Edita Navratilova

Interests

Research

Neurobiology of pain,Migraine,Post-traumatic headache

Teaching

Opioid pharmacology,Receptor binding,G protein coupled receptors,Autonomic nervous system,Pain

Courses

Scholarly Contributions

Chapters

• Navratilova, E., Hruby, V. J., Hruby, V. J., & Porreca, F. (2011). Delta Opioid Receptor Function. In The Opiate Receptors. Humana Press, Totowa, NJ. doi:10.1007/978-1-60761-993-2_12
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  Delta (δ) opioid receptors (DORs) are part of circuits involved in many physiological functions including the modulation of pain. Activation of DORs appears to mediate antinociception and antihyperalgesia in many conditions including stress, chronic pain, opioid-induced hyperalgesia (OIH), and opiate tolerance. δ Opioid signaling also extensively interacts with the mu (μ) opioid system, resulting in modulation of pain transmission. Multiple mechanisms may underlie DOR modulation of pain including the synergistic interaction between μ and δ opioid receptors and between spinal and supraspinal sites of action, enhanced endogenous enkephalinergic tone, potential μ-δ oligomers and modulation of plasma membrane receptor trafficking.

Journals/Publications

• Chen, Y., Khanna, R., Watanabe, M., Stratton, J., Ross, S., Porreca, F., Navratilova, E., Moutal, A., Mackenzie, K., Kopruszinski, C. M., Ikegami, D., & Dodick, D. W. (2022). Dysregulation of serum prolactin links the hypothalamus with female nociceptors to promote migraine.. Brain : a journal of neurology, 145(8), 2894-2909. doi:10.1093/brain/awac104
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  Migraine headache results from activation of meningeal nociceptors, however, the hypothalamus is activated many hours before the emergence of pain. How hypothalamic neural mechanisms may influence trigeminal nociceptor function remains unknown. Stress is a common migraine trigger that engages hypothalamic dynorphin/kappa opioid receptor (KOR) signaling and increases circulating prolactin. Prolactin acts at both long and short prolactin receptor isoforms (PRLR-L and PRLR-S) that are expressed in trigeminal afferents. Following down-regulation of PRLR-L, prolactin signaling at PRLR-S sensitizes nociceptors selectively in females. We hypothesized that stress may activate KOR on tuberoinfundibular dopaminergic neurons to increase circulating prolactin leading to female-selective sensitization of trigeminal nociceptors through dysregulation of PRLR isoforms. A mouse two-hit hyperalgesic priming model of migraine was used. Repeated restraint stress promoted vulnerability (i.e., first-hit priming) to a subsequent subthreshold (i.e., second-hit) stimulus from inhalational umbellulone, a TRPA1 agonist. Periorbital cutaneous allodynia served as a surrogate of migraine-like pain. Female and male KORCre; R26lsl-Sun1-GFP mice showed high percentage of KORCre labeled neurons co-localized in tyrosine hydroxylase positive cells in the hypothalamic arcuate nucleus (ARC). Restraint stress increased circulating prolactin to a greater degree in females. Stress-primed, but not control, mice of both sexes developed periorbital allodynia following inhalational umbellulone. Gi-DREADD activation (i.e., inhibition through Gi-coupled signaling) in KORCre neurons in the ARC also increased circulating prolactin and repeated chemogenetic manipulation of these neurons primed mice of both sexes to umbellulone. CRISPR/Cas9 deletion of ARC KOR prevented restraint stress-induced prolactin release in female mice and priming from repeated stress episodes in both sexes. Inhibition of circulating prolactin with systemic cabergoline, a dopamine D2 receptor agonist, blocked priming selectively in females. Repeated restraint stress down-regulated PRLR-L in the trigeminal ganglia of female mice. Deletion of PRLR in trigeminal ganglia by nasal CRISPR/Cas9 targeting both PRLR isoforms prevented stress-induced priming in female mice. Stress-induced activation of hypothalamic KOR increases circulating prolactin resulting in trigeminal downregulation of PRLR-L and pain responses to a normally innocuous TRPA1 stimulus. These are the first data that provide a mechanistic link between stress-induced hypothalamic activation and the trigeminal nociceptor effectors that produce trigeminal sensitization and migraine-like pain. This sexually dimorphic mechanism may help to explain female prevalence of migraine. KOR antagonists, currently in phase II clinical trials, may be useful as migraine preventives in both sexes, while dopamine agonists and prolactin/PRLR antibodies may improve therapy for migraine, and other stress-related neurological disorders, in women.
• Patwardhan, A., Porreca, F., Navratilova, E., Moutal, A., Yue, X., Porreca, F., Navratilova, E., Moutal, A., Kopruszinski, C. M., Khanna, R., Ikegami, D., & Dodick, D. W. (2022). A prolactin-dependent sexually dimorphic mechanism of migraine chronification.. Cephalalgia : an international journal of headache, 42(3), 197-208. doi:10.1177/03331024211039813
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  Determination of possible sex differences in mechanisms promoting migraine progression and the contribution of prolactin and the prolactin long (PRLR-L) and short (PRLR-S) receptor isoforms..The majority of patients with chronic migraine and medication overuse headache are female. Prolactin is present at higher levels in women and increases migraine. Prolactin signaling at the PRLR-S selectively sensitizes nociceptors in female rodents, while expression of the PRLR-L is protective..Medication overuse headache was modeled by repeated sumatriptan administration in male and female mice. Periorbital and hindpaw cutaneous allodynia served as a surrogate of migraine-like pain. PRLR-L and PRLR-S isoforms were measured in the trigeminal ganglion with western blotting. Possible co-localization of PRLR with serotonin 5HT1B and 5HT1D receptors was determined with RNAscope. Cabergoline, a dopamine receptor agonist that inhibits circulating prolactin, was co-administered with sumatriptan. Nasal administration of CRISPR/Cas9 plasmid was used to edit expression of both PRLR isoforms..PRLR was co-localized with 5HT1B or 5HT1D receptors in the ophthalmic region of female trigeminal ganglion. A single injection of sumatriptan increased serum PRL levels in female mice. Repeated sumatriptan promoted cutaneous allodynia in both sexes but down-regulated trigeminal ganglion PRLR-L, without altering PRLR-S, only in females. Co-administration of sumatriptan with cabergoline prevented allodynia and down-regulation of PRLR-L only in females. CRISPR/Cas9 editing of both PRLR isoforms in the trigeminal ganglion prevented sumatriptan-induced periorbital allodynia in females..We identified a sexually dimorphic mechanism of migraine chronification that involves down-regulation of PRLR-L and increased signaling of circulating prolactin at PRLR-S. These studies reveal a previously unrecognized neuroendocrine mechanism linking the hypothalamus to nociceptor sensitization that increases the risk of migraine pain in females and suggest opportunities for novel sex-specific therapies including gene editing through nasal delivery of CRISPR/Cas9 constructs.
• Porreca, F., Anderson, T., Navratilova, E., Oyarzo, J., Broide, R. S., Subramaniam, S. R., Vazquez-Cintron, E. J., Brin, M. F., Schwedt, T. J., & Dodick, D. W. (2022). Preclinical assessment of onabotulinumtoxinA for the treatment of mild traumatic brain injury-related acute and persistent post-traumatic headache. Cephalalgia, 42(11-12), 1194-1206. doi:10.1177/03331024221099841
• Porreca, F., Roberts, E., Rosen, H., Patwardhan, A., Moutal, A., Yue, X., Navratilova, E., Ito, H., Vagnerova, B., Watanabe, M., Kopruszinski, C., Moreira de Souza, L. H., Ikegami, D., Khanna, R., Yamazaki, M., Guerrero, M., Neugebauer, V., & Dodick, D. W. (2022). Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance. Brain. doi:10.1093/brain/awac153
• Yamanaka, A., Watanabe, M., Watanabe, D., Tanaka, K., Tamura, H., Suda, Y., Senba, E., Sato, D., Porreca, F., Navratilova, E., Narita, M., Narita, M., Mori, T., Matsui, R., Kuzumaki, N., & Hamada, Y. (2022). Relief of neuropathic pain by cell-specific manipulation of nucleus accumbens dopamine D1- and D2-receptor-expressing neurons.. Molecular brain, 15(1), 10. doi:10.1186/s13041-021-00896-2
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  Emerging evidence suggests that the mesolimbic dopaminergic network plays a role in the modulation of pain. As chronic pain conditions are associated with hypodopaminergic tone in the nucleus accumbens (NAc), we evaluated the effects of increasing signaling at dopamine D1/D2-expressing neurons in the NAc neurons in a model of neuropathic pain induced by partial ligation of sciatic nerve. Bilateral microinjection of either the selective D1-receptor (Gs-coupled) agonist Chloro-APB or the selective D2-receptor (Gi-coupled) agonist quinpirole into the NAc partially reversed nerve injury-induced thermal allodynia. Either optical stimulation of D1-receptor-expressing neurons or optical suppression of D2-receptor-expressing neurons in both the inner and outer substructures of the NAc also transiently, but significantly, restored nerve injury-induced allodynia. Under neuropathic pain-like condition, specific facilitation of terminals of D1-receptor-expressing NAc neurons projecting to the VTA revealed a feedforward-like antinociceptive circuit. Additionally, functional suppression of cholinergic interneurons that negatively and positively control the activity of D1- and D2-receptor-expressing neurons, respectively, also transiently elicited anti-allodynic effects in nerve injured animals. These findings suggest that comprehensive activation of D1-receptor-expressing neurons and integrated suppression of D2-receptor-expressing neurons in the NAc may lead to a significant relief of neuropathic pain.
• Yue, X., Ujcikova, H., Svoboda, P., Robles, D., Navratilova, E., & Lee, Y. S. (2022). Time-Dependent Changes in Protein Composition of Medial Prefrontal Cortex in Rats with Neuropathic Pain.. International journal of molecular sciences, 23(2), 955. doi:10.3390/ijms23020955
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  Chronic pain is associated with time-dependent structural and functional reorganization of the prefrontal cortex that may reflect adaptive pain compensatory and/or maladaptive pain-promoting mechanisms. However, the molecular underpinnings of these changes and whether there are time-dependent relationships to pain progression are not well characterized. In this study, we analyzed protein composition in the medial prefrontal cortex (mPFC) of rats at two timepoints after spinal nerve ligation (SNL) using two-dimensional gel electrophoresis (2D-ELFO) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). SNL, but not sham-operated, rats developed persistent tactile allodynia and thermal hyperalgesia, confirming the presence of experimental neuropathic pain. Two weeks after SNL (early timepoint), we identified 11 proteins involved in signal transduction, protein transport, cell homeostasis, metabolism, and apoptosis, as well as heat-shock proteins and chaperones that were upregulated by more than 1.5-fold compared to the sham-operated rats. Interestingly, there were only four significantly altered proteins identified at 8 weeks after SNL (late timepoint). These findings demonstrate extensive time-dependent modifications of protein expression in the rat mPFC under a chronic neuropathic pain state that might underlie the evolution of chronic pain characterized by early pain-compensatory and later aberrant mechanisms.
• Hein, M., Ji, G., Tidwell, D., D'Souza, P., Kiritoshi, T., Yakhnitsa, V., Navratilova, E., Porreca, F., & Neugebauer, V. (2021). Kappa opioid receptor activation in the amygdala disinhibits CRF neurons to generate pain-like behaviors. Neuropharmacology, 185, 108456.
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  Recent evidence suggests that kappa opioid receptors (KOR) in limbic brain regions such as the amygdala contribute to pain conditions, but underlying mechanisms remain to be determined. The amygdala is an important player in averse-affective aspects of pain and pain modulation. The central nucleus (CeA) serves output functions through projection neurons that include corticotropin releasing factor (CRF) expressing neurons. The CeA is also rich in KOR. Here we tested the novel hypothesis that KOR activation in the CeA generates pain-like behaviors through a mechanism that involves inhibition of synaptic inhibition (disinhibition) of CRF neurons. Intra-CeA administration of a KOR agonist (U-69,593) increased vocalizations of naïve rats to noxious stimuli, and induced anxiety-like behaviors in the open field test (OFT) and avoidance in the conditioned place preference test, without affecting mechanosensory thresholds. Optogenetic silencing of CeA-CRF neurons blocked the facilitatory effects of systemically applied U-69,593 in naïve rats. Patch-clamp recordings of CRF neurons in rat brain slices found that U-69,593 decreased feedforward inhibitory transmission evoked by optogenetic stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. U-69,593 decreased frequency, but not amplitude, of inhibitory synaptic currents, suggesting a presynaptic action. Multiphoton imaging of CeA-CRF neurons in rat brain slices showed that U-69,593 increased calcium signals evoked by electrical stimulation of presumed parabrachial input. This study shows for the first time that KOR activation increases activity of amygdala CRF neurons through synaptic disinhibition, resulting in averse-affective pain-like behaviors. Blocking KOR receptors may therefore represent a novel therapeutic strategy.
• Porreca, F., Navratilova, E., & Fillingim, R. B. (2021). Sexual dimorphism in functional pain syndromes.. Science translational medicine, 13(619), eabj7180. doi:10.1126/scitranslmed.abj7180
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  Injury-free pain conditions, defined as functional pain syndromes, are more prevalent and more disabling in women. Mechanisms of sexual dimorphism in functional pain are now emerging from preclinical studies, suggesting an opportunity to advance the development of sex-specific therapies that may improve treatment of pain in women.
• Porreca, F., Phelps, C. E., & Navratilova, E. (2021). Chronic Pain Produces Reversible Memory Deficits That Depend on Task Difficulty in Rats.. The journal of pain. doi:10.1016/j.jpain.2021.04.016
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  Cognitive impairment associated with chronic pain remains relatively poorly understood. Use of analgesic drugs and often present co-morbidities in patients can preclude conclusions of causative relationships between chronic pain and cognitive deficits. Here, the impact of pain resulting from spinal nerve ligation (SNL) injury in rats on short and long-term memory was assessed in the novel object recognition task. To understand if chronic pain seizes the limited cognitive resources that are available at any given time, task difficulty was varied by using either very different (ie, easy task) or similar (ie, difficult task) pairs of objects. Nerve-injured, male rats exhibited no short or long-term memory deficits under easy task conditions. However, unlike sham-operated controls, injured rats showed deficits in both short and long-term memory by failing to differentiate similar objects in the difficult task version. In SNL rats, duloxetine produced anti-allodynic effects and ameliorated long-term memory deficits in the difficult task suggesting benefits of pain relief possibly complemented by noradrenergic mediated cognitive enhancement. Together these data suggest chronic pain reversibly takes up a significant amount of limited cognitive resources, leaving sufficient available for easy, but not difficult, tasks. Perspective: Memory deficits in a rat model of chronic pain were only seen when the cognitive load was high, ie, in a difficult task. Acute treatment with duloxetine was sufficient to relieve memory deficits, suggesting chronic pain induces memory deficits by seizing limited cognitive resources to the detriment of task-related stimuli.
• Porreca, F., Phelps, C. E., & Navratilova, E. (2021). Cognition in the Chronic Pain Experience: Preclinical Insights.. Trends in cognitive sciences, 25(5), 365-376. doi:10.1016/j.tics.2021.01.001
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  Acutely, pain is protective. It promotes escape from, and future avoidance of, noxious stimuli through strong and often lifetime associative memories. However, with persistent acute pain or when pain becomes chronic, these memories can promote negative emotions and poor decisions often associated with deleterious behaviors. In this review, we discuss how preclinical studies can provide insights into the relationship between cognition and chronic pain. We also discuss the concept of pain as a cognitive disorder and new strategies for treating chronic pain that emphasize inhibiting the formation of pain memories or promoting 'forgetting' of established pain memories.
• Seguela, P., Qu, C., Porreca, F., Navratilova, E., & Lancon, K. (2021). Decreased dopaminergic inhibition of pyramidal neurons in anterior cingulate cortex maintains chronic neuropathic pain.. Cell reports, 37(9), 109933. doi:10.1016/j.celrep.2021.109933
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  Pyramidal neurons in the anterior cingulate cortex (ACC), a prefrontal region involved in processing the affective components of pain, display hyperexcitability in chronic neuropathic pain conditions, and their silencing abolishes hyperalgesia. We show that dopamine, through D1 receptor (D1R) signaling, inhibits pyramidal neurons of mouse ACC by modulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Activation of Gs-coupled D1R by dopamine induces the opening of HCN channels at physiological membrane potentials, driving a significant decrease in input resistance and excitability. Systemic L-DOPA in chronic neuropathic mice rescues HCN channel activity, normalizes pyramidal excitability in ACC, and blocks mechanical and thermal allodynia. Moreover, microinjection of a selective D1R agonist in the ACC relieves the aversiveness of ongoing neuropathic pain, while an ACC D1R antagonist blocks gabapentin- and lidocaine-evoked antinociception. We conclude that dopaminergic inhibition via D1R in ACC plays an analgesic role in physiological conditions and is decreased in chronic pain.
• Swiokla, J., Porreca, F., Navratilova, E., Kopruszinski, C. M., Dodick, D. W., & Chessell, I. P. (2021). A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain.. Cephalalgia : an international journal of headache, 41(3), 305-317. doi:10.1177/0333102420959794
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  Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments..A "two-hit" hyperalgesic priming strategy was used to induce vulnerability to a normally subthreshold challenge with umbellulone, a transient receptor potential ankyrin 1 (TRPA1) activator, in uninjured female and male C57BL/6 mice. Priming (i.e. the first hit) was induced by three consecutive daily episodes of restraint stress; repeated umbellulone was also evaluated for potential priming effects. Sixteen days after the first restraint stress, mice received inhalational umbellulone (i.e. the second hit) to elicit migraine-like pain. Medications currently used for acute or preventive migraine therapy including propranolol (a beta blocker) and sumatriptan (5HT1B/D agonist), as well as olcegepant, an experimental calcitonin gene related peptide (CGRP) receptor antagonist and nor-Binaltorphimine (nor-BNI), an experimental long-acting kappa opioid receptor (KOR) antagonist, were investigated for their efficacy to block priming and prevent or reverse umbellulone-induced allodynia in primed animals. To assess migraine-like pain, cutaneous allodynia was determined by responses to periorbital or hindpaw probing with von Frey filaments..Repeated restraint stress, but not umbellulone exposure, produced transient cutaneous allodynia that resolved within 16 d. Restraint stress produced long-lasting priming that persisted beyond 16 d, as demonstrated by reinstatement of cutaneous allodynia following inhalational umbellulone challenge. Pretreatment with propranolol or nor-BNI prior to restraint stress prevented both transient cutaneous allodynia and priming, demonstrated by a lack of umbellulone-induced cutaneous allodynia. Following establishment of restraint stress priming, olcegepant, but not propranolol or nor-BNI, prevented umbellulone-induced cutaneous allodynia. When administered 1 h after umbellulone, sumatriptan, but not olcegepant, reversed umbellulone-induced cutaneous allodynia in restraint stress-primed rats..We have developed a novel injury-free model with translational relevance that can be used to study mechanisms relevant to migraine-like pain and to evaluate novel acute or preventive treatments. Restraint stress priming induced a state of vulnerability to a subthreshold stimulus that has been referred to as "latent sensitization". The development of latent sensitization could be prevented by blockade of stress pathways with propranolol or with a kappa opioid receptor antagonist. Following establishment of latent sensitization, subthreshold stimulation with umbellulone reinstated cutaneous allodynia, likely from activation of meningeal TRPA1-expressing nociceptors. Accordingly, in restraint stress-primed animals, sumatriptan reversed umbellulone-induced cutaneous allodynia, supporting peripheral sites of action, while propranolol and nor-BNI were not effective. Surprisingly, olcegepant was effective in mice with latent sensitization when given prior to, but not after, umbellulone challenge, suggesting time-dependent contributions of calcitonin gene-related peptide receptor signaling in promoting migraine-like pain in this model. Activation of the calcitonin gene-related peptide receptor participates in initiating, but has a more limited role in maintaining, pain responses, supporting the efficacy of small molecule calcitonin gene-related peptide antagonists as preventive medications. Additionally, the effectiveness of sumatriptan in reversal of established pain thus suggests modulation of additional, non-calcitonin gene-related peptide receptor-mediated nociceptive mechanisms. Kappa opioid receptor antagonists may represent a novel preventive therapy for stress-related migraine.
• Weinstein, T. J., Turnes, J. M., Swiokla, J., Schwedt, T. J., Porreca, F., Navratilova, E., Kopruszinski, C. M., Dodick, D. W., & Anderson, T. (2021). CGRP monoclonal antibody prevents the loss of diffuse noxious inhibitory controls (DNIC) in a mouse model of post-traumatic headache.. Cephalalgia : an international journal of headache, 41(6), 749-759. doi:10.1177/0333102420981688
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  Determine the role of calcitonin-gene related peptide in promoting post-traumatic headache and dysregulation of central pain modulation induced by mild traumatic brain injury in mice..Mild traumatic brain injury was induced in lightly anesthetized male C57BL/6J mice by a weight drop onto a closed and unfixed skull, which allowed free head rotation after the impact. We first determined possible alterations in the diffuse noxious inhibitory controls, a measure of net descending pain inhibition called conditioned pain modulation in humans at day 2 following mild traumatic brain injury. Diffuse noxious inhibitory control was assessed as the latency to a thermally induced tail-flick that served as the test stimulus in the presence of right forepaw capsaicin injection that provided the conditioning stimulus. Post-traumatic headache-like behaviors were assessed by the development of cutaneous allodynia in the periorbital and hindpaw regions after mild traumatic brain injury. We then determined if intraperitoneal fremanezumab, an anti-calcitonin-gene related peptide monoclonal antibody or vehicle administered 2 h after sham or mild traumatic brain injury induction could alter cutaneous allodynia or diffuse noxious inhibitory control responses on day 2 post mild traumatic brain injury..In naïve and sham mice, capsaicin injection into the forepaw elevated the latency to tail-flick, reflecting the antinociceptive diffuse noxious inhibitory control response. Periorbital and hindpaw cutaneous allodynia, as well as a loss of diffuse noxious inhibitory control, was observed in mice 2 days after mild traumatic brain injury. Systemic treatment with fremanezumab blocked mild traumatic brain injury-induced cutaneous allodynia and prevented the loss of diffuse noxious inhibitory controls in mice subjected to a mild traumatic brain injury..Sequestration of calcitonin-gene related peptide in the initial stages following mild traumatic brain injury blocked the acute allodynia that may reflect mild traumatic brain injury-related post-traumatic headache and, additionally, prevented the loss of net descending inhibition within central pain modulation pathways. As loss of conditioned pain modulation has been linked to multiple persistent pain conditions, dysregulation of descending modulatory pathways may contribute to the persistence of post-traumatic headache. Additionally, evaluation of the conditioned pain modulation/diffuse noxious inhibitory controls response may serve as a biomarker of vulnerability for chronic/persistent pain. These findings suggest that early anti-calcitonin-gene related peptide intervention has the potential to be effective both for the treatment of mild traumatic brain injury-induced post-traumatic headache, as well as inhibiting mechanisms that may promote post-traumatic headache persistence.
• Zalevsky, J., Yang, M., Vanderveen, L., Swiokla, J., Schmidt, W. K., Porreca, F., Navratilova, E., Miyazaki, T., Liu, Y., Lee, Y. S., & Kopruszinski, C. M. (2021). Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents.. Cellular and molecular neurobiology, 41(5), 949-960.
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  Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models..Graded noxious stimulus intensities were used in rats to establish the antinociceptive potency and efficacy of NKTR-181 relative to morphine, fentanyl, and oxycodone. Characteristics of MOR agonist actions, as measured by antinociceptive tolerance and cross-tolerance, as well as opioid-induced hyperalgesia (OIH) and naloxone-precipitated withdrawal in NKTR-181- and morphine-dependent in mice, were compared..NKTR-181 showed dose- and time-related antinociception with similar maximal effects to morphine in the rat and mouse hot-water tail-flick test. No sex or species differences were observed in NKTR-181 or morphine antinociception. Rats treated with NKTR-181 and morphine exhibited decreases in both potency and maximal efficacy as nociceptive stimulus intensity was increased from a water temperature of 50 °C to 54 °C. Evaluation of antinociception at a high stimulus intensity revealed that oxycodone and fentanyl exhibited greater efficacy than either NKTR-181 or morphine. The relative potency difference between NKTR-181 and morphine across all tail-flick studies was determined to be 7.6-fold (90% confidence interval, 2.6, 21.5). The peak antinociceptive effect of NKTR-181 was delayed compared to that of the other opioids and cumulative drug effects were not observed. Repeated treatment with escalating, approximately equi-analgesic doses of NKTR-181 or morphine, produced antinociceptive tolerance and cross-tolerance. Under these pharmacological conditions, OIH and naloxone-precipitated physical dependence were similar for NKTR-181 and morphine..NKTR-181 had a slower onset, but similar efficacy, to morphine in the models studied supporting reduced abuse potential while maintaining analgesic effect in comparison with current opioids.
• Chen, Y., Moutal, A., Navratilova, E., Kopruszinski, C., Yue, X., Ikegami, M., Chow, M., Kanazawa, I., Bellampalli, S. S., Xie, J., Patwardhan, A., Rice, K., Fields, H., Akopian, A., Neugebauer, V., Dodick, D., Khanna, R., & Porreca, F. (2020). The prolactin receptor long isoform regulates nociceptor sensitization and opioid-induced hyperalgesia selectively in females. Science translational medicine, 12(529).
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  Pain is more prevalent in women for reasons that remain unclear. We have identified a mechanism of injury-free nociceptor sensitization and opioid-induced hyperalgesia (OIH) promoted by prolactin (PRL) in females. PRL signals through mutually inhibitory long (PRLR-L) and short (PRLR-S) receptor isoforms, and PRLR-S activation induces neuronal excitability. PRL and PRLR expression were higher in females. CRISPR-mediated editing of PRLR-L promoted nociceptor sensitization and allodynia in naïve, uninjured female mice that depended on circulating PRL. Opioids, but not trauma-induced nerve injury, decreased PRLR-L promoting OIH through activation of PRLR-S in female mice. Deletion of both PRLR-L and PRLR-S (total PRLR) prevented, whereas PRLR-L overexpression rescued established OIH selectively in females. Inhibition of circulating PRL with cabergoline, a dopamine D2 agonist, up-regulated PRLR-L and prevented OIH only in females. The PRLR-L isoform therefore confers protection against PRL-promoted pain in females. Limiting PRL/PRLR-S signaling pharmacologically or with gene therapies targeting the PRLR may be effective for reducing pain in a female-selective manner.
• Chen, Y., Navratilova, E., Dodick, D. W., & Porreca, F. (2020). An Emerging Role for Prolactin in Female-Selective Pain. Trends in neurosciences, 43(8), 635-648.
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  Women experience many pain conditions more frequently when compared with men, but the biological mechanisms underlying sex differences in pain remain poorly understood. In particular, little is known about possible sex differences in peripheral nociceptors, the fundamental building blocks of pain transmission. Emerging evidence reveals that prolactin (PRL) signaling at its cognate prolactin receptor (PRLR) in primary afferents promotes nociceptor sensitization and pain in a female-selective fashion. In this review, we summarize recent progress in understanding the female-selective role of PRL/PRLR in nociceptor sensitization and in pathological pain conditions, including postoperative, inflammatory, neuropathic, and migraine pain, as well as opioid-induced hyperalgesia. The clinical implications of the peripheral PRL/PRLR system for the discovery of new therapies for pain control in women are also discussed.
• Dickenson, A. H., Navratilova, E., Patel, R., Porreca, F., & Bannister, K. (2020). Supraspinal Opioid Circuits Differentially Modulate Spinal Neuronal Responses in Neuropathic Rats. Anesthesiology.
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  Descending control from supraspinal neuronal networks onto spinal cord neurons can modulate nociceptionEndogenous opioids in these brain circuits participate in pain modulationA differential opioidergic role for brain nuclei involved in supraspinal pain modulation has not been previously reported WHAT THIS ARTICLE TELLS US THAT IS NEW: In vivo electrophysiologic recordings from the dorsal horn of the spinal cord in male rats reveal differential effects of morphine at the anterior cingulate cortex, right amygdala, and the ventromedial medulla on evoked pain responsesThese data differentiate supraspinal opioid circuit regulation of spinal nociceptive processing and suggest that the regulation of sensory and affective components of pain are likely separate BACKGROUND:: The anterior cingulate cortex and central nucleus of the amygdala connect widely with brainstem nuclei involved in descending modulation, including the rostral ventromedial medulla. Endogenous opioids in these circuits participate in pain modulation. The hypothesis was that a differential opioidergic role for the brain nuclei listed in regulation of spinal neuronal responses because separable effects on pain behaviors in awake animals were previously observed.
• Gee, T. A., Weintraub, N. C., Lu, D., Phelps, C. E., Navratilova, E., Heien, M. L., & Porreca, F. (2020). A pain-induced tonic hypodopaminergic state augments phasic dopamine release in the nucleus accumbens. Pain, 161(10), 2376-2384.
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  Diseases and disorders such as Parkinson disease, schizophrenia, and chronic pain are characterized by altered mesolimbic dopaminergic neurotransmission. Dopamine release in the nucleus accumbens influences behavior through both tonic and phasic signaling. Tonic dopamine levels are hypothesized to inversely regulate phasic signals through dopamine D2 receptor feedback inhibition. We tested this hypothesis directly in the context of ongoing pain. Tonic and phasic dopamine signals were measured using fast-scan controlled-adsorption voltammetry and fast-scan cyclic voltammetry, respectively, in the nucleus accumbens shell of male rats with standardized levels of anesthesia. Application of capsaicin to the cornea produced a transient decrease in tonic dopamine levels. During the pain-induced hypodopaminergic state, electrically evoked phasic dopamine release was significantly increased when compared to baseline, evoked phasic release. A second application of capsaicin to the same eye had a lessened effect on tonic dopamine suggesting desensitization of TRPV1 channels in that eye. Capsaicin treatment in the alternate cornea, however, again produced coincident decreased dopaminergic tone and increased phasic dopamine release. These findings occurred independently of stimulus lateralization relative to the hemisphere of dopamine measurement. Our data show that (1) the mesolimbic dopamine circuit reliably encodes acute noxious stimuli; (2) ongoing pain produces decreases in dopaminergic tone; and (3) pain-induced decreases in tonic dopamine correspond to augmented evoked phasic dopamine release. Enhanced phasic dopamine neurotransmission resulting from salient stimuli may contribute to increased impulsivity and cognitive deficits often observed in conditions associated with decreased dopaminergic tone, including Parkinson disease and chronic pain.
• Kopruszinski, C. M., Navratilova, E., Swiokla, J., Dodick, D. W., Chessell, I. P., & Porreca, F. (2020). A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain. Cephalalgia : an international journal of headache, 333102420959794.
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  Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments.
• Kopruszinski, C. M., Navratilova, E., Vagnerova, B., Swiokla, J., Patwardhan, A., Dodick, D., & Porreca, F. (2020). Cannabinoids induce latent sensitization in a preclinical model of medication overuse headache. Cephalalgia : an international journal of headache, 40(1), 68-78.
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  Evaluation of cannabinoid receptor agonists in a preclinical model of medication overuse headache.
• Kopruszinski, C. M., Swiokla, J., Lee, Y. S., Navratilova, E., VanderVeen, L., Yang, M., Liu, Y., Miyazaki, T., Schmidt, W. K., Zalevsky, J., & Porreca, F. (2020). Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents. Cellular and molecular neurobiology.
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  Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models.
• Kopruszinski, C. M., Thornton, P., Arnold, J., Newton, P., Lowne, D., Navratilova, E., Swiokla, J., Dodick, D. W., Dobson, C., Gurrell, I., Chessell, I. P., & Porreca, F. (2020). Characterization and preclinical evaluation of a protease activated receptor 2 (PAR2) monoclonal antibody as a preventive therapy for migraine. Cephalalgia : an international journal of headache, 40(14), 1535-1550.
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  Migraine pain is thought to result from activation of meningeal nociceptors that might involve dural mast cell degranulation and release of proteases and pronociceptive mediators. Tryptase, the most abundant dural mast cell protease, has been demonstrated to stimulate dural mast cells, as well as trigeminal nociceptors by activating the protease activated receptor 2. Mast cell or neuronal protease activated receptors 2 may therefore represent a novel target for migraine treatment. In this study, we characterized and evaluated a novel protease activated receptor 2 monoclonal antibody as a preventive anti-migraine pain therapy in preclinical models.
• Navratilova, E., Behravesh, S., Oyarzo, J., Dodick, D. W., Banerjee, P., & Porreca, F. (2020). Ubrogepant does not induce latent sensitization in a preclinical model of medication overuse headache. Cephalalgia : an international journal of headache, 40(9), 892-902.
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  Ubrogepant, a small-molecule calcitonin gene-related peptide receptor antagonist, was recently approved as an oral medication for the acute treatment of migraine. This study aimed to determine whether ubrogepant shows efficacy in a preclinical model of migraine-like pain and whether repeated oral administration of ubrogepant induces latent sensitization relevant to medication overuse headache in rats.
• Navratilova, E., Behravesh, S., Oyarzo, J., Dodick, D. W., Banerjee, P., & Porreca, F. (2020). Ubrogepant does not induce latent sensitization in a preclinical model of medication overuse headache. Cephalalgia, 40(9), 892-902. doi:10.1177/0333102420938652
• Navratilova, E., Rau, J. C., Oyarzo, J., Johnson, K. W., Aurora, S. K., Schwedt, T. J., Dodick, D. W., & Porreca, F. (2020). Evaluation of LY573144 (lasmiditan) in a preclinical model of medication overuse headache. Cephalalgia, 40(9), 903-912. doi:10.1177/0333102420920006
• Neugebauer, V., Mazzitelli, M., Cragg, B., Ji, G., Navratilova, E., & Porreca, F. (2020). Amygdala, neuropeptides, and chronic pain-related affective behaviors. Neuropharmacology, 170, 108052.
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  Neuropeptides play important modulatory roles throughout the nervous system, functioning as direct effectors or as interacting partners with other neuropeptide and neurotransmitter systems. Limbic brain areas involved in learning, memory and emotions are particularly rich in neuropeptides. This review will focus on the amygdala, a limbic region that plays a key role in emotional-affective behaviors and pain modulation. The amygdala is comprised of different nuclei; the basolateral (BLA) and central (CeA) nuclei and in between, the intercalated cells (ITC), have been linked to pain-related functions. A wide range of neuropeptides are found in the amygdala, particularly in the CeA, but this review will discuss those neuropeptides that have been explored for their role in pain modulation. Calcitonin gene-related peptide (CGRP) is a key peptide in the afferent nociceptive pathway from the parabrachial area and mediates excitatory drive of CeA neurons. CeA neurons containing corticotropin releasing factor (CRF) and/or somatostatin (SOM) are a source of long-range projections and serve major output functions, but CRF also acts locally to excite neurons in the CeA and BLA. Neuropeptide S (NPS) is associated with inhibitory ITC neurons that gate amygdala output. Oxytocin and vasopressin exert opposite (inhibitory and excitatory, respectively) effects on amygdala output. The opioid system of mu, delta and kappa receptors (MOR, DOR, KOR) and their peptide ligands (β-endorphin, enkephalin, dynorphin) have complex and partially opposing effects on amygdala function. Neuropeptides therefore serve as valuable targets to regulate amygdala function in pain conditions. This article is part of the special issue on Neuropeptides.
• Porreca, F., Navratilova, E., Dickenson, A. H., Patel, R., & Bannister, K. (2020). Supraspinal Opioid Circuits Differentially Modulate Spinal Neuronal Responses in Neuropathic Rats. Anesthesiology, 132(4), 881-894. doi:10.1097/aln.0000000000003120
• Porreca, F., Patwardhan, A., Yue, X., Navratilova, E., Moutal, A., Chen, Y., Kopruszinski, C., Ikegami, M., Chow, M., Kanazawa, I., Bellampalli, S. S., Xie, J., Rice, K., Fields, H., Akopian, A., Neugebauer, V., Dodick, D., & Khanna, R. (2020). The prolactin receptor long isoform regulates nociceptor sensitization and opioid-induced hyperalgesia selectively in females. Science Translational Medicine, 12(529). doi:10.1126/scitranslmed.aay7550
• Rau, J. C., Navratilova, E., Oyarzo, J., Johnson, K. W., Aurora, S. K., Schwedt, T. J., Dodick, D. W., & Porreca, F. (2020). Evaluation of LY573144 (lasmiditan) in a preclinical model of medication overuse headache. Cephalalgia : an international journal of headache, 40(9), 903-912.
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  Medication overuse is a significant issue that complicates the treatment of headache disorders. The most effective medications for the acute treatment of migraine all have the capacity to induce medication overuse headache (MOH). Novel acute migraine-specific treatments are being developed. However, because the mechanism(s) underlying medication overuse headache are not well understood, it is difficult to predict whether any particular acute medication will induce MOH in susceptible individuals. LY573144 (lasmiditan), a 5-HT receptor agonist, has recently been shown to be effective in the acute treatment of migraine in phase 3 trials. The aim of this study is to determine whether frequent administration of lasmiditan induces behaviors consistent with MOH in a pre-clinical rat model.
• Yue, X., Patwardhan, A. M., Chen, Y., Yue, X., Xie, J. Y., Rice, K. C., Porreca, F., Patwardhan, A. M., Neugebauer, V., Navratilova, E., Moutal, A., Kopruszinski, C. M., Khanna, R., Kanazawa, I., Ikegami, M., Fields, H. L., Dodick, D. W., Chow, M., Chen, Y., , Bellampalli, S. S., et al. (2020). Erratum for the Research Article: "The prolactin receptor long isoform regulates nociceptor sensitization and opioid-induced hyperalgesia selectively in females" by Y. Chen, A. Moutal, E. Navratilova, C. Kopruszinski, X. Yue, M. Ikegami, M. Chow, I. Kanazawa, S. S. Bellampalli, J. Xie, A. Patwardhan, K. Rice, H. Fields, A. Akopian, V. Neugebauer, D. Dodick, R. Khanna, F. Porreca.. Science translational medicine, 12(533), eabb4549. doi:10.1126/scitranslmed.abb4549
• Navratilova, E., & Porreca, F. (2019). Substance P and Inflammatory Pain: Getting It Wrong and Right Simultaneously. Neuron, 101(3), 353-355.
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  How do neuropeptides participate in the classic neuroinflammatory sequelae of tissue injury that includes pain, immune cell infiltration, and swelling? In this issue of Neuron, Green et al. (2019) reveal that MrgprB2/MrgprX2 is a mast cell substance-P-specific receptor that critically links neural and immune systems and offers new approaches for neuroinflammatory therapeutics.
• Navratilova, E., Nation, K., Remeniuk, B., Neugebauer, V., Bannister, K., Dickenson, A. H., & Porreca, F. (2020). Selective modulation of tonic aversive qualities of neuropathic pain by morphine in the central nucleus of the amygdala requires endogenous opioid signaling in the anterior cingulate cortex. Pain, 161(3), 609-618.
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  The amygdala is a key subcortical region believed to contribute to emotional components of pain. As opioid receptors are found in both the central (CeA) and basolateral (BLA) nuclei of the amygdala, we investigated the effects of morphine microinjection on evoked pain responses, pain-motivated behaviors, dopamine release in the nucleus accumbens (NAc), and descending modulation in rats with left-side spinal nerve ligation (SNL). Morphine administered into the right or left CeA had no effect on nerve injury-induced tactile allodynia or mechanical hyperalgesia. Right, but not left, CeA morphine produced conditioned place preference (CPP) and increased extracellular dopamine in the NAc selectively in SNL rats, suggesting relief of aversive qualities of ongoing pain. In SNL rats, CPP and NAc dopamine release following right CeA morphine was abolished by blocking mu opioid receptor signaling in the rostral anterior cingulate cortex (rACC). Right CeA morphine also significantly restored SNL-induced loss of the diffuse noxious inhibitory controls, a spino-bulbo-spinal pain modulatory mechanism, termed conditioned pain modulation in humans. Microinjection of morphine into the BLA had no effects on evoked behaviors and did not produce CPP in nerve-injured rats. These findings demonstrate that the amygdalar action of morphine is specific to the right CeA contralateral to the side of injury and results in enhancement of net descending inhibition. In addition, engagement of mu opioid receptors in the right CeA modulates affective qualities of ongoing pain through endogenous opioid neurotransmission within the rACC, revealing opioid-dependent functional connections from the CeA to the rACC.
• Navratilova, E., Patwardhan, A., & Porreca, F. (2019). Opioid analgesics pass the acid test. Lancet (London, England), 393(10181), 1579-1581.
• Navratilova, E., Rau, J., Oyarzo, J., Tien, J., Mackenzie, K., Stratton, J., Remeniuk, B., Schwedt, T., Anderson, T., Dodick, D., & Porreca, F. (2019). CGRP-dependent and independent mechanisms of acute and persistent post-traumatic headache following mild traumatic brain injury in mice. Cephalalgia : an international journal of headache, 39(14), 1762-1775.
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  Acute and persistent post-traumatic headache are often debilitating consequences of traumatic brain injury. Underlying physiological mechanisms of post-traumatic headache and its persistence remain unknown, and there are currently no approved therapies for these conditions. Post-traumatic headache often presents with a migraine-like phenotype. As calcitonin-gene related peptide promotes migraine headache, we explored the efficacy and timing of intervention with an anti- calcitonin-gene related peptide monoclonal antibody in novel preclinical models of acute post-traumatic headache and persistent post-traumatic headache following a mild traumatic brain injury event in mice.
• Phelps, C. E., Navratilova, E., Dickenson, A. H., Porreca, F., & Bannister, K. (2019). Kappa opioid signaling in the right central amygdala causes hind paw specific loss of diffuse noxious inhibitory controls in experimental neuropathic pain. Pain, 160(7), 1614-1621.
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  Diffuse noxious inhibitory controls (DNICs) is a pain-inhibits-pain phenomenon demonstrated in humans and animals. Diffuse noxious inhibitory control is diminished in many chronic pain states, including neuropathic pain. The efficiency of DNIC has been suggested to prospectively predict both the likelihood of pain chronification and treatment response. Little is known as to why DNIC is dysfunctional in neuropathic pain. Here, we evaluated DNIC in the rat L5/L6 spinal nerve ligation (SNL) model of chronic pain using both behavioral and electrophysiological outcomes. For behavior, nociceptive thresholds were determined using response to noxious paw pressure on both hind paws as the test stimulus before, and after, injection of a conditioning stimulus of capsaicin into the left forepaw. Functionally, the spike firing of spinal wide-dynamic-range neuronal activity was evaluated before and during noxious ear pinch, while stimulating the ipsilateral paw with von Frey hairs of increased bending force. In both assays, the DNIC response was significantly diminished in the ipsilateral (ie, injured) paw of SNL animals. However, behavioral loss of DNIC was not observed on the contralateral (ie, uninjured) paw. Systemic application of nor-binaltorphimine, a kappa opioid antagonist, did not ameliorate SNL-induced hyperalgesia but reversed loss of the behavioral DNIC response. Microinjection of nor-binaltorphimine into the right central amygdala (RCeA) of SNL rats did not affect baseline thresholds but restored DNIC both behaviorally and electrophysiologically. Cumulatively, these data suggest that net enhanced descending facilitations may be mediated by kappa opioid receptor signaling from the right central amygdala to promote diminished DNIC after neuropathy.
• Porreca, F., Navratilova, E., Rau, J., Oyarzo, J., Tien, J., Mackenzie, K., Stratton, J., Remeniuk, B., Schwedt, T., Anderson, T., & Dodick, D. (2019). CGRP-dependent and independent mechanisms of acute and persistent post-traumatic headache following mild traumatic brain injury in mice. Cephalalgia, 39(14), 1762-1775. doi:10.1177/0333102419877662
• Becker, S., Navratilova, E., Nees, F., & Van Damme, S. (2018). Emotional and Motivational Pain Processing: Current State of Knowledge and Perspectives in Translational Research. Pain research & management, 2018, 5457870.
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  Pain elicits fear and anxiety and promotes escape, avoidance, and adaptive behaviors that are essential for survival. When pain persists, motivational priority and attention shift to pain-related information. Such a shift often results in impaired functionality, leading to maladaptive pain-related fear and anxiety and escape and avoidance behaviors. Neuroimaging studies in chronic pain patients have established that brain activity, especially in cortical and mesolimbic regions, is different from activity observed during acute pain in control subjects. In this review, we discuss the psychophysiological and neuronal factors that may be associated with the transition to chronic pain. We review information from human studies on neural circuits involved in emotional and motivational pain processing and how these circuits are altered in chronic pain conditions. We then highlight findings from animal research that can increase our understanding of the molecular and cellular mechanisms underlying emotional-motivational pain processing in the brain. Finally, we discuss how translational approaches incorporating results from both human and animal investigations may aid in accelerating the discovery of therapies.
• Becker, S., Navratilova, E., Nees, F., & Van Damme, S. (2018). Shared Mechanisms of Chronic Pain and Emotional-Motivational Problems: From Basic Science to the Clinics. Pain research & management, 2018, 9305026.
• Cowen, S. L., Phelps, C. E., Navratilova, E., McKinzie, D. L., Okun, A., Husain, O., Gleason, S. D., Witkin, J. M., & Porreca, F. (2018). Chronic pain impairs cognitive flexibility and engages novel learning strategies in rats. Pain, 159(7), 1403-1412.
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  Cognitive flexibility, the ability to adapt behavior to changing outcomes, is critical to survival. The prefrontal cortex is a key site of cognitive control, and chronic pain is known to lead to significant morphological changes to this brain region. Nevertheless, the effects of chronic pain on cognitive flexibility and learning remain uncertain. We used an instrumental paradigm to assess adaptive learning in an experimental model of chronic pain induced by tight ligation of the spinal nerves L5/6 (spinal nerve ligation model). Naive, sham-operated, and spinal nerve ligation (SNL) rats were trained to perform fixed-ratio, variable-ratio, and contingency-shift behaviors for food reward. Although all groups learned an initial lever-reward contingency, learning was slower in SNL animals in a subsequent choice task that reversed reinforcement contingencies. Temporal analysis of lever-press responses across sessions indicated no apparent deficits in memory consolidation or retrieval. However, analysis of learning within sessions revealed that the lever presses of SNL animals occurred in bursts, followed by delays. Unexpectedly, the degree of bursting correlated positively with learning. Under a variable-ratio probabilistic task, SNL rats chose a less profitable behavioral strategy compared with naive and sham-operated animals. After extinction of behavior for learned preferences, SNL animals reverted to their initially preferred (ie, less profitable) behavioral choice. Our data suggest that in the face of uncertainty, chronic pain drives a preference for familiar associations, consistent with reduced cognitive flexibility. The observed burst-like responding may represent a novel learning strategy in animals with chronic pain.
• Gomtsian, L., Bannister, K., Eyde, N., Robles, D., Dickenson, A. H., Porreca, F., & Navratilova, E. (2018). Morphine effects within the rodent anterior cingulate cortex and rostral ventromedial medulla reveal separable modulation of affective and sensory qualities of acute or chronic pain. Pain, 159(12), 2512-2521.
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  Modulation of pain may result from engagement of opioid receptors in multiple brain regions. Whether sensory and affective qualities of pain are differentially affected by brain opioid receptor circuits remains unclear. We previously reported that opioid actions within the rostral anterior cingulate cortex (ACC) produce selective modulation of affective qualities of neuropathic pain in rodents, but whether such effects may occur in other areas of the ACC is not known. Here, morphine was microinjected into 3 regions of the ACC or into the rostral ventromedial medulla (RVM), and pain behaviors in naive, sham, or spinal nerve ligated (SNL) rats were evaluated. In naive animals, the tail-flick response was inhibited by RVM, but not ACC, morphine. Anterior cingulate cortex morphine did not affect tactile allodynia (the von Frey test) or mechanical (Randall-Selitto) or thermal (Hargreaves) hyperalgesia in spinal nerve ligated rats. In contrary, RVM morphine reduced tactile allodynia and produced both antihyperalgesic and analgesic effects against mechanical and thermal stimuli as well as conditioned place preference selectively in nerve-injured rats. Within the RVM, opioids inhibit nociceptive transmission reflected in both withdrawal thresholds and affective pain behaviors. Activation of mu opioid receptors within specific rostral ACC circuits, however, selectively modulates affective dimensions of ongoing pain without altering withdrawal behaviors. These data suggest that RVM and ACC opioid circuits differentially modulate sensory and affective qualities of pain, allowing for optimal behaviors that promote escape and survival. Targeting specific ACC opioid circuits may allow for treatment of chronic pain while preserving the physiological function of acute pain.
• Nation, K. M., DeFelice, M., Hernandez, P. I., Dodick, D. W., Neugebauer, V., Navratilova, E., & Porreca, F. (2018). Lateralized Kappa Opioid Receptor Signaling from the Amygdala Central Nucleus Promotes Stress-Induced Functional Pain. Pain.
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  The diffuse noxious inhibitory controls (DNIC) response is often decreased, or lost, in stress-related functional pain syndromes. As the dynorphin/kappa opioid receptor (KOR) pathway is activated by stress, we determined its role in DNIC using a model of stress-induced functional pain. Male, Sprague Dawley rats were primed for 7 days with systemic morphine resulting in opioid-induced hyperalgesia. Fourteen days after priming, when hyperalgesia was resolved, rats were exposed to environmental stress and DNIC was evaluated by measuring hindpaw response threshold to noxious pressure (test stimulus) following capsaicin injection in the forepaw (conditioning stimulus). Morphine priming without stress did not alter DNIC. However, stress produced a loss of DNIC in morphine-primed rats in both hindpaws that was abolished by systemic administration of the KOR antagonist, nor-binaltorphimine (nor-BNI). Microinjection of nor-BNI into the right, but not left, central nucleus of the amygdala (CeA) prevented the loss of DNIC in morphine-primed rats. DNIC was not modulated by bilateral nor-BNI in the rostral ventromedial medulla (RVM). Stress increased dynorphin content in both the left and right CeA of primed rats, reaching significance only in the right CeA; no change was observed in the RVM or hypothalamus. While morphine-priming alone is not sufficient to influence DNIC, it establishes a state of latent sensitization that amplifies the consequences of stress. Following priming, stress-induced dynorphin/KOR signaling from the right CeA inhibits DNIC in both hindpaws, likely reflecting enhanced descending facilitation that masks descending inhibition. KOR antagonists may provide a new therapeutic strategy for stress-related functional pain disorders.
• Nation, K. M., Dodick, D. W., Navratilova, E., & Porreca, F. (2018). Sustained exposure to acute migraine medications combined with repeated noxious stimulation dysregulates descending pain modulatory circuits: Relevance to medication overuse headache. Cephalalgia : an international journal of headache, 333102418804157.
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  Background Loss of conditioned pain modulation/diffuse noxious inhibitory controls has been demonstrated in patients with migraine and medication overuse headache. We hypothesized that exposure to acute migraine medications may lead to dysregulation of central pain modulatory circuits that could be revealed by evaluating diffuse noxious inhibitory controls and that prior noxious stimulus is required for a loss of the diffuse noxious inhibitory control response in rats exposed to these medications. Methods Rats were "primed" by continuous infusion of morphine or one of two doses of sumatriptan. Diffuse noxious inhibitory control was evaluated at the end of drug-priming (day 7) and again after sensory thresholds returned to baseline (day 21). The Randall-Selitto hindpaw pressure test was used as the test stimulus and forepaw capsaicin injection served as the conditioning stimulus. Results Morphine-primed rats showed opioid-induced hyperalgesia accompanied by a loss of diffuse noxious inhibitory controls on day 7. Sumatriptan-primed rats did not develop hyperalgesia or loss of diffuse noxious inhibitory controls on day 7. Morphine-primed and high-dose sumatriptan-primed rats only had a loss of diffuse noxious inhibitory control on day 21 if they received a capsaicin injection on day 7. Conclusions Prolonged exposure to migraine treatments followed by an acute nociceptive stimulation caused long-lasting alterations in descending pain modulation, shown by a loss of diffuse noxious inhibitory controls. Morphine was more detrimental than sumatriptan, consistent with clinical observations of higher medication overuse headache risk with opioids. These data suggest a mechanism of medication overuse headache by which migraine medications combined with repeated episodes of pain may amplify the consequences of nociceptor activation and increase the probability of future migraine attacks as well as risk of medication overuse headache.
• Nation, K., Porreca, F., Navratilova, E., Ikegami, M., & Ikegami, D. (2018). Engagement of kappa opioid system in the right amygdala diminishes diffuse noxious inhibitory controls (DNIC). Proceedings for Annual Meeting of The Japanese Pharmacological Society, WCP2018(0), PO3-2-19. doi:10.1254/jpssuppl.wcp2018.0_po3-2-19
• Navratilova, E., Becker, S., Nees, F., & Van Damme, S. (2018). Emotional and Motivational Pain Processing: Current State of Knowledge and Perspectives in Translational Research. Pain Research and Management, 2018, 1-12. doi:10.1155/2018/5457870
• Navratilova, E., Ji, G., Phelps, C., Qu, C., Hein, M., Yakhnitsa, V., Neugebauer, V., & Porreca, F. (2018). Kappa opioid signaling in the central nucleus of the amygdala promotes disinhibition and aversiveness of chronic neuropathic pain. Pain.
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  Chronic pain is associated with neuroplastic changes in the amygdala that may promote hyper-responsiveness to mechanical and thermal stimuli (allodynia and hyperalgesia) and/or enhance emotional and affective consequences of pain. Stress promotes dynorphin-mediated signaling at the kappa opioid receptor (KOR) in the amygdala and mechanical hypersensitivity in rodent models of functional pain. Here, we tested the hypothesis that KOR circuits in the central nucleus of the amygdala (CeA) undergo neuroplasticity in chronic neuropathic pain resulting in increased sensory and affective pain responses. After spinal nerve ligation (SNL) injury in rats, pretreatment with a long-acting KOR antagonist, nor-binaltorphimine (nor-BNI), subcutaneously or through microinjection into the right CeA, prevented conditioned place preference (CPP) to intravenous gabapentin, suggesting that nor-BNI eliminated the aversiveness of ongoing pain. By contrast, systemic or intra-CeA administration of nor-BNI had no effect on tactile allodynia in SNL animals. Using whole-cell patch-clamp electrophysiology, we found that nor-BNI decreased synaptically evoked spiking of CeA neurons in brain slices from SNL but not sham rats. This effect was mediated through increased inhibitory postsynaptic currents, suggesting tonic disinhibition of CeA output neurons due to increased KOR activity as a possible mechanism promoting ongoing aversive aspects of neuropathic pain. Interestingly, this mechanism is not involved in SNL-induced mechanical allodynia. Kappa opioid receptor antagonists may therefore represent novel therapies for neuropathic pain by targeting aversive aspects of ongoing pain while preserving protective functions of acute pain.
• Navratilova, E., Watanabe, M., Narita, M., Hamada, Y., Yamashita, A., Tamura, H., Ikegami, D., Kondo, T., Shinzato, T., Shimizu, T., Fukuchi, Y., Muto, A., Okano, H., Yamanaka, A., Tawfik, V. L., Kuzumaki, N., Porreca, F., & Narita, M. (2018). Activation of ventral tegmental area dopaminergic neurons reverses pathological allodynia resulting from nerve injury or bone cancer. Molecular Pain, 14, 174480691875640. doi:10.1177/1744806918756406
• Porreca, F., Navratilova, E., Nation, K. M., & Dodick, D. W. (2018). Sustained exposure to acute migraine medications combined with repeated noxious stimulation dysregulates descending pain modulatory circuits: Relevance to medication overuse headache. Cephalalgia, 39(5), 617-625. doi:10.1177/0333102418804157
• Watanabe, M., Narita, M., Hamada, Y., Yamashita, A., Tamura, H., Ikegami, D., Kondo, T., Shinzato, T., Shimizu, T., Fukuchi, Y., Muto, A., Okano, H., Yamanaka, A., Tawfik, V., Kuzumaki, N., Navratilova, E., Porreca, F., & Narita, M. (2018). [EXPRESS] Activation of ventral tegmental area dopaminergic neurons reverses pathological allodynia resulting from nerve injury or bone cancer. Molecular pain, 1744806918756406.
• Watanabe, M., Sugiura, Y., Sugiyama, E., Narita, M., Navratilova, E., Kondo, T., Uchiyama, N., Yamanaka, A., Kuzumaki, N., Porreca, F., & Narita, M. (2018). [EXPRESS] Extracellular N-acetylaspartylglutamate released in the nucleus accumbens modulates the pain sensation: analysis using a microdialysis/mass spectrometry integrated system. Molecular pain, 1744806918754934.
• Xie, J. Y., Porreca, F., Navratilova, E., Kopruszinski, C. M., Ikegami, M., Ikegami, D., & Felice, M. D. (2018). Sensitization of kappa opioid system in the amygdala in rat model of stress-induced medication overuse headache. Proceedings for Annual Meeting of The Japanese Pharmacological Society, WCP2018(0), PO3-2-43. doi:10.1254/jpssuppl.wcp2018.0_po3-2-43
• Bannister, K., Qu, C., Navratilova, E., Oyarzo, J., Xie, J. Y., King, T., Dickenson, A. H., & Porreca, F. (2017). Multiple sites and actions of gabapentin-induced relief of ongoing experimental neuropathic pain. Pain, 158(12), 2386-2395.
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  Gabapentin (GBP) is a first-line therapy for neuropathic pain, but its mechanisms and sites of action remain uncertain. We investigated GBP-induced modulation of neuropathic pain following spinal nerve ligation (SNL) in rats. Intravenous or intrathecal GBP reversed evoked mechanical hypersensitivity and produced conditioned place preference (CPP) and dopamine (DA) release in the nucleus accumbens (NAc) selectively in SNL rats. Spinal GBP also significantly inhibited dorsal horn wide-dynamic-range neuronal responses to a range of evoked stimuli in SNL rats. By contrast, GBP microinjected bilaterally into the rostral anterior cingulate cortex (rACC), produced CPP, and elicited NAc DA release selectively in SNL rats but did not reverse tactile allodynia and had marginal effects on wide-dynamic-range neuronal activity. Moreover, blockade of endogenous opioid signaling in the rACC prevented intravenous GBP-induced CPP and NAc DA release but failed to block its inhibition of tactile allodynia. Gabapentin, therefore, can potentially act to produce its pain relieving effects by (a) inhibition of injury-induced spinal neuronal excitability, evoked hypersensitivity, and ongoing pain and (b) selective supraspinal modulation of affective qualities of pain, without alteration of reflexive behaviors. Consistent with previous findings of pain relief from nonopioid analgesics, GBP requires engagement of rACC endogenous opioid circuits and downstream activation of mesolimbic reward circuits reflected in learned pain-motivated behaviors. These findings support the partial separation of sensory and affective dimensions of pain in this experimental model and suggest that modulation of affective-motivational qualities of pain may be the preferential mechanism of GBP's analgesic effects in patients.
• Porreca, F., & Navratilova, E. (2017). Reward, motivation, and emotion of pain and its relief. Pain.
• Porreca, F., Roberts, E., Navratilova, E., Streicher, J. M., Yue, X., LaVigne, J., Xie, J. Y., De Felice, M., Kopruszinski, C. M., Eyde, N., Remeniuk, B., Hernandez, P., Goshima, N., Ossipov, M., King, T., Dodick, D., & Rosen, H. (2017). Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention. Cephalalgia, 37(8), 780-794. doi:10.1177/0333102417702120
• Xie, J. Y., De Felice, M., Kopruszinski, C. M., Eyde, N., LaVigne, J., Remeniuk, B., Hernandez, P., Yue, X., Goshima, N., Ossipov, M., King, T., Streicher, J. M., Navratilova, E., Dodick, D., Rosen, H., Roberts, E., & Porreca, F. (2017). Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention. Cephalalgia : an international journal of headache, 37(8), 780-794.
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  Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.
• Xie, J. Y., Rice, F. L., Porreca, F., Navratilova, E., Dodick, D. W., Bourgeois, J. R., Albrecht, P. J., & Acker, E. (2017). Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine.. Cephalalgia : an international journal of headache, 37(14), 1350-1372. doi:10.1177/0333102416677051
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  Objective The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey. Methods We used a comprehensive integrated multi-molecular immunofluorescence labeling strategy to extensively profile the rat DDI and to a lesser extent that of the Rhesus monkey. Results The DDI was distributed across a dense, pervasive capillary network and included free nerve endings of peptidergic CGRP-expressing C fibers that were closely intertwined with noradrenergic (NA) sympathetic fibers and thin-caliber nonpeptidergic "C/Aδ" fibers. These newly identified C/Aδ fibers were unmyelinated, like C fibers, but expressed NF200, usually indicative of Aδ fibers, and uniquely co-labeled for the CGRP co-receptor, RAMP1. Slightly-larger caliber NF200-positive fibers co-labeled for myelin basic protein (MBP) and terminated as unbranched corpuscular endings. The DDI peptidergic fibers co-labeled for the lectin IB4 and expressed presumably excitatory α1-adrenergic receptors, as well as inhibitory 5HT1D receptors and the delta opioid receptor (δOR), but rarely the mu opioid receptor (µOR). Labeling for P2X3, TRPV1, TRPA1, and parasympathetic markers was not observed in the DDI. Interpretation These results suggest potential functional interactions, wherein peptidergic DDI fibers may be activated by stress-related sympathetic activity, resulting in CGRP release that could be detected in the circulation. CGRP may also activate nonpeptidergic C/Aδ fibers that are likely mechanosensitive or polymodal, leading to activation of post-synaptic pain transmission circuits. The distribution of α1-adrenergic receptors, RAMP1, and the unique expression of the δOR on CGRP-expressing DDI fibers suggest strategies for functional modulation and application to therapy.
• Becerra, L., Bishop, J., Barmettler, G., Xie, Y., Navratilova, E., Porreca, F., & Borsook, D. (2016). Triptans disrupt brain networks and promote stress-induced CSD-like responses in cortical and subcortical areas. Journal of neurophysiology, 115(1), 208-17.
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  A number of drugs, including triptans, promote migraine chronification in susceptible individuals. In rats, a period of triptan administration over 7 days can produce "latent sensitization" (14 days after discontinuation of drug) demonstrated as enhanced sensitivity to presumed migraine triggers such as environmental stress and lowered threshold for electrically induced cortical spreading depression (CSD). Here we have used fMRI to evaluate the early changes in brain networks at day 7 of sumatriptan administration that may induce latent sensitization as well as the potential response to stress. After continuous infusion of sumatriptan, rats were scanned to measure changes in resting state networks and the response to bright light environmental stress. Rats receiving sumatriptan, but not saline infusion, showed significant differences in default mode, autonomic, basal ganglia, salience, and sensorimotor networks. Bright light stress produced CSD-like responses in sumatriptan-treated but not control rats. Our data show the first brain-related changes in a rat model of medication overuse headache and suggest that this approach could be used to evaluate the multiple brain networks involved that may promote this condition.
• Okun, A., McKinzie, D. L., Witkin, J. M., Remeniuk, B., Husein, O., Gleason, S. D., Oyarzo, J., Navratilova, E., McElroy, B., Cowen, S., Kennedy, J. D., & Porreca, F. (2016). Hedonic and motivational responses to food reward are unchanged in rats with neuropathic pain. Pain, 157(12), 2731-2738.
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  Rewards influence responses to acute painful stimuli, but the relationship of chronic pain to hedonic or motivational aspects of reward is not well understood. We independently evaluated hedonic qualities of sweet or bitter tastants and motivation to seek food reward in rats with experimental neuropathic pain induced by L5/6 spinal nerve ligation. Hedonic response was measured by implantation of intraoral catheters to allow passive delivery of liquid solutions, and "liking/disliking" responses were scored according to a facial reactivity scale. Spinal nerve ligation rats did not differ from controls in either "liking" or "disliking" reactions to intraoral sucrose or quinine, respectively, at postsurgery day 21, suggesting no differences in perceived hedonic value of sweet or bitter tastants. To assess possible motivational deficits during acute and chronic pain, we used fixed- and progressive-ratio response paradigms of sucrose pellet presentation in rats with transient inflammatory or chronic neuropathic pain. Assessment of response acquisition and break points under the progressive ratio schedule revealed no differences between sham and spinal nerve ligation rats for up to 120 days after injury. However, rats with inflammation showed decrements in lever pressing and break points on days 1 and 2 after complete Freund adjuvant injection that normalized by day 4, consistent with transient ongoing pain. Thus, although acute ongoing inflammatory pain may transiently reduce reward motivation, we did not detect influences of chronic neuropathic pain on hedonic or motivational responses to food rewards. Adaptations that allow normal reward responding to food regardless of chronic pain may be of evolutionary benefit to promote survival.
• Porreca, F., Navratilova, E., Xie, Y., Becerra, L., Bishop, J., Barmettler, G., & Borsook, D. (2016). Triptans disrupt brain networks and promote stress-induced CSD-like responses in cortical and subcortical areas. Journal of Neurophysiology, 115(1), 208-217. doi:10.1152/jn.00632.2015
• Rice, F. L., Xie, J. Y., Albrecht, P. J., Acker, E., Bourgeois, J., Navratilova, E., Dodick, D. W., & Porreca, F. (2016). Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine. Cephalalgia : an international journal of headache.
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  The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey.
• Xie, J. Y., Porreca, F., Ossipov, M. H., Navratilova, E., Morimura, K., & Atcherley, C. W. (2016). Positive emotions and brain reward circuits in chronic pain.. The Journal of comparative neurology, 524(8), 1646-52. doi:10.1002/cne.23968
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  Chronic pain is an important public health problem that negatively impacts the quality of life of affected individuals and exacts enormous socioeconomic costs. Chronic pain is often accompanied by comorbid emotional disorders including anxiety, depression, and possibly anhedonia. The neural circuits underlying the intersection of pain and pleasure are not well understood. We summarize recent human and animal investigations and demonstrate that aversive aspects of pain are encoded in brain regions overlapping with areas processing reward and motivation. We highlight findings revealing anatomical and functional alterations of reward/motivation circuits in chronic pain. Finally, we review supporting evidence for the concept that pain relief is rewarding and activates brain reward/motivation circuits. Adaptations in brain reward circuits may be fundamental to the pathology of chronic pain. Knowledge of brain reward processing in the context of pain could lead to the development of new therapeutics for the treatment of emotional aspects of pain and comorbid conditions.
• Nair, P., Yamamoto, T., Cowell, S., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Ma, S., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2015). Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities. Bioorganic & medicinal chemistry letters, 25(17), 3716-20.
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  Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively.
• Navratilova, E., Atcherley, C. W., & Porreca, F. (2015). Brain Circuits Encoding Reward from Pain Relief. Trends in neurosciences, 38(11), 741-50.
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  Relief from pain in humans is rewarding and pleasurable. Primary rewards, or reward-predictive cues, are encoded in brain reward/motivational circuits. While considerable advances have been made in our understanding of reward circuits underlying positive reinforcement, less is known about the circuits underlying the hedonic and reinforcing actions of pain relief. We review findings from electrophysiological, neuroimaging, and behavioral studies supporting the concept that the rewarding effect of pain relief requires opioid signaling in the anterior cingulate cortex (ACC), activation of midbrain dopamine neurons, and the release of dopamine in the nucleus accumbens (NAc). Understanding of circuits that govern the reward of pain relief may allow the discovery of more effective and satisfying therapies for patients with acute or chronic pain.
• Navratilova, E., Xie, J. Y., Meske, D., Qu, C., Morimura, K., Okun, A., Arakawa, N., Ossipov, M., Fields, H. L., & Porreca, F. (2015). Endogenous opioid activity in the anterior cingulate cortex is required for relief of pain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 35(18), 7264-71.
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  Pain is aversive, and its relief elicits reward mediated by dopaminergic signaling in the nucleus accumbens (NAc), a part of the mesolimbic reward motivation pathway. How the reward pathway is engaged by pain-relieving treatments is not known. Endogenous opioid signaling in the anterior cingulate cortex (ACC), an area encoding pain aversiveness, contributes to pain modulation. We examined whether endogenous ACC opioid neurotransmission is required for relief of pain and subsequent downstream activation of NAc dopamine signaling. Conditioned place preference (CPP) and in vivo microdialysis were used to assess negative reinforcement and NAc dopaminergic transmission. In rats with postsurgical or neuropathic pain, blockade of opioid signaling in the rostral ACC (rACC) inhibited CPP and NAc dopamine release resulting from non-opioid pain-relieving treatments, including peripheral nerve block or spinal clonidine, an α2-adrenergic agonist. Conversely, pharmacological activation of rACC opioid receptors of injured, but not pain-free, animals was sufficient to stimulate dopamine release in the NAc and produce CPP. In neuropathic, but not sham-operated, rats, systemic doses of morphine that did not affect withdrawal thresholds elicited CPP and NAc dopamine release, effects that were prevented by blockade of ACC opioid receptors. The data provide a neural explanation for the preferential effects of opioids on pain affect and demonstrate that engagement of NAc dopaminergic transmission by non-opioid pain-relieving treatments depends on upstream ACC opioid circuits. Endogenous opioid signaling in the ACC appears to be both necessary and sufficient for relief of pain aversiveness.
• Remeniuk, B., Sukhtankar, D., Okun, A., Navratilova, E., Xie, J. Y., King, T., & Porreca, F. (2015). Behavioral and neurochemical analysis of ongoing bone cancer pain in rats. Pain, 156(10), 1864-73.
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  Cancer-induced bone pain is described as dull, aching ongoing pain. Ongoing bone cancer pain was characterized after intratibial injection of breast cancer cells in rats. Cancer produced time-dependent bone remodeling and tactile hypersensitivity but no spontaneous flinching. Conditioned place preference (CPP) and enhanced dopamine (DA) release in the nucleus accumbens (NAc) shell was observed after peripheral nerve block (PNB) selectively in tumor-bearing rats revealing nociceptive-driven ongoing pain. Oral diclofenac reversed tumor-induced tactile hypersensitivity but did not block PNB-induced CPP or NAc DA release. Tumor-induced tactile hypersensitivity, and PNB-induced CPP and NAc DA release, was blocked by prior subcutaneous implantation of a morphine pellet. In sham rats, morphine produced a modest but sustained increase in NAc DA release. In contrast, morphine produced a transient 5-fold higher NAc DA release in tumor bearing rats compared with sham morphine rats. The possibility that this increased NAc DA release reflected the reward of pain relief was tested by irreversible blockade of rostral anterior cingulate cortex (rACC) μ-opioid receptors (MORs). The rACC MOR blockade prevented the morphine-induced transient increased NAc DA release in tumor bearing rats but did not affect morphine-induced effects in sham-operated animals. Consistent with clinical experience, ongoing cancer pain was controlled by morphine but not by a dose of diclofenac that reversed evoked hypersensitivity. Additionally, the intrinsic reward of morphine can be dissociated from the reward of relief of cancer pain by blockade of rACC MOR. This approach allows mechanistic and therapeutic assessment of ongoing cancer pain with likely translation relevance.
• Navratilova, E., & Porreca, F. (2014). Reward and motivation in pain and pain relief. Nature neuroscience, 17(10), 1304-12.
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  Pain is fundamentally unpleasant, a feature that protects the organism by promoting motivation and learning. Relief of aversive states, including pain, is rewarding. The aversiveness of pain, as well as the reward from relief of pain, is encoded by brain reward/motivational mesocorticolimbic circuitry. In this Review, we describe current knowledge of the impact of acute and chronic pain on reward/motivation circuits gained from preclinical models and from human neuroimaging. We highlight emerging clinical evidence suggesting that anatomical and functional changes in these circuits contribute to the transition from acute to chronic pain. We propose that assessing activity in these conserved circuits can offer new outcome measures for preclinical evaluation of analgesic efficacy to improve translation and speed drug discovery. We further suggest that targeting reward/motivation circuits may provide a path for normalizing the consequences of chronic pain to the brain, surpassing symptomatic management to promote recovery from chronic pain.
• Xie, J. Y., Qu, C., Patwardhan, A., Ossipov, M. H., Navratilova, E., Becerra, L., Borsook, D., & Porreca, F. (2014). Activation of mesocorticolimbic reward circuits for assessment of relief of ongoing pain: a potential biomarker of efficacy. Pain, 155(8), 1659-66.
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  Preclinical assessment of pain has increasingly explored operant methods that may allow behavioral assessment of ongoing pain. In animals with incisional injury, peripheral nerve block produces conditioned place preference (CPP) and activates the mesolimbic dopaminergic reward pathway. We hypothesized that activation of this circuit could serve as a neurochemical output measure of relief of ongoing pain. Medications commonly used clinically, including gabapentin and nonsteroidal anti-inflammatory drugs (NSAIDs), were evaluated in models of post-surgical (1 day after incision) or neuropathic (14 days after spinal nerve ligation [SNL]) pain to determine whether the clinical efficacy profile of these drugs in these pain conditions was reflected by extracellular dopamine (DA) release in the nucleus accumbens (NAc) shell. Microdialysis was performed in awake rats. Basal DA levels were not significantly different between experimental groups, and no significant treatment effects were seen in sham-operated animals. Consistent with clinical observation, spinal clonidine produced CPP and produced a dose-related increase in net NAc DA release in SNL rats. Gabapentin, commonly used to treat neuropathic pain, produced increased NAc DA in rats with SNL but not in animals with incisional, injury. In contrast, ketorolac or naproxen produced increased NAc DA in animals with incisional but not neuropathic pain. Increased extracellular NAc DA release was consistent with CPP and was observed selectively with treatments commonly used clinically for post-surgical or neuropathic pain. Evaluation of NAc DA efflux in animal pain models may represent an objective neurochemical assay that may serve as a biomarker of efficacy for novel pain-relieving mechanisms.
• Becerra, L., Navratilova, E., Porreca, F., & Borsook, D. (2013). Analogous responses in the nucleus accumbens and cingulate cortex to pain onset (aversion) and offset (relief) in rats and humans. Journal of neurophysiology, 110(5), 1221-6.
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  In humans, functional magnetic resonance imaging (fMRI) activity in the anterior cingulate cortex (ACC) and the nucleus accumbens (NAc) appears to reflect affective and motivational aspects of pain. The responses of this reward-aversion circuit to relief of pain, however, have not been investigated in detail. Moreover, it is not clear whether brain processing of the affective qualities of pain in animals parallels the mechanisms observed in humans. In the present study, we analyzed fMRI blood oxygen level-dependent (BOLD) activity separately in response to an onset (aversion) and offset (reward) of a noxious heat stimulus to a dorsal part of a limb in both humans and rats. We show that pain onset results in negative activity change in the NAc and pain offset produces positive activity change in the ACC and NAc. These changes were analogous in humans and rats, suggesting that translational studies of brain circuits modulated by pain are plausible and may offer an opportunity for mechanistic investigation of pain and pain relief.
• Nair, P., Yamamoto, T., Largent-Milnes, T. M., Cowell, S., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Ma, S., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2013). Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities. Bioorganic & medicinal chemistry letters, 23(17), 4975-8.
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  The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr(1)-DAla(2)-Gly(3)-Phe(4)-Gly(5)-Trp(6)-O-[3',5'-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.
• Navratilova, E., Xie, J. Y., King, T., & Porreca, F. (2013). Evaluation of reward from pain relief. Annals of the New York Academy of Sciences, 1282, 1-11.
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  The human experience of pain is multidimensional and comprises sensory, affective, and cognitive dimensions. Preclinical assessment of pain has been largely focused on the sensory features that contribute to nociception. The affective (aversive) qualities of pain are clinically significant but have received relatively less mechanistic investigation in preclinical models. Recently, operant behaviors such as conditioned place preference, avoidance, escape from noxious stimulus, and analgesic drug self-administration have been used in rodents to evaluate affective aspects of pain. An important advance of such operant behaviors is that these approaches may allow the detection and mechanistic investigation of spontaneous neuropathic or ongoing inflammatory/nociceptive (i.e., nonevoked) pain that is otherwise difficult to assess in nonverbal animals. Operant measures may allow the identification of mechanisms that contribute differentially to reflexive hypersensitivity or to pain affect and may inform the decision to progress novel mechanisms to clinical trials for pain therapy. Additionally, operant behaviors may allow investigation of the poorly understood mechanisms and neural circuits underlying motivational aspects of pain and the reward of pain relief.
• Porreca, F., Navratilova, E., Becerra, L., & Borsook, D. (2013). Analogous responses in the nucleus accumbens and cingulate cortex to pain onset (aversion) and offset (relief) in rats and humans. Journal of Neurophysiology, 110(5), 1221-1226. doi:10.1152/jn.00284.2013
• Porreca, F., Navratilova, E., Xie, J. Y., & King, T. (2013). Evaluation of reward from pain relief: Evaluation of reward from pain relief. Annals of the New York Academy of Sciences, 1282(1), 1-11. doi:10.1111/nyas.12095
• King, T., Qu, C., Okun, A., Melemedjian, O. K., Mandell, E. K., Maskaykina, I. Y., Navratilova, E., Dussor, G. O., Ghosh, S., Price, T. J., & Porreca, F. (2012). Contribution of PKMζ-dependent and independent amplification to components of experimental neuropathic pain. Pain, 153(6), 1263-73.
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  Injuries can induce adaptations in pain processing that result in amplification of signaling. One mechanism may be analogous to long-term potentiation and involve the atypical protein kinase C, PKMζ. The possible contribution of PKMζ-dependent and independent amplification mechanisms to experimental neuropathic pain was explored in rats with spinal nerve ligation (SNL) injury. SNL increased p-PKMζ in the rostral anterior cingulate cortex (rACC), a site that mediates, in part, the unpleasant aspects of pain. Inhibition of PKMζ within the rACC by a single administration of ζ-pseudosubstrate inhibitory peptide (ZIP) reversed SNL-induced aversiveness within 24 hours, whereas N-methyl-d-aspartate receptor blockade with MK-801 had no effects. The SNL-induced aversive state (reflecting "spontaneous" pain), was re-established in a time-dependent manner, with full recovery observed 7 days post-ZIP administration. Neither rACC ZIP nor MK-801 altered evoked responses. In contrast, spinal ZIP or MK-801, but not scrambled peptide, transiently reversed evoked hypersensitivity, but had no effect on nerve injury-induced spontaneous pain. PKMζ phosphorylation was not altered by SNL in the spinal dorsal horn. These data suggest that amplification mechanisms contribute to different aspects of neuropathic pain at different levels of the neuraxis. Thus, PKMζ-dependent amplification contributes to nerve injury-induced aversiveness within the rACC. Moreover, unlike mechanisms maintaining memory, the consequences of PKMζ inhibition within the rACC are not permanent in neuropathic pain, possibly reflecting the re-establishment of amplification mechanisms by ongoing activity of injured nerves. In the spinal cord, however, both PKMζ-dependent and independent mechanisms contribute to amplification of evoked responses, but apparently not spontaneous pain.
• Navratilova, E., Xie, J. Y., Okun, A., Qu, C., Eyde, N., Ci, S., Ossipov, M. H., King, T., Fields, H. L., & Porreca, F. (2012). Pain relief produces negative reinforcement through activation of mesolimbic reward-valuation circuitry. Proceedings of the National Academy of Sciences of the United States of America, 109(50), 20709-13.
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  Relief of pain is rewarding. Using a model of experimental postsurgical pain we show that blockade of afferent input from the injury with local anesthetic elicits conditioned place preference, activates ventral tegmental dopaminergic cells, and increases dopamine release in the nucleus accumbens. Importantly, place preference is associated with increased activity in midbrain dopaminergic neurons and blocked by dopamine antagonists injected into the nucleus accumbens. The data directly support the hypothesis that relief of pain produces negative reinforcement through activation of the mesolimbic reward-valuation circuitry.
• Porreca, F., Navratilova, E., Xie, J. Y., Okun, A., Qu, C., Eyde, N., Ci, S., Ossipov, M. H., King, T., & Fields, H. L. (2012). Pain relief produces negative reinforcement through activation of mesolimbic reward–valuation circuitry. Proceedings of the National Academy of Sciences, 109(50), 20709-20713. doi:10.1073/pnas.1214605109
• Xie, J. Y., Porreca, F., Ossipov, M. H., Okun, A., Navratilova, E., Eyde, N., Deeny, T. K., & Ci, S. (2012). Pain relief activates the mesolimbic dopamine reward pathway. The FASEB Journal, 26.
• Yamamoto, T., Nair, P., Jacobsen, N. E., Kulkarni, V., Davis, P., Ma, S., Navratilova, E., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2010). Biological and conformational evaluation of bifunctional compounds for opioid receptor agonists and neurokinin 1 receptor antagonists possessing two penicillamines. Journal of medicinal chemistry, 53(15), 5491-501.
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  Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, lead to antiopioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[d-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.
• Hruby, V. J., Vanderah, T. W., Navratilova, E., Vagner, J., Jacobsen, N. E., Yamamoto, T., Nair, P., Kulkarni, V., Davis, P., Ma, S., Yamamura, H. I., Porreca, F., & Lai, J. (2009). Improving Metabolic Stability by Glycosylation: Bifunctional Peptide Derivatives That Are Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists. Journal of Medicinal Chemistry, 52(16), 5164-5175. doi:10.1021/jm900473p
• Nair, P., Yamamoto, T., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Largent, T., Ma, S., Yamamura, H. I., Vanderah, T., Lai, J., Porreca, F., & Hruby, V. J. (2009). Novel bifunctional peptides as opioid agonists and NK-1 antagonists. Advances in experimental medicine and biology, 611, 537-8.
• Salamon, Z., Fitch, J., Cai, M., Tumati, S., Navratilova, E., & Tollin, G. (2009). Plasmon-waveguide resonance studies of ligand binding to integral proteins in membrane fragments derived from bacterial and mammalian cells. Analytical biochemistry, 387(1), 95-101.
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  A procedure has been developed for directly depositing membrane fragments derived from bacterial cells (chromatophores from Rhodopseudomonas sphaeroides) and mammalian cells (mu-opioid receptor- and MC4 receptor-transfected human embryonic kidney (HEK) cells and rat trigeminal ganglion cells) on the silica surface of a plasmon-waveguide resonance (PWR) spectrometer. Binding of ligands (cytochrome c(2) for the chromatophores, the peptide agonists DAMGO and melanotan-II that are specific for the mu-opioid and MC4 receptors, and two nonpeptide agonists that are specific for the CB1 receptor) to these membrane fragments has been observed and characterized with high sensitivity using PWR spectral shifts. The K(D) values obtained are in excellent agreement with conventional pharmacological assays and with prior PWR studies using purified receptors inserted into deposited lipid bilayer membranes. These studies provide a new tool for obtaining useful biological information about receptor-mediated processes in real biological membranes.
• Yamamoto, T., Nair, P., Jacobsen, N. E., Vagner, J., Kulkarni, V., Davis, P., Ma, S., Navratilova, E., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2009). Improving metabolic stability by glycosylation: bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists. Journal of medicinal chemistry, 52(16), 5164-75.
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  In order to obtain a metabolically more stable analgesic peptide derivative, O-beta-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3',5'-Bzl(CF(3))(2)) which was a previously reported bifunctional compound with delta/micro opioid agonist and neurokinin-1 receptor antagonist activities and with a half-life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-d-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3',5'-Bzl(CF(3))(2)) was found to have effective bifunctional activities with a well-defined conformation with two beta-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 + or - 9% of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.
• Vanderah, T. W., Navratilova, E., Vagner, J., Yamamoto, T., Nair, P., Largent-Milnes, T., Davis, P., Ma, S., Moye, S., Tumati, S., Lai, J., Yamamura, H. I., Porreca, F., & Hruby, V. J. (2008). A Structure–Activity Relationship Study and Combinatorial Synthetic Approach of C-Terminal Modified Bifunctional Peptides That Are δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists. Journal of Medicinal Chemistry, 51(5), 1369-1376. doi:10.1021/jm070332f
• Yamamoto, T., Nair, P., Jacobsen, N. E., Davis, P., Ma, S., Navratilova, E., Moye, S., Lai, J., Yamamura, H. I., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2008). The importance of micelle-bound states for the bioactivities of bifunctional peptide derivatives for delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists. Journal of medicinal chemistry, 51(20), 6334-47.
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  To provide new insight into the determining factors of membrane-bound peptide conformation that might play an important role in peptide-receptor docking and further biological behaviors, the dodecylphosphocholine (DPC) micelle-bound conformations of bifunctional peptide derivatives of delta-preferring opioid agonists and NK1 antagonists (1: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF 3) 2; 2: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3,5-Bzl(CF 3) 2; 3: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bzl) were determined based on 2D NMR studies. Although the differences in the primary sequence were limited to the C-terminus, the obtained NMR conformations were unexpectedly different for each compound. Moreover, their biological activities showed different trends in direct relation to the compound-specific conformations in DPC micelles. The important result is that not only were the NK1 antagonist activities different (the pharmacophore located at the C-terminus)but the opioid agonist activities (this pharmacophore was at the structurally preserved N-terminus) also were shifted, suggesting that a general conformational change in the bioactive state was induced due to relatively small and limited structural modifications.
• Yamamoto, T., Nair, P., Vagner, J., Largent-Milnes, T., Davis, P., Ma, S., Navratilova, E., Moye, S., Tumati, S., Lai, J., Yamamura, H. I., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2008). A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists. Journal of medicinal chemistry, 51(5), 1369-76.
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  A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.
• Yue, X., Tumati, S., Navratilova, E., Strop, D., St John, P. A., Vanderah, T. W., Roeske, W. R., Yamamura, H. I., & Varga, E. V. (2008). Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner. European journal of pharmacology, 584(2-3), 272-7.
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  Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.
• Navratilova, E., Waite, S., Stropova, D., Eaton, M. C., Alves, I. D., Hruby, V. J., Roeske, W. R., Yamamura, H. I., & Varga, E. V. (2007). Quantitative evaluation of human delta opioid receptor desensitization using the operational model of drug action. Molecular pharmacology, 71(5), 1416-26.
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  Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human delta opioid receptors (hDOR) by measuring guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of beta-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of beta-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced beta-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization.
• Yamamoto, T., Nair, P., Davis, P., Ma, S., Navratilova, E., Moye, S., Tumati, S., Lai, J., Vanderah, T. W., Yamamura, H. I., Porreca, F., & Hruby, V. J. (2007). Design, synthesis, and biological evaluation of novel bifunctional C-terminal-modified peptides for delta/mu opioid receptor agonists and neurokinin-1 receptor antagonists. Journal of medicinal chemistry, 50(12), 2779-86.
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  A series of bifunctional peptides that act as agonists for delta and mu opioid receptors with delta selectivity and as antagonist for neurokinin-1 (NK1) receptors were designed and synthesized for potential application as analgesics in various pain states. The peptides were characterized using radioligand binding assays and functional assays using cell membrane and animal tissue. Optimization was performed on the fifth residue which serves as an address moiety for both receptor recognitions. It had critical effects on both activities at delta/mu opioid receptors and NK1 receptors. Among the synthesized peptides, H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF3) 2 (5) and H-Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 (7) had excellent agonist activity for both delta opioid and mu opioid receptors and excellent antagonist activity for NK1 receptors. These results indicate that the rational design of multifunctional ligands with opioid agonist and neurokinin-1 antagonist activities can be accomplished and may provide a new tool for treatment of chronic and several pain states.
• Gu, X., Ying, J., Min, B., Cain, J. P., Davis, P., Willey, P., Navratilova, E., Yamamura, H. I., Porreca, F., & Hruby, V. J. (2005). Parallel synthesis and biological evaluation of different sizes of bicyclo[2,3]-Leu-enkephalin analogues. Biopolymers, 80(2-3), 151-63.
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  Parallel synthesis of peptides and peptidomimetics has been an important approach to search for biologically active ligands. A novel systematic synthesis of different size bicyclic dipeptide mimetics was developed on solid-phase supports. By taking advantage of the enantioselective synthesis of omega-unsaturated amino acids and their N-methylated derivatives, the hemiaminal problem was prevented in the pathway to thiazolidine formation. The bicyclic dipeptide was generated on the solid-phase support in three steps by an unconventional method. By inserting this bicyclic scaffold into the synthesis of a larger bioactive peptide, 11 different sizes of bicyclo[2,3]-Leu-enkephalin analogues were synthesized in a fast and efficient way. Modeling studies show that a reversed turn structure at positions 2-3 was favored when an L- and L-bicyclic scaffold was used, and that an extended conformation at the N-terminal was favored when a D- and L-bicyclic scaffold was inserted. Binding affinities and bioassay studies show ligands with micromolar binding affinities and antagonist bioactivities for the [6,5]- and [7,5]-bicyclo-Leu-enkephalin analogues.
• Navratilova, E., Eaton, M. C., Stropova, D., Varga, E. V., Vanderah, T. W., Roeske, W. R., & Yamamura, H. I. (2005). Morphine promotes phosphorylation of the human delta-opioid receptor at serine 363. European journal of pharmacology, 519(3), 212-4.
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  After prolonged stimulation, the delta-opioid receptor becomes desensitized by regulatory mechanisms such as receptor phosphorylation, internalization and down-regulation. In this study, we demonstrate that morphine treatment causes phosphorylation of S363 in the C-terminus of the human delta-opioid receptor. Morphine-mediated phosphorylation reached 53+/-8% of maximum deltorphin II-mediated phosphorylation. Phosphorylation of S363 may contribute to delta-opioid receptor desensitization by morphine.
• Varga, E. V., Hosohata, K., Borys, D., Navratilova, E., Nylen, A., Vanderah, T. W., Porreca, F., Roeske, W. R., & Yamamura, H. I. (2005). Antinociception depends on the presence of G protein gamma2-subunits in brain. European journal of pharmacology, 508(1-3), 93-8.
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  We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein gamma2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the gamma2 subunit (Ggamma2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a delta-opioid receptor agonist. In the present study, we examined the involvement of Ggamma2 in antinociception mediated by other (mu- or kappa-opioid, cannabinoid, alpha2-adrenoreceptor) analgesic agents in a warm (55 degrees C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein gamma2 subunits in supraspinal antinociception.
• Elmagbari, N. O., Egleton, R. D., Palian, M. M., Lowery, J. J., Schmid, W. R., Davis, P., Navratilova, E., Dhanasekaran, M., Keyari, C. M., Yamamura, H. I., Porreca, F., Hruby, V. J., Polt, R., & Bilsky, E. J. (2004). Antinociceptive structure-activity studies with enkephalin-based opioid glycopeptides. The Journal of pharmacology and experimental therapeutics, 311(1), 290-7.
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  Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase 9-fluorenylmethyloxy carbamate methods. The compounds differed in the sixth and subsequent amino acid residues (Ser or Thr) and in the attached carbohydrate moiety. In vitro binding and functional smooth muscle bioassays indicated that the addition of mono- or disaccharides did not significantly affect the opioid receptor affinity or agonist activity of the glycopeptides compared with their unglycosylated parent peptides. All of the glycopeptides tested produced potent antinociceptive effects in male ICR mice following intracerebroventricular injection in the 55 degrees C tail-flick test. The calculated A(50) values for the Ser/Thr and monosaccharide combinations were all very similar with values ranging from 0.02 to 0.09 nmol. Selected compounds were administered to mice intravenously and tested for antinociception to indirectly assess serum stability and BBB penetration. All compounds tested produced full antinociceptive effects with calculated A (50) values ranging from 2.2 to 46.4 micromol/kg with the disaccharides having potencies that equaled or exceeded that of morphine on a micromoles per kilogram basis. Substitution of a trisaccharide or bis- and tris-monosaccharides resulted in a decrease in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase central nervous system bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.
• Gu, X., Ying, J., Agnes, R. S., Navratilova, E., Davis, P., Stahl, G., Porreca, F., Yamamura, H. I., & Hruby, V. J. (2004). Novel design of bicyclic beta-turn dipeptides on solid-phase supports and synthesis of [3.3.0]-Bicyclo([2,3])-leu-enkephalin analogues. Organic letters, 6(19), 3285-8.
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  [structure: see text] External bicyclic beta-turn dipeptide mimetics provide an excellent design approach that can offer a rich chiral ensemble of structures with different backbone conformations. We report herein a novel design of a convergent combinatorial synthetic methodology, which is illustrated by the solid-phase synthesis of a series of [3.3.0]-bicyclo([2,3])-Leu-enkephalin analogues. The reactions were optimized and the epimeric configurations were determined by 2D NMR spectroscopy. Biological assays show that these analogues have more potent delta binding affinity and bioactivity for delta vs micro opioid receptor, which may be related to the different conformations preferred by these analogues in our modeling studies.
• Hruby, V. J., Porreca, F., Navratilova, E., Gu, X., Ying, J., Agnes, R. S., Davis, P., Stahl, G., & Yamamura, H. I. (2004). Novel Design of Bicyclic β-Turn Dipeptides on Solid-Phase Supports and Synthesis of [3.3.0]-Bicyclo^[2,3]-Leu-enkephalin Analogues. Organic Letters, 6(19), 3285-3288. doi:10.1021/ol0488183
• Navratilova, E., Varga, E. V., Stropova, D., Jambrosic, J. C., Roeske, W. R., & Yamamura, H. I. (2004). Mutation S363A in the human delta-opioid receptor selectively reduces down-regulation by a peptide agonist. European journal of pharmacology, 485(1-3), 341-3.
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  Chemically distinct opioid agonists have different abilities to down-regulate opioid receptors. The present study investigated the role of Ser(363) in human delta-opioid receptor down-regulation by a delta-selective peptide- and non-peptide agonist. Cyclic[D-Pen(2),D-Pen(5)]enkephalin (DPDPE)-mediated down-regulation was significantly attenuated by a S363A mutation. In contrast, this mutation had no effect on down-regulation by (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80). These results demonstrate that the molecular mechanism of the human delta-opioid receptor down-regulation is agonist-specific.
• Varga, E. V., Navratilova, E., Stropova, D., Jambrosic, J., Roeske, W. R., & Yamamura, H. I. (2004). Agonist-specific regulation of the delta-opioid receptor. Life sciences, 76(6), 599-612.
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  Delta opioid receptor (DOR) agonists are attractive potential analgesics, since these compounds exhibit strong antinociceptive activity with relatively few side effects. In the past decade, several novel classes of delta-opioid agonists have been synthesized. Recent experimental data indicate that structurally distinct opioid agonists interact differently with the delta-opioid receptor. Consequently, individual agonist-bound DOR conformations may interact differently with intracellular proteins. In the present paper, after a brief review of the cellular processes that contribute to homologous desensitization of the DOR signaling, we shall focus on experimental data demonstrating that chemically different agonists differ in their ability to phosphorylate, internalize, and/or down-regulate the DOR. Homologous regulation of the opioid receptor signaling is thought to play an important role in the development of opioid tolerance. Therefore, agonist-specific differences in DOR regulation suggest that by further chemical modification, delta-selective opioid analgesics can be designed that exhibit a reduced propensity for analgesic tolerance.
• Yamamura, H. I., Varga, E. V., Tourwe, D., Toth, G., Tomboly, C., Szucs, M., Roeske, W. R., Peter, A., Navratilova, E., Keresztes, A., Fulop, F., & Borsodi, A. (2004). New endomorphin analogs with mu-agonist and delta-antagonist properties. Pure and Applied Chemistry, 76(5), 951-957. doi:10.1351/pac200476050951
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  Endomorphins (endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2, endomorphin-2, Tyr-Pro-Phe-Phe-NH 2 ) are potent and selective μ-opioid receptor agonists. In order to improve the affinity and chemical stability of endomorphins, we have designed, synthesized, and characterized novel analogs with unnatural (2', 6'-dimethyltyrosine, Dmt) and/or i3-alicyclic amino acids (ACPC and ACHC). Radioligand binding assay indicated that several of the novel analogs exhibit high affinity for both μ- and δ-opioid receptors in rat- or mouse-brain membrane preparations. The most promising derivatives-such as Dmt-Pro-Trp/Phe-Phe-NH 2 , Dmt-(1S,2R)-ACPC-Phe-Phe-NH2, and Dmt-(1S,2R)-ACHC-Phe-Phe-NH 2 )-were characterized in recombinant cell lines expressing human μ- or δ-opioid receptors. Interestingly, while these novel peptides were potent opioid agonists in the functional [ 3 5 S]GTPγS binding assays in Chinese hamster ovary cells expressing the μ-opioid receptors, some behaved as antagonist or inverse agonist in the human δ-opioid receptor-expressing CHO cells. Since it has previously been demonstrated that the coadministration of 6-antagonists with μ-analgesics attenuates the development of analgesic tolerance, introduction of high-affinity δ-antagonist properties into the μ-agonist endomorphins is expected to lead to potent analgesics that produce limited tolerance.
• Okura, T., Varga, E. V., Hosohata, Y., Navratilova, E., Cowell, S. M., Rice, K., Nagase, H., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2003). Agonist-specific down-regulation of the human delta-opioid receptor. European journal of pharmacology, 459(1), 9-16.
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  Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.
• Smith, T. L., & Navratilova, E. (2003). The effect of ethanol exposure on mitogen-activated protein kinase activity and expression in cultured rat astrocytes. Neuroscience letters, 341(2), 91-4.
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  The effects of ethanol exposures on mitogen-activated protein kinase (MAPK) activity were determined in confluent astrocyte monolayers prepared from neonatal rat cerebral cortex. Acute 30 min exposure to 50 mM ethanol had no significant effect on MAPK activity. However, chronic exposure to ethanol for 4 days elicited a concentration-dependent increase in the basal level of this enzyme activity with no parallel increase in its protein expression. In addition, the magnitude of MAPK activation by epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor was significantly increased above corresponding control values in cells chronically exposed to ethanol. Immunolabeling experiments indicated that the protein expression of receptors for these growth factors was unaffected by ethanol treatment. Our results suggest that even after chronic ethanol treatment, MAPK phosphorylation and, hence, activation remains elevated.
• Varga, E. V., Yamamura, H. I., Rubenzik, M. K., Stropova, D., Navratilova, E., & Roeske, W. R. (2003). Molecular mechanisms of excitatory signaling upon chronic opioid agonist treatment. Life sciences, 74(2-3), 299-311.
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  Opioid receptor agonists mediate their analgesic effects by interacting with Gi/o protein-coupled opioid receptors. Acute treatment with opioid agonists is thought to mediate analgesia by hyperpolarization of presynatic neurons, leading to the inhibition of excitatory (pain) neurotransmitters release. After chronic treatment however, the opioid receptors gradually become less responsive to agonists, and increased drug doses become necessary to maintain the therapeutic effect (tolerance). Analgesic tolerance is the result of two, partially overlapping processes: a gradual loss of inhibitory opioid function is accompanied by an increase in excitatory signaling. Recent data indicate that chronic opioid agonist treatment simultaneously desensitizes the inhibitory-, and augments the stimulatory effects of the opioids. In the present paper we review the molecular mechanisms that may have a role in the augmentation of the excitatory signaling upon chronic opioid agonist treatment. We also briefly review our recent experimental data on the molecular mechanism of chronic opioid agonist-mediated functional sensitization of forskolin-stimulated cAMP formation, in a recombinant Chinese hamster ovary cell line stably expressing the human delta-opioid receptor (hDOR/CHO). To interpret the experimental data, we propose that chronic hDOR activaton leads to activation of multiple redundant signaling pathways that converge to activate the protein kinase, Raf-1. Raf-1 in turn phosphorylates and sensitizes the native adenylyl cyclase VI isoenzyme in hDOR/CHO cells, causing a rebound increase in forskolin-stimulated cAMP formation upon agonist withdrawal.
• Smith, T. L., & Navratilova, E. (1999). Increased calcium/calmodulin protein kinase activity in astrocytes chronically exposed to ethanol: influences on glutamate transport. Neuroscience letters, 269(3), 145-8.
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  The effects of ethanol exposures on calcium/calmodulin-dependent protein kinase activity as well as its influence on glutamate uptake were determined in astrocytes prepared from neonatal rat cerebral cortex. Acute 15-min exposure to 100 mM ethanol had no effect on Ca2+/CaM-dependent protein kinase activity. However, chronic exposure to 100 mM ethanol for 4 days elicited a significant increase in the activity of this enzyme with no parallel increase in its expression. Ca2+/CaM-independent kinase activity was less than 1% of the Ca2+/CaM-dependent kinase activity and was unaffected by any of the ethanol exposures. Exposure to 100 mM ethanol for four days also resulted in a significant increase in Na+-dependent [3H]glutamate uptake which was reversed when ethanol-exposed astrocytes were co-incubated with KN-93, a specific inhibitor of Ca2+/CaM kinase. These results suggest that the effects of ethanol on glutamate transport may be mediated in part, by the level of Ca2+/CaM kinase activity.
• van Antwerpen, R., La Belle, M., Navratilova, E., & Krauss, R. M. (1999). Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy. Journal of lipid research, 40(10), 1827-36.
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  Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized. The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.

Reviews

• Navratilova, E., Morimura, K., Xie, J. Y., Atcherley, C. W., Ossipov, M. H., & Porreca, F. (2016. Positive emotions and brain reward circuits in chronic pain.
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  Chronic pain is an important public health problem that negatively impacts quality of life of affected individuals and exacts enormous socio-economic costs. Chronic pain is often accompanied by comorbid emotional disorders including anxiety, depression and possibly anhedonia. The neural circuits underlying the intersection of pain and pleasure are not well understood. We summarize recent human and animal investigations demonstrating that aversive aspects of pain are encoded in brain regions overlapping with areas processing reward and motivation. We highlight findings revealing anatomical and functional alterations of reward/motivation circuits in chronic pain. Finally, we review supporting evidence for the concept that pain relief is rewarding and activates brain reward/motivation circuits. Adaptations in brain reward circuits may be fundamental to the pathology of chronic pain. Knowledge of brain reward processing in the context of pain could lead to the development of new therapeutics for the treatment of emotional aspects of pain and comorbid conditions. This article is protected by copyright. All rights reserved.