Jose A. Moron-Concepcion, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): Relapse to drug use in abstinent human opioid addicts is the major obstacle impeding success of opioid addiction treatment. Relapse can be triggered by exposure to environmental cues associated with drug use; as such, disruption of the learned associations between the opioid and environmental cues may be an effective approach for reducing relapse and extending abstinence. Synaptic plasticity in the hippocampus, a key neural substrate of learning and memory, is an integral component of the development of context-dependent associations. Relatively stable changes in gene expression at the postsynaptic density (PSD), which receives and transduces synaptic information, are associated with synaptic plasticity. Thus changes in the protein expression profile within the hippocampal PSD may be integral in mediating synaptic plasticity that underlies the expression and extinction of a conditioned response to an opioid-paired environment. In the present proposal, we will test the hypothesis that the expression and extinction of a conditioned behavioral response to a previously morphine-paired environment are associated with differential protein expression profiles in the hippocampal PSD. In Specific Aim 1, we will establish a paradigm for the study of extinction of morphine conditioned place preference (CPP) via repeated exposure of the rats to the previously morphine-paired CPP chambers in the absence of morphine. In Specific Aim 2, we will characterize the protein expression profiles in hippocampal PSD that track with the expression and extinction of morphine CPP. Relative levels of protein expression will be analyzed through isotope-coded affinity tag (ICAT) followed by tandem mass spectrometry (LC-MS/MS). In Specific Aim 3, we will validate the levels of expression of hippocampal PSD-associated proteins identified as being differentially expressed in animals that express morphine CPP compared to those in which morphine CPP has been extinguished; biochemical and immunohistochemical techniques will be employed. Proteins altered following extinction will be the basis for further analyses of the mechanisms of extinction of drug-associated conditioned responses, and will provide novel targets for pharmacotherapeutic intervention in the quest to rapidly and effectively disrupt learned drug-environment conditioned associations to reduce the risk of relapse and promote abstinence in opioid addicts. Opioid addiction is a chronic, relapsing behavioral disorder and exposure to environmental "cues" associated with opioid consumption triggers relapse in opioid addicts. The present proposal will examine the protein changes linked to disruption of the behavioral response to morphine-associated environmental cues in order to identify new targets for developing therapeutic medications to reduce the risk of relapse in opioid addicts. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): While abuse and addiction to opiates has been a long-standing problem, the recent surge in abuse of opiate analgesics foreshadows the potential for rising rates of addiction to opiates. Repeated administration of drugs of abuse, such as morphine, causes a progressive and persistent sensitization of its locomotor stimulant and positive reinforcing effects. Sensitization to morphine can be sustained for several months after drug cessation and serves as a useful animal model of plasticity and the neuroadaptations associated with repeated administration of opioids having abuse potential. Studies show that sensitization has a close relationship with relapse, compulsive drug-seeking, and drug-taking behavior. Recent evidence suggests a role for the hippocampus in controlling these long-lasting behavioral adaptations. Investigation of an opiate-induced sensitization may help us to better understand the relapse mechanisms and provide new strategies for the treatment of drug addiction. Additionally, the key role of hippocampal synapses in learning and memory suggests that an understanding of the role of its specialized subcellular compartments in addictive processes is essential. Glutamatergic systems are thought to be involved in opiate-induced neuronal and behavioral plasticity although the mechanisms underlying these effects are only beginning to be understood. We propose to analyze the role of synaptic AMPA glutamate receptors in the neuronal adaptations associated with repeated administration of morphine. The proposed experiments will test the hypothesis that repeated morphine administration modulates synaptic transmission and plasticity at hippocampal synapses by altering the expression and composition of AMPA glutamate receptors;and that these adaptive effects will persist over time leading to neuroadaptations in glutamatergic synaptic function which could be responsible for the long-term behavioral sensitization induced by repeated morphine administration. In Specific Aim 1 we will analyze the synaptic mechanisms underlying the neuroadaptations initiated by repeated morphine administration which drive dynamic changes in the expression and composition of GluR subunits (GluR1/2/3) of AMPA glutamate receptors at hippocampal synapses and determine their correlation with long-term behavioral sensitization. In Specific Aim 2 we will characterize the electrophysiological mechanisms contributing to GluR subunit composition at glutamatergic synapses during basal synaptic transmission and plasticity in the hippocampus following repeated morphine administration, and determine their persistence. These studies are significant because they elucidate key mechanisms underlying neuroadaptive changes in synaptic neurotransmission at hippocampal synapses and behavioral responses that occur upon repeated morphine exposure;in addition, they will provide insight into the neuronal adaptations that may lead to novel approaches for pharmacotherapeutic intervention of opiate addiction. PUBLIC HEALTH RELEVANCE: The long-term effects of repeated exposure to drugs of abuse are a major point of interest in the study of the pathophysiology of drug addiction. The repeated administration of a variety of potentially addictive drugs, such as morphine, produces increases in their motor-stimulant effects (called behavioral sensitization) and their incentive-motivational properties that persist many months after cessation of drug administration, thus mimicking long-term sensitivity to drugs observed in human addicts. The present proposal will analyze the mechanisms underlying morphine-induced sensitization by characterizing the modulation and alteration of hippocampus neurotransmission at the synaptic level upon repeated morphine administration.

DESCRIPTION (provided by applicant): Abrupt abstinence or withdrawal from opiate drugs causes a series of severe adverse symptoms, which keep drug-dependent individuals craving continued opiates. One of the core of withdrawal symptoms is an increase in pain sensitivity (pain sensitization or hyperalgesia). This pain sensitization is due to synaptic plasticity, particularly in the spinal cord and primary afferents. Recent evidence suggests that mechanisms underlying synaptic plasticity in the hippocampus may also occur in the spinal cord. Although acute exposure to opiates may induce hyperalgesia, chronic or repeated administration of the drug facilitate the magnitude and duration of opiate-induced hyperalgesia, and may expand the anatomical sites at which hyperalgesia is induced. Persistence of chronic opiate-induced hyperalgesia is consistent with cellular mechanisms of opiate dependence that only develop after repeated exposure to the drug. It has been demonstrated that AMPA glutamate receptor trafficking within the spinal cord is involved in the development of pain sensitivity. Similarly, hippocampal glutamatergic systems are thought to be involved in opiate-induced neuronal and behavioral plasticity. We propose to document the parallel changes in AMPA receptor expression occurring in opiate dependence and pain sensitivity at three different levels: 1) hippocampus, 2) spinal cord and 3) primary afferent neurons. The proposed experiments will test the hypothesis that acute withdrawal from repeated morphine administration results in sensory sensitization by altering synaptic expression and composition of AMPA receptors and that this neuroplasticity in AMPA receptors is similar to that underlying chronic inflammatory pain. In Specific Aim 1 we will characterize the sensory sensitization that develops in a model of morphine withdrawal and compare it to the sensitization that develops in a model of chronic inflammatory pain. In Specific Aim 2 we will analyze the dynamic changes in expression and composition of AMPA receptor subunits (GluR1/2/3) in morphine withdrawal-associated sensitization and compare them to those occurring in chronic inflammatory pain. In Specific Aim 3 we will characterize the neuroplasticity in AMPA receptor synapses underlying morphine withdrawal-associated sensory sensitization and compare it to the AMPA receptor neuroplasticity underlying chronic inflammatory pain. In Specific Aim 4 we will demonstrate that treatment with AMPA antagonists relieves both morphine withdrawal-associated sensory sensitization as well as that associated with chronic inflammatory pain. These studies are significant because they will elucidate key glutamatergic maladaptive changes that opiate addicts and inflammatory pain patients have in common. Overall, these studies will provide insight into the neuroplasticity that may lead to novel approaches for pharmacotherapeutic intervention for pain treatment in opiate addicts. PUBLIC HEALTH RELEVANCE: The experiments proposed in this application will address the question of how drug-induced alterations in the hippocampus, spinal cord and primary afferents contribute to enhanced sensory sensitivity in opiate addicts. These results will enhance our understanding of the synaptic molecular and membrane mechanisms underlying this adverse effect of drug dependence and will help us to improve pain management in opiate addicts.

DESCRIPTION (provided by applicant): Chronic pain represents one of the most significant societal burdens in terms of the number of Americans affected, its impact on the health care system and lost productivity. Classical opiates, such as morphine, remain the gold standard of care for the management of moderate to severe post-operative and cancer pain as well as for the treatment of chronic non-malignant and inflammatory pain. However, the long-term use of mu opioid receptor (MOP) agonists such as morphine, in the setting of chronic pain, is limited by the development of tolerance and physical dependence. Opiate tolerance is the gradual loss of drug potency or efficacy, and reduced duration of action. The opioid receptor subfamilies include mu, delta, and kappa opioid receptors (MOP, DOP, and KOP). While it is clear that morphine-induced analgesia is mediated by MOP activation, the role of DOP in analgesia remains unclear. It has been reported that morphine-induced analgesic tolerance in acute pain is reduced upon administration of DOP antagonists and in mice lacking functional DOP. Thus, it seems that MOP-DOP interactions play an important role in modulating morphine-induced analgesic tolerance. Surprisingly, there are no studies regarding the role of DOP or MOP-DOP interactions in the development of morphine-induced analgesic tolerance in chronic pain. We have recently reported that pretreatment with the DOP2 antagonist, naltriben, disrupts morphine conditioned place preference and that this effect is associated with an increase in the levels of DOP dimer at the synapse. In addition, our preliminary studies show that morphine tolerance in the presence of chronic inflammatory pain is associated with an increased expression of the DOP at the synapse and with increased levels of the MOP-DOP heteromer in the spinal cord dorsal horn. Based on these data, we propose that pretreatment with DOP antagonists or disruption of the MOP-DOP heteromer will result in an attenuation of the analgesic tolerance that develops after repeated morphine injections during chronic pain. We also propose that morphine-induced analgesic tolerance is mediated by increased DOP function and MOP-DOP heteromer abundance, which in turn reduces MOP-mediated inhibition of excitatory transmission. This results in a loss of opiate analgesic potential, at the primary afferent, spinal cord and at brain sites implicated in opioid control of nociception such as the midbrain periaqueductal gray (PAG). In Specific Aim 1 we will conduct behavioral and biochemical analyses to investigate the role of MOP-DOP interactions in the attenuation of morphine-induced analgesic tolerance during chronic inflammatory pain. In Specific Aim 2 using in vitro recordings, we will characterize the role of DOP and MOP-DOP interactions in the control of excitatory transmission during morphine-induced analgesic tolerance in the presence of chronic inflammatory pain. The outcomes of the present studies will have a sustained, powerful impact on the fields of the biology and pharmacology of opioid receptors with the prospects of novel, safer and more effective pharmacotherapeutic strategies for the treatment of chronic pain.

Although significant advances in the treatment of opiate addiction have been made, relapse to opiate use after abstinence continues to impede successful treatment, highlighting the need for efforts to dissect the mechanism of opiate-dependent changes in brain plasticity. Recent studies have attempted to determine the role of structural plasticity in drug-induced behavior with conflicting findings which may result from the complexity of the intracellular signaling mechanisms underlying structural plasticity of dendritic spines caused by drugs of abuse. In this application we propose to develop novel in vivo approaches that will allow us to image the dynamic structural and functional plasticity that is triggered following opiate exposure and that may play a role in the mechanisms underlying reinstatement of drug seeking. To determine the relationship between dendrite structure and the formation and retrieval of drug associated memories, we propose to investigate how morphine exposure in a novel context modifies hippocampal dendritic spine morphology. In vivo imaging is a powerful tool to track rewiring in the hippocampal neural network, at the level of individual spines, throughout the learning, acquisition, expression, extinction and subsequent reinstatement of morphine conditioned place preference (CPP). Ideally one would be able to observe neural networks, in real time, as morphine CPP and reinstatement take place. The advent of virtual reality training paradigms during two-photon imaging makes this combination of behavior and network surveillance possible. In vivo imaging will enable us to follow the dynamics of spine remodeling and allow us to determine whether spine changes are cause or consequence of CPP. In addition, we will be able to visualize whether alterations in dendritic spines persist even following extinction which would indicate that spine remodeling may help store the latent memory driving drug-context associations. Therefore, the goals proposed in this application are: 1) to use in vivo 2-photon imaging in the dorsal hippocampus to follow structural changes in dendritic spines in CA1 neurons during the acquisition and expression of morphine CPP, and following its reinstatement; 2) to implement novel virtual reality spatial navigation protocols that enable us to conduct structural and functional imaging analyses of hippocampal cells in vivo during the formation of morphine-context associations. Overall, in this proposal we will conduct in vivo imaging analyses in awake mice to elucidate the temporal dynamics of hippocampal dendritic spine remodeling and its relationship to the formation of drug-context associations that may play a role in the mechanisms underlying reinstatement of drug seeking. In addition we will implement novel virtual navigation approaches to examine hippocampal circuit dynamics during the formation of morphine-context associations. The imaging methods and behavioral training protocols pioneered in this grant will be widely disseminated to the addiction field to advance the boundaries of current research.

? DESCRIPTION: This application, submitted in response to RFA-DA-16-004, proposes to functionally validate genetic variants identified in a NIDA-funded R01 (DA17305; PI E. Nelson) in which GWAS analyses in a sample limited to opioid misusers tested the hypothesis that genetic polymorphisms influence progression from exposure to severe opioid dependence. The strongest association was for SNPs in cornichon family AMPA receptor auxiliary protein 3 (CNIH3). Confirmation analyses performed in two samples with non-dependent opioid misusers yielded a genome-wide significant meta-analytic p of <4.3E-9 for rs10799590, an intronic CNIH3 SNP associated with reduced risk [OR 0.64 (0.54-0.74)].The large effect accounts for between 1.2 and 5.8% of the phenotypic variance. Furthermore, the association of CNIH3 is consistent with studies of opioids in animal models highlighting the importance of glutamate signaling. This application's R21 phase proposes to generate transgenic mice into which human CNIH3 has been introduced (both the risk and protective haplotypes) using innovative adaptations of CRISPR technology. The R33 phase proposes to use these mice to test our overarching hypothesis that CNIH3 polymorphisms alter gene expression impacting AMPA signaling and behavioral response to opioids. We will use behavioral assays related to opioid dependence (conditioned place preference, extinction, and reinstatement; context-dependent sensitization), which previously connected AMPA signaling to opioids. We will determine gene expression in response to morphine, focusing on addiction-related brain regions in which AMPA receptor subunit composition changes have been reported, to determine whether significant differences are found between mice with the protective and risk-associated haplotypes. We will also examine potential routes by which haplotype-associated effects may be mediated. The R21 component of the project's specific aim is: AIM 1 To generate humanized Cnih3 mice carrying the identified protective and risk-associated haplotypes The specific aims of the R33 component of the project are: AIM 1 To examine phenotypic differences between groups of transgenic mice with the protective and risk- associated haplotypes (with wild type and Cnih3 knockout mice as controls) in the expression of morphine CPP, its extinction, and reinstatement and context-dependent sensitization (CDS) AIM 2 To determine whether the protective haplotype alters Cnih3 expression at baseline (pre-opioids) or in response to morphine in these behavioral paradigms AIM 3 To examine whether these haplotypes alter Cnih3 and related protein content in the post-synaptic density of brain areas associated with morphine-induced behavioral responses AIM 4 To examine evidence for epigenetic mediation of altered gene expression associated with these SNPs in response to morphine CPP, including identifying SNPs that alter the activity of enhancers

Maladaptive pain-induced dysfunction in motivational circuits are the likely critical factors that lead to pathological alterations in natural and drug reward seeking behaviors, yet the neural circuit mechanisms for these effects are largely unknown. The mesolimbic system is a key network node that integrates pain and reward. Dopamine (DA) transmission in the mesolimbic system, via the VTA to NAc has long been recognized for its role in motivated behavior. Alterations in DA signaling within the mesolimbic pathway are associated with motivational deficits, and animals in pain show impaired motivated responses to natural and drug reward. Importantly, from a translational perspective, negative correlations between pain and mesolimbic DA activity in humans have been often reported. Mu opioid receptor (MOPR) agonists are positively reinforcing and remain the predominant opioids used for alleviating clinical pain and recreational use/abuse. We recently found that persistent inflammatory pain downregulates function of MOPR in the VTA with a concomitant loss of opioid- induced DA release in the NAc in a dose-dependent manner leading to increase intake of higher doses of opioids which are known to contribute to abuse-associated phenotypes. In addition, our preliminary data suggest that persistent inflammation causes an increase in endogenous opioid tone which is likely leading to desensitization of MOPR in the VTA. Collectively, these findings suggest that pain suppresses VTA?NAc neural circuit activity through suppression of mesolimbic DA release. We predict that this effect is mediated by an increase in endogenous opioid tone and reduction of MOPR function in the VTA which negatively impact the animal's motivational state contributing to an opioid abuse-associated phenotype. In this application we propose cross disciplinary cutting-edge approaches to dissect the neuronal and cellular mechanisms underlying the downregulation of mu-opioid circuits in the presence of inflammatory pain. In three specific aims we will: i) determine the mechanisms for downregulation of mu-opioid-containing GABAergic circuits in inflammatory pain ii) determine whether increases in endogenous mu-opioid agonist tone changes mu-opioid function, and whether these processes are necessary and sufficient for the effects of inflammatory pain on motivated behavior. Finally, in a third aim we will directly visualize endogenous opioid-containing neuronal ensembles during the development of inflammatory pain and decreased motivational states. Here we will determine whether mu-opioid receptors, their endogenous agonists, within discrete mesolimbic neural circuits, are necessary and sufficient to mediate pain-induced alterations in opioid intake and motivated behavior.

Although significant advances in the treatment of opiate addiction have been made, relapse to opiate use after abstinence continues to impede successful treatment, highlighting the need for efforts to dissect the mechanism of opiate-dependent changes in brain function. Long-lasting associations between opiates and the context in which they are taken result in cues that lead to drug craving and ultimately relapse. The hippocampus has traditionally been recognized for its role in learning and memory but recent evidence has shown its critical role in the behavioral effects of opiates, probably via activation of hippocampal excitatory inputs to the reward pathway (i.e. Nucleus Accumbens, NAc). Several lines of evidence suggest that drug- induced contextual/cue memories are associated with molecular changes in hippocampal function and that these changes may be necessary for some behavioral effects of opiates. Results from our laboratory have revealed that AMPAR and NMDAR play a crucial role in opiate-induced contextual learning. In addition SK2 channels, which are functionally linked to NMDAR, are critically involved in coding contextual memories. Furthermore, we have recently found that context-dependent sensitization to morphine leads to the activation of SK2 channels in the hippocampus. SK2-mediated functional effects on activated spines may be responsible for the expression and reinstatement of morphine place preference behavior. We also have preliminary data showing that the intrahippocampal administration of the SK2 blocker, apamin, following conditioning training, blocks the expression of morphine CPP. However, there are no reports dissecting how SK2 channels influence learning for opiate-paired contextual/cue behavior. Our central hypothesis is that enhancement of SK2 function, via increased Ca2+ influx through NMDAR, in the hippocampus plays a critical role in the formation of the association between the drug reward experience and the context/cue. Our overall objective is to uncover the cellular mechanisms underlying morphine-induced activation of SK2 channels, and to investigate how the activation of hippocampal SK2 channels are integral for morphine-induced contextual/cue learned associations, using both the CPP and i.v. drug self-administration models. In Aim 1, we will determine the mechanisms by which SK2 channel function is increased following morphine contextual/cue associations. In Aim 2, we will define the requirement for enhanced SK2 channel function in the expression and reinstatement of morphine contextual/cue associations in vivo. In Aim 3, we will determine whether hippocampal excitatory inputs to the NAc are enhanced during morphine-contextual/cue associations and to measure the underlying SK2-mediated changes in hippocampal pyramidal neuronal networks. Studies proposed here offer potentially novel drug addiction treatments in targeting SK2 channels to prevent drug-context/cue associations and thus prevent relapse to opiate use.

Project Summary The purpose of this application is to request a further five years of support for an Institutional National Research Service Award (years 26-30) which we have held for 25 years to support multidisciplinary, interdepartmental post-doctoral training focused on the neurobiology of substance abuse with a strong emphasis on neuroimaging, molecular and familial genetics, pharmacoepidemiology, behavior, neural circuits, and pharmacology. We request support for six postdoctoral fellows, as we have had for the past five years ? filling all slots consistently. The fellowship will usually last two years for PhDs and often three for MDs due to their lack of research training in pursuit of their medical degrees. Fellows with a wide variety of backgrounds will be recruited including: Psychology, Psychiatry, Genetics, Medicine, Anthropology, Sociology, Biology, Neuropharmacology and Neuroscience. The primary research training experience is the apprenticeship model ? conduct of supervised research under the tutelage of one or more preceptors who are well established researchers, and experienced mentors. The trainee's work is usually focused on a single but broad ranging research project developed jointly by the preceptor and trainee. Mentored research takes up approximately 70%-80% of the trainee's effort. The remainder of the training program is made up of lectures, seminars, reading courses, individual tutorials and, when appropriate, formal didactic course work available in our graduate and medical school. Tutorials in neurobiology and genetics are tailored for trainees with specific interests that may or may not fall into their major area of focus. To arrange these tutorials, the program coordinator and preceptor identify areas of specific interest and set up the tutorial for the trainees with one of our preceptors or tutors who best meets the training objective. The main research interests of the five Departments involved in this grant (Psychiatry [as the central, administrative department], Anesthesiology, Psychological & Brain Sciences, Neurology and Genetics) are broad yet all have a heavy focus on neurobiology in its broadest context and/or pain and pain management (i.e., anesthesiology). These Departments as a whole offer more than 30 hours of relevant seminars per week dealing with the subject matter of this grant and are open to any of our interested postdoctoral fellows, although to be sure we cannot envision any circumstances that would encourage attendance at all, or most, of these seminars. Some careful selection must be made to ensure that trainees focus on their main research and career objective, mentored research. None-the-less, it is reassuring to have that much ancillary training available to amplify or to broaden the perspectives of our trainees.

Pain and reward are considered opponent processes but are processed within overlapping brain structures. It has been demonstrated that rewarding stimuli can decrease pain sensitivity, whereas pain can impair reward processing leading to an anhedonic state. However, it is not yet known how the presence of pain modifies the reinforcing properties of natural rewards and opioids. The mesolimbic pathway is a critical brain nuclei that is altered in opioid addiction making it an ideal neural circuit to investigate the mechanistic basis for opioid abuse in the presence of pain. Opioid-induced released of dopamine (DA) in the nucleus accumbens (NAc) contributes to their abuse potential, where an allostatic shift in reward signaling leads to the pathological state of addiction. Mu opioid receptor (MOPR) agonists are positively reinforcing and remain the predominant opioids used for clinical and recreational-abuse purposes. In contrast, the activation of brain kappa opioid receptors (KOPR) causes dysphoria via suppression of mesolimbic DA and 5HT activity within the nucleus NAc reward circuitry. It is thought that these two opposing opioid-receptor systems work together to partially maintain the balance of affective state, however dysregulation of one or the other system can lead to dramatic changes in reward processing behaviors. We recently reported that persistent inflammatory pain negatively impacts function of MOPR in the ventral tegmental area (VTA) with a concomitant loss of mu-opioid-induced DA release in the NAc which may partially underlie the observed increase in the intake of very high doses of the opioid. Interestingly, our initial findings indicate that persistent inflammatory pain enhances KOPR function in the NAc, promoting negative affect states (i.e. decrease in the overall motivational state and enhanced aversive behavior) which may be crucially involved in driving increased opioid consumption when high doses are accessible, as recently proposed to maintain drug seeking and escalation of intake. Taken together, these preliminary findings strongly support the central hypothesis of this multidisplinary proposal that pain reduces the activity of the VTA-NAc dopamine reward circuit, via an enhancement of the dynorphin-KOPR system activity to decrease motivation and promote dysphoria. We hypothesize that this pain-induced KOPR-mediated negative affective state drives the intake of high dose opioids leading to misuse and drug escalation. Using a series of multidisplinary approaches including electrophysiology, microdialysis, voltammetry, optogenetics, chemogenetics, mouse genetics, and rodent PET imaging tools, we propose to determine whether in vivo manipulation of dynorphin-KOPR system in the VTA-NAc circuit prevents pain-induced negative affect which drives opioid dose escalation.