neurodegenerative disorders. More recently, they have also been implicated in the regulation of peripheral and spinal nociceptive processing. Role, regulation and therapeutic potential of SK channels in pain-related brain functions and plasticity are unknown, and the concept of SK channel dysfunction as a pain mechanism is novel. A comprehensive multidisciplinary approach will be used that integrates state-of-the-art behavioral assays, brain slice physiology, pharmacology, optogenetics, viral vector strategies, and molecular biology for mechanistic loss and gain of function analyses of SK channels in the amygdala output region (central nucleus, CeA) in the well-established spinal nerve ligation (SNL) rat model of neuropathic pain. We will use posthoc analysis of biocytin-labelled CeA neurons and a novel transgenic Crh-Cre rat model to study SK dysfunction in corticotropin releasing factor (CRF) containing CeA neurons that are known to project to brain centers for behavioral regulation (Pomrenze et al., 2015). CRF plays an important role in amygdala plasticity (Neugebauer, 2015). Aim 1 will determine the behavioral significance of loss and rescue of SK2 channel function in neuropathic pain. Sensory thresholds, emotional responses, and anxiety- and depression-like behaviors will be measured. Aim 2 will determine electrophysiological mechanisms of SK2 channel dysfunction and rescue in neuropathic pain, using patch-clamp recordings of CeA neurons in brain slices from behaviorally tested rats. Aim 3 will determine molecular mechanisms of SK2 channel dysfunction and regulation, and validate gene transfer rescue, using CeA tissue from behaviorally tested rats. Successful completion of these conceptually innovative studies will significantly advance our knowledge of brain plasticity in chronic pain, provide novel targets, and validate a gene transfer rescue strategy to mitigate chronic neuropathic pain.

Project Summary Many patients suffer from chronic pain in the absence of identifiable injury. Such pains are termed ?functional? and include irritable bowel syndrome, temporomandibular joint disorder, fibromyalgia, migraine and others. Functional pain patients experience pain free periods that are interrupted by attacks of pain that can persist for variable periods of time. The chronification of these pain disorders has been linked to the number and frequency of attacks suggesting that repeated nociceptive episodes promote and maintain a state of central sensitization that reflects increased vulnerability to future attacks. Functional pain patients commonly identify stress as a key trigger of pain episodes but neurobiological mechanisms remain to be determined. In this project, we test the novel hypothesis that in sensitized states, stress-induced kappa opioid receptor (KOR) signaling in the amygdala promotes functional pain responses. We have developed an injury-free rodent model of stress-related functional pain based on hyperalgesic priming with opioids. Opioids have been shown to produce opioid-induced hyperalgesia (OIH) in humans and in animals. OIH is characterized by generalized tactile and thermal hyperalgesia, decreased nociceptive thresholds, increase temporal summation, and a loss of descending noxious inhibitory controls (DNIC). Following resolution of OIH, and in the absence of stress, animals have normal pain responses. Hyperalgesic priming, however, produces a state of latent sensitization so that animals previously exposed to morphine are now prone to stress-induced hyperalgesia and a loss of DNIC that is prevented by blockade of KOR signaling within the central nucleus of the amygdala (CeA). Our electrophysiological data support a KOR-mediated disinhibition of CeA neurons that promote pain. We will use advanced behavioral and electrophysiological approaches with optogenetic and chemogenetic methods to demonstrate that activation of CeA KOR neurons in control, unprimed mice promotes pain-related responses (Specific Aim 1). These studies will establish the neural circuitry within the amygdala that may underlie a novel KOR-mediated pronociceptive CeA output that is engaged through disinhibition. Specific Aim 2 will determine if exogenous activation of the CeA KOR circuit results in amplified pain responses following priming- induced latent sensitization. In Specific Aim 3 we will determine whether blockade of stress-induced endogenous CeA KOR signaling reduces pain responses following priming-induced latent sensitization. The proposed studies will characterize a previously unknown stress-related KOR mediated hyperalgesic circuit from CeA and determine how this circuit may promote decreased resilience to stress. Importantly, these studies may unravel mechanisms for therapeutic interventions in stress-related functional pain disorders through an actionable molecular target. KOR antagonists are currently in development.

Summary: Pain is a serious clinical problem that affects more than 100 million Americans. The economic costs of pain have been estimated to be more than several hundred billion dollars including healthcare costs and lost productivity. Persistent pain may produce long-term disability and lead to precipitation of depression, anxiety and cognitive impairment. Currently used medications for chronic pain are not always effective and have limitations in terms of tolerance and abuse liability. Thus, identifying novel therapeutic targets is essential to address this clinical burden. Peripheral and central pathways that encode, transmit, and amplify or reduce pain signals have been identified, including the spinothalamic and spinoparabrachial pathways. Plasticity of glutamatergic synapses along key nodes in the spinoparabrachial-amygdala pathway plays an important role in pain modulation and in the transition from subacute to chronic pain. However, the mechanisms governing the development, maintenance and plasticity of this system and their role in persistence of pain behaviors remain poorly understood. The proposed research will advance the concept that the trans-synaptic signaling complex centered on glutamate delta 1 receptor regulates function of synapses in the laterocapsular region of central amygdala also known as ?nociceptive amygdala? and contributes to persistent pain mechanisms. Specific Aim1 will define the cell type- and projection-specific distribution of these receptors and their role in regulating amygdala circuitry and nocifensive and averse-affective behavior under normal conditions. Specific Aim 2 will determine persistent/chronic pain-related changes in glutamate delta 1 signaling using inflammatory and neuropathic pain models and test the effect of a rescue strategy on synaptic neuroplasticity in pain models. Changes in ultrastructure of amygdala synapses in pain models will be evaluated using 3D-electron microscopy. Specific Aim 3 will determine the effect of restoring trans- synaptic signaling through the glutamate delta 1 receptor in mitigating nocifensive and averse-affective behaviors in pain models. Complementary experiments will address the effect of cell-type specific manipulation of central amygdala circuitry in mitigating pain. To accomplish these aims we will utilize a combination of brain slice electrophysiology, behavior, chemo- and opto-genetics, confocal and electron microscopy (immuno and 3D), and genetic approaches to determine the functional and structural mechanisms through which the glutamate delta 1 signaling complex regulates pain-related neuroplasticity and behaviors. This project is significant because it would identify a novel brain mechanism of pain that could be targeted for pain management. Scientific rigor of research design is established by the use of multiple methods and approaches, replication of experiments in independent laboratories, use of validated models and reagents, consideration of blinding, biological variables and sex in addition to other aspects of experimental design.

Project Summary: Many patients suffer from chronic pain in the absence of identifiable injury. Such pains are termed ?functional? and include irritable bowel syndrome, temporomandibular joint disorder, fibromyalgia, migraine and others. For reasons that are not understood, almost all functional pain syndromes (FPS) are female prevalent. FPS patients experience pain-free interictal periods punctuated by attacks of pain. The frequency of attacks is predictive of risk of chronification. Pain episodes thus produce a priming effect, establishing a state of increased vulnerability to future attacks, likely reflecting peripheral and central sensitization. FPS patients commonly identify stress as a key trigger of pain. Repeated stress may thus promote vulnerability and pain in a sexually dimorphic fashion. We have developed an injury-free rodent model of FPS based on hyperalgesic priming with repeated stress. Hyperalgesic priming produces a pain-free state of increased vulnerability that has been termed ?latent sensitization? (LS). Following induction of LS, normally subthreshold triggers can produce pain attacks, modeling the interictal and ictal periods of FPS. We will use this model to test the novel hypothesis that repeated stress activates kappa opioid receptor (KOR) signaling in the hypothalamus resulting in release of prolactin (PRL) and dysregulation of prolactin receptor (PRLR) isoform expression selectively in female nociceptors. PRL signals through homodimers of PRLR long and short (i.e., PRLR-L and PRLR-S) isoforms that respectively regulate transcription and pain. Repeated stress down-regulates PRLR-L promoting female-selective pain through stress-induced PRL/PRLR-S signaling. The balance of PRLR isoforms may therefore ?tune? female nociceptors to promote LS and pain from normally subthreshold stimuli. We will use genetic and chemogenetic manipulations along with anatomical, neurochemical, electrophysiological, pharmacological and behavioral studies in male and female mice to evaluate the role of dorsal root ganglion (DRG) PRLR-L down-regulation and stress-related hypothalamic KOR activation as essential mechanisms of LS and stress-related pain in females. Aim 1 will establish the effects of repeated stress on hypothalamic KOR signaling and PRL release. Aim 2 will establish a potential causal relationship of repeated stress or hypothalamic KOR activation on DRG PRLR isoform expression, neural excitability, LS and stress-related pain. Aim 3 will determine if KOR antagonists, DA agonists or a PRL antibody will prevent LS and FPS-like pain selectively in females. The proposed studies will characterize a previously unknown stress-related neuroendocrine link between hypothalamic KOR and PRL/PRLR signaling to promote female selective functional pain. Importantly, these studies will advance knowledge about previously unknown biological mechanisms and may unravel mechanisms for therapeutic interventions allowing improved therapy of FPS in women.