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The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbEs antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments.

The μ-opioid receptor (μOR) represents an important target of therapeutic and abused drugs. So far, most understanding of μOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet μOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated μOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the μOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new μOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.

Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both in vivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.

Traditional methods for site-specific drug delivery in the brain are slow, invasive, and difficult to interface with recordings of neural activity. Here, we demonstrate the feasibility and experimental advantages of in vivo photopharmacology using caged opioid drugs that are activated in the brain with light after systemic administration in an inactive form. To enable bidirectional manipulations of endogenous opioid receptors in vivo, we developed photoactivatable oxymorphone (PhOX) and photoactivatable naloxone (PhNX), photoactivatable variants of the mu opioid receptor agonist oxymorphone and the antagonist naloxone. Photoactivation of PhOX in multiple brain areas produced local changes in receptor occupancy, brain metabolic activity, neuronal calcium activity, neurochemical signaling, and multiple pain- and reward-related behaviors. Combining PhOX photoactivation with optical recording of extracellular dopamine revealed adaptations in the opioid sensitivity of mesolimbic dopamine circuitry in response to chronic morphine administration. This work establishes a general experimental framework for using in vivo photopharmacology to study the neural basis of drug action.

BACKGROUND: Mu opioid receptors (MORs) are key for reward processing, mostly studied in dopaminergic pathways. MORs are also expressed in the dorsal raphe nucleus (DRN), which is central for the modulation of reward and mood, but MOR function in the DRN remains underexplored. Here, we investigated whether MOR-expressing neurons of the DRN (DRN-MOR neurons) participate in reward and emotional responses. METHODS: We characterized DRN-MOR neurons anatomically using immunohistochemistry and functionally using fiber photometry in responses to morphine and rewarding/aversive stimuli. We tested the effect of opioid uncaging on the DRN on place conditioning. We examined the effect of DRN-MOR neuron optostimulation on positive reinforcement and mood-related behaviors. We mapped their projections and selected DRN-MOR neurons projecting to the lateral hypothalamus for a similar optogenetic experimentation. RESULTS: DRN-MOR neurons form a heterogeneous neuronal population essentially composed of GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons. Calcium activity of DRN-MOR neurons was inhibited by rewarding stimuli and morphine. Local photo-uncaging of oxymorphone in the DRN produced conditioned place preference. DRN-MOR neuron optostimulation triggered real-time place preference and was self-administered, promoted social preference, and reduced anxiety and passive coping. Finally, specific optostimulation of DRN-MOR neurons projecting to the lateral hypothalamus recapitulated the reinforcing effects of total DRN-MOR neuron stimulation. CONCLUSIONS: Our data show that DRN-MOR neurons respond to rewarding stimuli and that their optoactivation has reinforcing effects and promotes positive emotional responses, an activity which is partially mediated by their projections to the lateral hypothalamus. Our study also suggests a complex regulation of DRN activity by MOR opioids, involving mixed inhibition/activation mechanisms that fine-tune DRN function.

Sarath S Nair,1 Jyothi Nagesh,1 Shambhavi C,2 Anshul Singh,1 Shirley Lewis,1 Umesh Velu,1 Deepika Chenna3 1Department of Radiation Oncology, Kasturba Medical College Manipal MAHE, Manipal, Karnataka, India; 2Department of Medical Radiation Physics Program, MCHP Manipal, MAHE, Manipal, Karanataka, India; 3Department of Immunohematology and Blood Transfusion, Kasturba Medical College Manipal MAHE, Manipal, Karnataka, IndiaCorrespondence: Jyothi Nagesh, Email jyothi.nagesh@manipal.eduObjective: To identify a suitable approach for blood irradiation other than the commonly used water medium and to study the impact of different algorithm dose computations.Methods: Water is the commonly used medium for blood irradiation. In this study computed tomography scans were taken with locally made blood irradiation phantoms other than water, by using air, rice powder and thermocole using parallel beam for 25 Gy. Plans were recalculated for different algorithms such as collapsed cone (CC), Monte Carlo (MC) and pencil beam (PB). The dose–volume parameters and measured doses were collected and analyzed for each medium and algorithm.Findings: The monitor unit (MU) for rice powder and water are close (2461± 57 and 2469± 61, respectively), with a maximum dose of 28.0± 1.8 and 28.0± 1.9 Gy. The PB algorithm resulted in lower monitor unit values regardless of the medium used, generating values of 2418, 2406, 2382, and 2362 for water, rice powder, air, and Thermocol, respectively. A significant increase in dose was observed irrespective of the medium used when the MC algorithm was employed, with a maximum of 30.26 Gy in rice powder; a smaller dose was used when the CC algorithm was employed, with 26.3 Gy in water medium. The average maximum doses of all groups were equal using the one-way Anova statistical test. Regarding the impact of field size, rice powder appears to have consistent doses across various field sizes, with slight increases as field size grows, which is similar to water.Novelty/Applications: While water is the conventional medium, this study highlights the potential benefits of rice powder, such as eliminating the risks associated with bubble formation and water spillage, which can lead to equipment malfunction and safety hazards. Although previous studies have explored rice powder as a bolus and tissue-equivalent material, this study uniquely applies this knowledge to blood irradiation, an area where rice powder has not been thoroughly investigated.Keywords: computed tomography, collapsed cone, Monte Carlo, pencil beam, monitor units, MU, treatment planning station.

Photoactivatable drugs and peptides can drive quantitative studies into receptor signaling with high spatiotemporal precision, yet few are compatible with behavioral studies in mammals. We developed CNV-Y-DAMGO-a caged derivative of the mu opioid receptor-selective peptide agonist DAMGO. Photoactivation in the mouse ventral tegmental area produced an opioid-dependent increase in locomotion within seconds of illumination. These results demonstrate the power of in vivo photopharmacology for dynamic studies into animal behavior.

AbstractThe anterior cingulate cortex (ACC) processes the affective component of pain, whereas the primary somatosensory cortex (S1) is involved in its sensory-discriminative component. Injection of morphine in the ACC has been reported to be analgesic, and endogenous opioids in this area are required for pain relief. Mu opioid receptors (MORs) are expressed in both ACC and S1; however, the identity of MOR-expressing cortical neurons remains unknown. Using the Oprm1-mCherry mouse line, we performed selective patch clamp recordings of MOR+ neurons, as well as immunohistochemistry with validated neuronal markers, to determine the identity and laminar distribution of MOR+ neurons in ACC and S1. We found that the electrophysiological signatures of MOR+ neurons differ significantly between these 2 areas, with interneuron-like firing patterns more frequent in ACC. While MOR+ somatostatin interneurons are more prominent in ACC, MOR+ excitatory neurons and MOR+ parvalbumin interneurons are more prominent in S1. Our results suggest a differential contribution of MOR-mediated modulation to ACC and S1 outputs. We also found that females had a greater density of MOR+ neurons compared with males in both areas. In summary, we conclude that MOR-dependent opioidergic signaling in the cortex displays sexual dimorphisms and likely evolved to meet the distinct function of pain-processing circuits in limbic and sensory cortical areas.

The most common equine tapeworm, Anoplocephala perfoliata, has often been neglected amongst molecular investigations and has been faced with limited treatment options. However, the recent release of a transcriptome dataset has now provided opportunities for in-depth analysis of A. perfoliata protein expression. Here, global, and sub-proteomic approaches were utilized to provide a comprehensive characterization of the A. perfoliata soluble glutathione transferases (GST) (ApGST). Utilizing both bioinformatics and gel-based proteomics, GeLC and 2D-SDS PAGE, the A. perfoliata ‘GST-ome’ was observed to be dominated with Mu class GST representatives. In addition, both Sigma and Omega class GSTs were identified, albeit to a lesser extent and absent from affinity chromatography approaches. Moreover, 51 ApGSTs were localized across somatic (47 GSTs), extracellular vesicles (EVs) (Whole: 1 GST, Surface: 2 GSTs) and EV depleted excretory secretory product (ESP) (9 GSTs) proteomes. In related helminths, GSTs have shown promise as novel anthelmintic or vaccine targets for improved helminth control. Thus, provides potential targets for understanding A. perfoliata novel infection mechanisms, host–parasite relationships and anthelmintic treatments.

Purpose: The present study comparatively evaluates the impact of energy-matched flattening filter-free (FFF) photon beams with different energy levels on the physical-dosimetric quality of lung and liver stereotactic body radiotherapy (SBRT) treatment plans. Methods: For this purpose, 54 different lung and liver lesions from 44 patients who had already received SBRT combined with volumetric modulated arc therapy (VMAT) were included in this retrospective planning study. Planning computed tomography scans already available were used for the renewed planning with 6 MV, 6 MV-FFF, 10 MV, and 10 MV-FFF under constant planning objectives. The treatment delivery data, dosimetric distributions, and dose–volume histograms as well as parameters such as the conformity index and gradient indices were the basis for the evaluation and comparison of treatment plans. Results: A significant reduction of beam-on time (BOT) was achieved due to the high dose rates of FFF beams. In addition, we showed that for FFF beams compared to flattened beams of the same energy level, smaller planning target volumes (PTV) require fewer monitor units (MU) than larger PTVs. An equal to slightly superior target volume coverage and sparing of healthy tissue as well as organs at risk in both lung and liver lesions were found. Significant differences were seen mainly in the medium to lower dose range. Conclusion: We found that FFF beams together with VMAT represent an excellent combination for SBRT of lung or liver lesions with shortest BOT for 10 MV-FFF but significant dose savings for 6 MV-FFF in lung lesions. [ABSTRACT FROM AUTHOR]

Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.

Functional interactions between G protein-coupled receptors are poised to enhance neuronal sensitivity to neuromodulators and therapeutic drugs. Mu and delta opioid receptors (MORs and DORs) can interact when overexpressed in the same cells, but whether co-expression of endogenous MORs and DORs in neurons leads to functional interactions is unclear. Here, in mice, we show that both MORs and DORs inhibit parvalbumin-expressing basket cells (PV-BCs) in hippocampal CA1 through partially occlusive signaling pathways that terminate on somato-dendritic potassium channels and presynaptic calcium channels. Using photoactivatable opioid neuropeptides, we find that DORs dominate the response to enkephalin in terms of both ligand sensitivity and kinetics, which may be due to relatively low expression levels of MOR. Opioid-activated potassium channels do not show heterologous desensitization, indicating that MORs and DORs signal independently. In a direct test for heteromeric functional interactions, the DOR antagonist TIPP-Psi does not alter the kinetics or potency of either the potassium channel or synaptic responses to photorelease of the MOR agonist [d-Ala2, NMe-Phe4, Gly-ol5]enkephalin (DAMGO). Thus, aside from largely redundant and convergent signaling, MORs and DORs do not functionally interact in PV-BCs in a way that impacts somato-dendritic potassium currents or synaptic transmission. These findings imply that cross-talk between MORs and DORs, either in the form of physical interactions or synergistic intracellular signaling, is not a preordained outcome of co-expression in neurons.

There is a need to develop a novel analgesic for pain associated with interstitial cystitis/painful bladder syndrome (IC/PBS). The use of the conventional μ-opioid receptor agonists to manage IC/PBS pain is controversial due to adverse CNS effects. These effects are attenuated in benzylideneoxymorphone (BOM), a low-efficacy μ-opioid receptor agonist/δ-opioid receptor antagonist that attenuates thermal pain and is devoid of reinforcing effects. We hypothesize that BOM will inhibit bladder pain by attenuating responses of urinary bladder distension (UBD)-sensitive afferent fibers. Therefore, the effect of BOM was tested on responses of UBD-sensitive afferent fibers in L6 dorsal root from inflamed and non-inflamed bladder of rats. Immunohistochemical (IHC) examination reveals that following the induction of inflammation there were significant high expressions of μ, δ, and μ-δ heteromer receptors in DRG. BOM dose-dependently (1-10 mg/kg, i.v) attenuated mechanotransduction properties of these afferent fibers from inflamed but not from non-inflamed rats. In behavioral model of bladder pain, BOM significantly attenuated visceromotor responses (VMRs) to UBD only in inflamed group of rats when injected either systemically (10 mg/kg, i.v.) or locally into the bladder (0.1 ml of 10 mg/ml). Furthermore, oxymorphone (OXM), a high-efficacy μ-opioid receptor agonist, attenuated responses of mechanosensitive bladder afferent fibers and VMRs to UBD. Naloxone (10 mg/kg, i.v.) significantly reversed the inhibitory effects of BOM and OXM on responses of bladder afferent fibers and VMRs suggesting μ-opioid receptor-related analgesic effects of these compounds. The results reveal that a low-efficacy, bifunctional opioid-based compound can produce analgesia by attenuating mechanotransduction functions of afferent fibers innervating the urinary bladder.

Temel bilgi edinme yollarından biri olan haber; tefsir, hadis, fıkıh, kelâm ve belâgat gibi farklı ilmî disiplinlerin konusu olmuştur. Özellikle Kur’an’ın Hz. Peygamber’e Allah (c.c.) tarafından vahiy yoluyla indirilen haber topluluğu olması, haberin İslâmî ilimlerin ortak payesi olmasına yol açmıştır. İslâmî ilimlerin tamamında haberden bir şekilde söz edilse de fıkıh usûlü ve kelâm âlimleri onu tanım, mahiyet, nitelik ve nicelik bakımından derinlemesine incelemişlerdir. Öyle ki usûlcüler ve kelâmcılar bütün fertlerini kapsayabilecek tanımlar oluşturmaya çalışmanın yanında birtakım itikadî kaygıları da gözetmişler ve bunu benimsedikleri haber tanımına yansıtmaya çalışmışlardır. Mu‘tezilî ve Eş‘arî usûlcülerde bu durum çok net bir şekilde görülmektedir. Mu‘tezilî ve Eş‘arî usûlcülerin haberin içeriği ve kısımlarıyla ilgili görüşlerini inceleyen çalışmalar bulunmakla birlikte onların haber tanımları ilmî bir terim olarak incelenmemiştir. İşte bu araştırmada Cübbâîlerden Fahreddin er-Râzî’ye kadarki dönemde yaşayan Mu‘tezilî ve Eş‘arî usûlcülerin haber tanımları analiz edilmiş ve onların bu tanımlarda gözettiği itikadî ve dilbilimsel hususlar tespit edilmeye çalışılmıştır. Araştırma giriş ve sekiz bölümden oluşmaktadır. Girişte haberin sözlük anlamından ve İslâmî ilimler içindeki özel konumundan söz edilmiş, sekiz bölümde ise Cübbâîlerden Fahreddin er-Râzî’ye kadarki dönemde yaşayan Mu‘tezilî ve Eş‘arî usûlcülerin haber tanımları analiz edilmiştir.

Temel bilgi edinme yollarından biri olan haber tefsir, hadis, fıkıh, kelâm ve belâgat gibi farklı ilmî disiplinlerin konusu olmuştur. Özellikle Kur’an’ın Hz. Peygamber’e Allah (c.c.) tarafından vahiy yoluyla indirilen haber topluluğu olması, haberin İslâmî ilimlerin ortak payesi olmasına yol açmıştır. İslâmî ilimlerin tamamında haberden bir şekilde söz edilse de fıkıh usûlü ve kelâm âlimleri onu tanım, mahiyet, nitelik ve nicelik bakımından derinlemesine incelemişlerdir. Öyle ki usûlcüler ve kelâmcılar bütün fertlerini kapsayabilecek tanımlar oluşturmaya çalışmanın yanında birtakım itikadî kaygıları da gözetmişler ve bunu benimsedikleri haber tanımına yansıtmaya çalışmışlardır. Mu‘tezilî ve Eş‘arî usûlcülerde bu durum çok net bir şekilde görülmektedir. Mu‘tezilî ve Eş‘arî usûlcülerin haberin içeriği ve kısımlarıyla ilgili görüşlerini inceleyen çalışmalar bulunmakla birlikte onların haber tanımları ilmî bir terim olarak incelenmemiştir. İşte bu araştırmada 6./12. ve 7./13. yüzyıllarda yaşayan Mu‘tezilî ve Eş‘arî usûlcülerden Fahreddin er-Râzî, Sekkâkî ve Seyfeddin el-Âmidî’nin haber tanımları analiz edilmiş ve onların bu tanımlarda gözettiği itikâdî ve dilbilimsel hususlar tespit edilmeye çalışılmıştır. Daha önceki yüzyıllarda Mu‘tezilî ve Eş‘arî usûlcüler nezdinde genel itibariyle üç haber tanımı şöhret bulmuştur. Bunlardan biri Ebü’l-Hüseyin el-Basrî’ye aittir. Diğer iki tanım ise “Kendisine doğru ve yalan denilebilen söz” şeklindeki tanımın çeşitli formlarıdır. Bu tanımlarda “onaylama ve “yalanlama” ifadelerinin yanı sıra “ve” ve “veya” bağlaçlarından hangisinin kullanılmasının gerektiği tartışılmış ve çoğunlukla bütün fertlerini kapsamına alamaması, kapsamadığı bazı fertleri çıkaramaması, bilinmeyen kavramlarla açıklanması ve kısır döngüye yol açması gibi nedenlerle eleştirmiştir. Araştırmada Râzî, Sekkâkî ve Âmidî’nin sözü geçen tanımlara yönelttikleri eleştiriler aktarılmış, sonra da her birinin benimsediği görüş yahut haber tanımı açıklanmıştır. Usûlcülerin konuyla ilgili görüşleri vefat sırasına göre ve ayrı başlıklar altında ele alınmış, bunun bir sonucu olarak önce Râzî’nin, sonra Sekkâkî’nin en sonunda da Âmidî’nin yaklaşımı incelenmiştir. Yine bu başlıklarda adı anılan üç usûlcünün görüşü tıpkı eserlerindeki gibi maddeler halinde açıklanmış, yanı sıra eleştirdiği kişi ve fırkalar ile kullandığı yöntem de tespit edilmeye çalışılmıştır. Ayrıca araştırmada Râzî, Sekkâkî ve Âmidî’nin konuyla ilgili yaklaşımı mukayese edilmiş, arka planda hangi kaygıları taşıdıkları, birbirlerini ve kendilerinden sonraki usûlcüleri ne şekilde etkiledikleri tespit edilmiştir. Böylece Râzî, Sekkâkî ve Âmidî’nin haberin tanımı özelinde görüşleri, kendilerinden önceki ve sonraki dönemle birlikte incelenmiş olmakta ve okuyucuya geniş bir bakış açısı sunmaktadır. Fıkıh usûlü, kelâm ve belâgat ilişkisinin vurgulandığı araştırmanın en önemli hedeflerinden biri, haberin tanımı dolayısıyla İslâmî ilimler arasındaki ilişkiyi ortaya koymaktır. Görüşlerini açıkladığımız âlimlerin birden fazla alanda derinleşmiş olması bu hedefi gerçekleştirmeyi mümkün kılan en önemli sâiktir. Öyle ki Râzî ve Sekkâkî, belâgat eserlerinde dahi usûlcülerin ve kelâmcıların haber tanımlarını incelemiş ve bu konudaki görüşünü yazdığı eserin ait olduğu ilim dalına göre değil, kendi bilgi anlayışına göre oluşturmuş âlimlerdir. Dahası, 6./12. ve 7./13. yüzyıllarda kaleme alına eserlerdeki bir kavramın tartışılması nedeniyle araştırmada mantık ilminin fıkıh usûlündeki kullanımından misaller de bulunmaktadır. Böylece fıkıh usûlü, kelâm ve belâgat disiplinleri arasındaki ilişki somut misaller üzerinden gösterilmiş olmaktadır ki bu, günümüz araştırmacıları için yeni bir araştırma sahası olabilir. Sırf bundan ötürü araştırmada Râzî, Sekkâkî ve Âmidî’nin fıkıh usûlü ve belâgat eserlerinin yanında önceki asırlarda yazılan usûl eserleri ile bazı kelâm eserlerinden de faydalanılmıştır. Araştırma giriş ve beş bölümden oluşmaktadır. Girişte haberin sözlük anlamından, İslâmî ilimler içindeki özel konumundan ve araştırmanın içeriğinden söz edilmiştir. Birinci bölümde Râzî’nin, ikinci bölümde Sekkâkî’nin, üçüncü bölümde ise Âmidî’nin haberin tanımı ve mahiyeti hakkındaki görüşleri, eleştirileri ve sundukları çözüm açıklanmıştır. Dördüncü bölümde sözü geçen üç usûlcünün konuyla ilgili görüşü ve metodolojisi genel bir değerlendirmeyle mukayese edilmiş, son bölümde ise sonraki dönemlerdeki usûlcülere etkisi ortaya koyulmaya çalışılmıştır.

Opioid-induced respiratory depression (OIRD) causes death following an opioid overdose, yet the neurobiological mechanisms of this process are not well understood. Here, we show that neurons within the lateral parabrachial nucleus that express the µ-opioid receptor (PBL Oprm1 neurons) are involved in OIRD pathogenesis. PBL Oprm1 neuronal activity is tightly correlated with respiratory rate, and this correlation is abolished following morphine injection. Chemogenetic inactivation of PBL Oprm1 neurons mimics OIRD in mice, whereas their chemogenetic activation following morphine injection rescues respiratory rhythms to baseline levels. We identified several excitatory G protein-coupled receptors expressed by PBL Oprm1 neurons and show that agonists for these receptors restore breathing rates in mice experiencing OIRD. Thus, PBL Oprm1 neurons are critical for OIRD pathogenesis, providing a promising therapeutic target for treating OIRD in patients.

Clinical µ-opioid receptor (MOR) agonists produce hyperalgesic priming, a form of maladaptive nociceptor neuroplasticity, resulting in pain chronification. We have established an in vitro model of opioid-induced hyperalgesic priming (OIHP), in male rats, to identify nociceptor populations involved and its maintenance mechanisms. OIHP was induced in vivo by systemic administration of fentanyl and confirmed by prolongation of prostaglandin E2 (PGE2) hyperalgesia. Intrathecal cordycepin, which reverses Type I priming, or the combination of Src and mitogen-activated protein kinase (MAPK) inhibitors, which reverses Type II priming, both partially attenuated OIHP. Parallel in vitro experiments were performed on small-diameter (

Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs, antidepressants and SSRI’s. Within the opioid family mu and kappa opioid receptor are commonly targeted. However, activation of the mu opioid receptor has side effects of constipation, tolerance, dependence, euphoria, and respiratory depression; activation of the kappa opioid receptor leads to dysphoria and sedation. The side effects of mu opioid receptor activation have led to mu receptor drugs being widely abused with great overdose risk. For these reasons, newer safer opioid analgesics are in high demand. For many years a focus within the opioid field was finding drugs that activated the G protein pathway at mu opioid receptor, without activating the β-arrestin pathway, known as biased agonism. Recent advances have shown that this may not be the way forward to develop safer analgesics at mu opioid receptor, though there is still some promise at the kappa opioid receptor. Here we discuss recent novel approaches to develop safer opioid drugs including efficacy vs bias and fine-tuning receptor activation by targeting sub-pockets in the orthosteric site, we explore recent works on the structural basis of bias, and we put forward the suggestion that Gα subtype selectivity may be an exciting new area of interest.

The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.

μ-Opioid receptors (MORs) are densely expressed in different brain regions known to mediate reward. One such region is the striatum where MORs are densely expressed, yet the role of these MOR populations in modulating reward is relatively unknown. We have begun to address this question by using a series of genetically engineered mice based on the Cre recombinase/loxP system to selectively delete MORs from specific neurons enriched in the striatum: dopamine 1 (D1) receptors, D2 receptors, adenosine 2a (A2a) receptors, and choline acetyltransferase (ChAT). We first determined the effects of each deletion on opioid-induced locomotion, a striatal and dopamine-dependent behavior. We show that MOR deletion from D1 neurons reduced opioid (morphine and oxycodone)-induced hyperlocomotion, whereas deleting MORs from A2a neurons resulted in enhanced opioid-induced locomotion, and deleting MORs from D2 or ChAT neurons had no effect. We also present the effect of each deletion on opioid intravenous self-administration. We first assessed the acquisition of this behavior using remifentanil as the reinforcing opioid and found no effect of genotype. Mice were then transitioned to oxycodone as the reinforcer and maintained here for 9 d. Again, no genotype effect was found. However, when mice underwent 3 d of extinction training, during which the drug was not delivered, but all cues remained as during the maintenance phase, drug-seeking behavior was enhanced when MORs were deleted from A2a or ChAT neurons. These findings show that these selective MOR populations play specific roles in reward-associated behaviors.

BackgroundHuman laboratory paradigms are a pillar in medication development for alcohol use disorders (AUD). Neuroimaging paradigms, in which individuals are exposed to cues that elicit neural correlates of alcohol craving (e.g., mesocorticolimbic activation), are increasingly utilized to test the effects of AUD medications. Elucidation of the translational effects of these neuroimaging paradigms on human laboratory paradigms, such as self-administration, is warranted. The current study is a secondary analysis examining whether alcohol cue-induced activation in the ventral striatum is predictive of subsequent alcohol self-administration in the laboratory.MethodsNon-treatment-seeking heavy drinkers of East Asian descent (n = 41) completed a randomized, placebo-controlled, double-blind, crossover experiment on the effects of naltrexone on neuroimaging and human laboratory paradigms. Participants completed 5 days of study medication (or placebo); on day 4, they completed a neuroimaging alcohol taste cue-reactivity task. On the following day (day 5), participants completed a 60-minute alcohol self-administration paradigm.ResultsMultilevel Cox regressions indicated a significant effect of taste cue-elicited ventral striatum activation on latency to first drink, Wald χ2  = 2.88, p = 0.05, such that those with higher ventral striatum activation exhibited shorter latencies to consume their first drink. Similarly, ventral striatum activation was positively associated with total number of drinks consumed, F(1, 38) = 5.90, p = 0.02. These effects were significant after controlling for alcohol use severity, OPRM1 genotype, and medication. Other potential regions of interest (anterior cingulate, thalamus) were not predictive of self-administration outcomes.ConclusionsNeuroimaging alcohol taste cue paradigms may be predictive of laboratory paradigms such as self-administration. Elucidation of the relationships among different paradigms will inform how these paradigms may be used synergistically in experimental medicine and medication development.

Since the isolation of morphine from the opium poppy over 200 years ago, the molecular basis of opioid action has remained the subject of intense inquiry. The identification of specific receptors responsible for opioid function and the discovery of many chemically diverse molecules with unique opioid-like efficacies have provided glimpses into the molecular logic of opioid action. Recent revolutions in the structural biology of transmembrane proteins have, for the first time, yielded high-resolution views into the 3-dimensional shapes of all 4 opioid receptors. These studies have begun to decode the chemical logic that enables opioids to specifically bind and activate their receptor targets. A combination of spectroscopic experiments and computational simulations has provided a view into the molecular movements of the opioid receptors, which itself gives rise to the complex opioid pharmacology observed at the cellular and behavioral levels. Further diversity in opioid receptor structure is driven by both genetic variation and receptor oligomerization. These insights have enabled computational drug discovery efforts, with some evidence of success in the design of completely novel opioids with unique efficacies. The combined progress over the past few years provides hope for new, efficacious opioids devoid of the side effects that have made them the scourge of humanity for millennia.

How mammalian neural circuits generate rhythmic activity in motor behaviors, such as breathing, walking, and chewing, remains elusive. For breathing, rhythm generation is localized to a brainstem nucleus, the preBötzinger Complex (preBötC). Rhythmic preBötC population activity consists of strong inspiratory bursts, which drive motoneuronal activity, and weaker burstlets, which we hypothesize reflect an emergent rhythmogenic process. If burstlets underlie inspiratory rhythmogenesis, respiratory depressants, such as opioids, should reduce burstlet frequency. Indeed, in medullary slices from neonatal mice, the μ-opioid receptor (μOR) agonist DAMGO slowed burstlet generation. Genetic deletion of μORs in a glutamatergic preBötC subpopulation abolished opioid-mediated depression, and the neuropeptide Substance P, but not blockade of inhibitory synaptic transmission, reduced opioidergic effects. We conclude that inspiratory rhythmogenesis is an emergent process, modulated by opioids, that does not rely on strong bursts of activity associated with motor output. These findings also point to strategies for ameliorating opioid-induced depression of breathing.

Morphologically variable species complexes can present significant challenges to conservation when taxonomic boundaries, and therefore conservation units, are ambiguous. In such cases, the definition of Evolutionarily Significant Units (ESUs) based on high-resolution genomic data can be an effective method to clarify genetic divergence and inform conservation actions. Here, we employ a population genomic approach using SNP data to delineate conservation units in a morphologically ambiguous plant species complex (Conospermum caeruleum; Proteaceae) from a global biodiversity hotspot. Over 200 individuals representing five subspecies and three informal morphological forms were sampled across a wide geographic range. Phylogenomic (ML tree and SplitsTree network) and population genomic (STRUCTURE, PCoA, FST) analyses resolved three divergent genetic groups that were incongruent with the current taxonomy, but consistent with geographic distribution. One ESU was comprised of three genetic subgroups, and these can be considered Management Units (MUs) to conserve population genetic structure and diversity. These MUs were surprisingly incongruent with the current subspecies-level taxonomy, but one MU was consistent with a recently documented morphological form, and all were consistent with ecogeographic distribution. We recommend a full taxonomic revision of the Conospermum genus based on a phylogenomic approach to assess any wider incongruence with morphology, and that conservation strategies should be informed by genetic data. Our study exemplifies the application of genomics to the conservation of a morphologically ambiguous species complex, enabling and enhancing the practical conservation of appropriate units of biodiversity. [ABSTRACT FROM AUTHOR]

BACKGROUND AND AIMS: The gut immune, cannabinoid, and opioid systems constitute an integrated network contributing to visceral sensation and pain modulation. We aimed to assess the expression of the µ-opioid receptor (MOR), its ligand β-endorphin (β-END), and cannabinoid receptor-2 (CB2 ) in patients with irritable bowel syndrome (IBS) and asymptomatic controls (AC) and their correlation with sex and symptom perception. METHODS: Mucosal biopsies were obtained from the left colon of 31 IBS patients (45% women) with predominant constipation (IBS-C, 9) or diarrhea (IBS-D, 10) or with mixed bowel habits (IBS-M, 12) and 32 AC (44% women) and processed for qRT-PCR, Western blotting, and immunohistochemistry. KEY RESULTS: µ-opioid receptor and CB2 mRNA and protein expression and β-END protein levels were increased in patients with IBS compared to AC (all Ps=0.021). A significant sex by IBS interaction was found in relation to CB2 mRNA expression (P = .003) with women showing a markedly higher expression to men (P = .035). In contrast, in AC, men had higher expression than women (P = .033). β-END, MOR, and CB2 immunoreactivities (IR) were localized to CD4+T cells including EMR-1+ eosinophils and CD31+ T cells but not to mast cells. CONCLUSIONS: The increased expression of MOR, β-END, and CB2 in the mucosa of IBS patients, where they are localized to immune cells, suggests that opioid and cannabinoid systems play an immune-related compensatory role in visceral pain in IBS patients. Further work is necessary to support this hypothesis.

Opioid overdoses, many of which are attributed to use of illicit fentanyl, are currently one of the leading causes of death in the U.S. Although fentanyl has been used safely for decades in clinical settings, the widespread use of illicit fentanyl is a recent phenomenon. Starting in 2013, illicitly manufactured fentanyl and its analogs began to appear on the streets. These substances were added to or sold as heroin, often unbeknownst to the user. Because fentanyl is so potent, only small amounts are needed to produce pharmacological effects, but the margin between safe and toxic doses is narrow. Surprisingly little is known about the exact signaling mechanisms underlying fentanyl-related respiratory depression or the effectiveness of naloxone in reversing this effect. Similarly, little is known about the ability of treatment medications such as buprenorphine, methadone, or naltrexone to reduce illicit fentanyl use. The present article reviews the receptor, preclinical and clinical pharmacology of fentanyl, and how its pharmacology may predict the effectiveness of currently approved medications for treating illicit fentanyl use.

Opioid overdoses, many of which are attributed to use of illicit fentanyl, are currently one of the leading causes of death in the U.S. Although fentanyl has been used safely for decades in clinical settings, the widespread use of illicit fentanyl is a recent phenomenon. Starting in 2013, illicitly manufactured fentanyl and its analogs began to appear on the streets. These substances were added to or sold as heroin, often unbeknownst to the user. Because fentanyl is so potent, only small amounts are needed to produce pharmacological effects, but the margin between safe and toxic doses is narrow. Surprisingly little is known about the exact signaling mechanisms underlying fentanyl-related respiratory depression or the effectiveness of naloxone in reversing this effect. Similarly, little is known about the ability of treatment medications such as buprenorphine, methadone, or naltrexone to reduce illicit fentanyl use. The present article reviews the receptor, preclinical and clinical pharmacology of fentanyl, and how its pharmacology may predict the effectiveness of currently approved medications for treating illicit fentanyl use.

We investigated the dose dependence of the role of nociceptors in opioid-induced side-effects, hyperalgesia and pain chronification, in the rat. Systemic morphine produced a dose-dependent biphasic change in mechanical nociceptive threshold. At lower doses (0.003-0.03 mg/kg, s.c.) morphine induced mechanical hyperalgesia, while higher doses (1-10 mg/kg, s.c.) induced analgesia. Intrathecal (i.t.) oligodeoxynucleotide (ODN) antisense to mu-opioid receptor (MOR) mRNA, attenuated both hyperalgesia and analgesia. 5 days after systemic morphine (0.03-10 mg/kg s.c.), mechanical hyperalgesia produced by intradermal (i.d.) prostaglandin E2 (PGE2) was prolonged, indicating hyperalgesic priming at the peripheral terminal of the nociceptor. The hyperalgesia induced by i.t. PGE2 (400 ng/10 µl), in groups that received 0.03 (that induced hyperalgesia) or 3 mg/kg (that induced analgesia) morphine, was also prolonged, indicating priming at the central terminal of the nociceptor. The prolongation of the hyperalgesia induced by i.d. or i.t. PGE2, in rats previously treated with either a hyperalgesic (0.03 mg/kg, s.c.) or analgesic (3 mg/kg, s.c.) dose, was reversed by i.d. or i.t. injection of the protein translation inhibitor cordycepin (1 µg), indicative of Type I priming at both terminals. Although pretreatment with MOR antisense had no effect on priming induced by 0.03 mg/kg morphine, it completely prevented priming by 3 mg/kg morphine, in both terminals. Thus, the induction of hyperalgesia, but not priming, by low-dose morphine, is MOR-dependent. In contrast, induction of both hyperalgesia and priming by high-dose morphine is MOR-dependent. The receptor at which low-dose morphine acts to produce priming remains to be established.

The ability to anticipate, attend and respond appropriately tospecific stimuli is involved in the execution of everyday tasks.The current investigation examined the relations betweencognitive skills measured by the NIH Toolbox and changes inthe power of mu oscillations during anticipation of and inresponse to a tactile stimulus. Electroencephalographic (EEG)activity was measured after a visuospatial cue directed adults(n=40) to monitor their right or left hand for upcoming tactilestimulation. In the 500 ms prior to the onset of the tactilestimulus, a desynchronization was apparent 8 – 14 Hz atcontralateral central sites, consistent with prior investigationsof mu rhythm; a widespread synchronization was apparent inthe 250 ms proceeding delivery of the tactile stimulus. Theextent of contralateral reduction in mu power was associatedwith speed processing ability, while ipsilateral mu power wasassociated with flanker performance and marginally correlatedwith card sort performance. Regression further probe thesignificance and specificity of these effects. Increases in mupower following onset of the tactile stimulus were notassociated with any behavioral measures. Mu modulationduring attention to a specific bodily location appears related tovariability in the broader ability to regulate behavior in a goal-directed manner, and perhaps to speed of stimulus processing.

Stimulation of the mu-opioid receptor (MOR) on nociceptors with fentanyl can produce hyperalgesia (opioid-induced hyperalgesia, OIH) and hyperalgesic priming, a model of transition to chronic pain. We investigated if local and systemic administration of biased MOR agonists (PZM21 and TRV130 [oliceridine]), which preferentially activate G-protein over β-arrestin translocation, and have been reported to minimize some opioid side effects, also produces OIH and priming. Injected intradermally (100 ng), both biased agonists induced mechanical hyperalgesia and, when injected at the same site, 5 days later, prostaglandin E2 (PGE2) produced prolonged hyperalgesia (priming). OIH and priming were both prevented by intrathecal treatment with an oligodeoxynucleotide (ODN) antisense (AS) for MOR mRNA. Agents that reverse Type I (the protein translation inhibitor cordycepin) and Type II (combination of Src and mitogen-activated protein kinase [MAPK] inhibitors) priming, or their combination, did not reverse priming induced by local administration of PZM21 or TRV130. While systemic PZM21 at higher doses (1 and 10 mg/kg) induced analgesia, lower doses (0.001, 0.01, 0.1, and 0.3 mg/kg) induced hyperalgesia; all doses induced priming. Hyperalgesia, analgesia and priming induced by systemic administration of PZM21 were also prevented by MOR AS-ODN. And, priming induced by systemic PZM21 was also not reversed by intradermal cordycepin or the combination of Src and MAPK inhibitors. Thus, maintenance of priming induced by biased MOR agonists, in the peripheral terminal of nociceptors, has a novel mechanism.

Recently, numerous studies have revealed 4–12 Hz fluctuations of behavioral performance in a multitude of tasks. The majority has utilized stimuli near detection threshold and observed related fluctuations in hit‐rates, attributing these to perceptual or attentional processes. As neural oscillations in the 8–20 Hz range also feature prominently in cortical motor areas, they might cause fluctuations in the ability to induce responses, independent of attentional capabilities. Additionally, different effectors (e.g., the left versus right hand) might be cyclically prioritized in an alternating fashion, similar to the attentional sampling of distinct locations, objects, or memory templates. Here, we investigated these questions via a behavioral dense‐sampling approach. Twenty‐six participants performed a simple visual discrimination task using highly salient stimuli. We varied the interval between each motor response and the subsequent target from 330 to 1040 ms, and analyzed performance as a function of this interval. Our data show significant fluctuations of both RTs and sensitivity between 12.5 and 25 Hz, but no evidence for an alternating prioritization of left‐ versus right‐hand responses. While our results suggest an impact of motor‐related signals on performance oscillations, they might additionally be influenced by perceptual processes earlier in the processing hierarchy. In summary, we demonstrate that behavioral oscillations generalize to situations involving highly salient stimuli, closer to everyday life. Moreover, our work adds to the literature by showing fluctuations at a high speed, which might be a consequence of both low task difficulty and the involvement of sensorimotor rhythms. Numerous studies have revealed 4‐12 Hz fluctuations in performance, using stimuli close to detection threshold. Here we report oscillations in response times and sensitivity at 12‐25 Hz, employing a simple task and highly salient stimuli. We conclude that the phenomenon can be generalized to situations closer to everyday life, while the higher frequency likely reflects both reduced task difficulty and the involvement of sensorimotor neural oscillations. [ABSTRACT FROM AUTHOR]

Background: The aim was to develop in-house software that is able to calculate and generate the biological plan evaluation of the esophagus treatment plan using the Niemierko model for normal tissue complication probability and tumor control probability. The Niemierko model can be applied for esophagus cancer treatment plan to estimate the tumor control probability (TCP) and the normal tissue complication probability (NTCP) using different planning techniques. The equivalent uniform dose (EUD) and effective volume parameters were compared with organ at risk. Subsequently, EUD and TCP parameter were compared with tumor volume for all five different planning techniques. Materials and methods: Ten cases for esophageal cancer were included in this study. For each patient, five treatment plans were generated. The Anisotropic analytical algorithms (AAA) were used for dose calculation for the three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) techniques. The in-house developed radiobiological plan evaluation software using python programming is used for this study which takes a dose volume histogram (DVH) text file as an input file for biological plan evaluation. Results and Conclusion: EUD, NTCP, TCP and effective volume were calculated from the Niemierko model using the in-house developed python based software and compared with treatment monitor units (MU) with all five different treatment plan. The best technique is quantified as benchmarked out of other different qualities of treatment. The four field 3D-CRT treatment plan is found to be the best suited from the perspective of biological plan index evaluation among the other planning techniques. [ABSTRACT FROM AUTHOR]

Daily life demands that we differentiate between a multitude of emotional facial expressions (EFEs). The mirror neuron system (MNS) is becoming increasingly implicated as a neural network involved with understanding emotional body expressions. However, the specificity of the MNS's involvement in emotion recognition has remained largely unexplored. This study investigated whether six basic dynamic EFEs (anger, disgust, fear, happiness, sadness, and surprise) would be differentiated through event-related desynchronisation (ERD) of sensorimotor alpha and beta oscillatory activity, which indexes sensorimotor MNS activity. We found that beta ERD differentiated happy, fearful, and sad dynamic EFEs at the central region of interest, but not at occipital regions. Happy EFEs elicited significantly greater central beta ERD relative to fearful and sad EFEs within 800 - 2,000 ms after EFE onset. These differences were source-localised to the primary somatosensory cortex, which suggests they are likely to reflect differential sensorimotor simulation rather than differential attentional engagement. Furthermore, individuals with higher trait anxiety showed less beta ERD differentiation between happy and sad faces. Similarly, individuals with higher trait autism showed less beta ERD differentiation between happy and fearful faces. These findings suggest that the differential simulation of specific affective states is attenuated in individuals with higher trait anxiety and autism. In summary, the MNS appears to support the skills needed for emotion processing in daily life, which may be influenced by certain individual differences. This provides novel evidence for the notion that simulation-based emotional skills may underlie the emotional difficulties that accompany affective disorders, such as anxiety. [ABSTRACT FROM AUTHOR]

Several SARS-CoV-2 variants of concern (VOC) and interest (VOI) co-circulate in Colombia, and determining the neutralizing antibody (nAb) responses is useful to improve the efficacy of COVID-19 vaccination programs. Thus, nAb responses against SARS-CoV-2 isolates from the lineages B.1.111, P.1 (Gamma), B.1.621 (Mu), AY.25.1 (Delta), and BA.1 (Omicron), were evaluated in serum samples from immunologically naïve individuals between 9 and 13 weeks after receiving complete regimens of CoronaVac, BNT162b2, ChAdOx1, or Ad26.COV2.S, using microneutralization assays. An overall reduction of the nAb responses against Mu, Delta, and Omicron, relative to B.1.111 and Gamma was observed in sera from vaccinated individuals with BNT162b2, ChAdOx1, and Ad26.COV2.S. The seropositivity rate elicited by all the vaccines against B.1.111 and Gamma was 100%, while for Mu, Delta, and Omicron ranged between 32 to 87%, 65 to 96%, and 41 to 96%, respectively, depending on the vaccine tested. The significant reductions in the nAb responses against the last three dominant SARS-CoV-2 lineages in Colombia indicate that booster doses should be administered following complete vaccination schemes to increase the nAb titers against emerging SARS-CoV-2 lineages. [ABSTRACT FROM AUTHOR]

The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs.

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR.

Repeated stimulation of mu-opioid receptors (MORs), by an MOR-selective agonist DAMGO induces type II priming, a form of nociceptor neuroplasticity, which has 2 components: opioid-induced hyperalgesia (OIH) and prolongation of prostaglandin-E2 (PGE2)-induced hyperalgesia. We report that intrathecal antisense knockdown of the MOR in nociceptors, prevented the induction of both components of type II priming. Type II priming was also eliminated by SSP-saporin, which destroys the peptidergic class of nociceptors. Because the epidermal growth factor receptor (EGFR) participates in MOR signaling, we tested its role in type II priming. The EGFR inhibitor, tyrphostin AG 1478, prevented the induction of prolonged PGE2-induced hyperalgesia, but not OIH, when tested out to 30 days after DAMGO. However, even when repeatedly injected, an EGFR agonist did not induce hyperalgesia or priming. A phosphopeptide, which blocks the interaction of Src, focal adhesion kinase (FAK), and EGFR, also prevented DAMGO-induced prolongation of PGE2 hyperalgesia, but only partially attenuated the induction of OIH. Inhibitors of Src and mitogen-activated protein kinase (MAPK) also only attenuated OIH. Inhibitors of matrix metalloproteinase, which cleaves EGF from membrane protein, markedly attenuated the expression, but did not prevent the induction, of prolongation of PGE2 hyperalgesia. Thus, although the induction of prolongation of PGE2-induced hyperalgesia at the peripheral terminal of peptidergic nociceptor is dependent on Src, FAK, EGFR, and MAPK signaling, Src, FAK, and MAPK signaling is only partially involved in the induction of OIH.

Physiological responses to the opioid neuropeptide enkephalin often involve both mu and delta opioid receptors. To facilitate quantitative studies into opioid signaling, we previously developed a caged [Leu5]-enkephalin that responds to ultraviolet irradiation, but its residual activity at delta receptors confounds experiments that involve both receptors. To reduce residual activity, we evaluated side-chain, N-terminus, and backbone caging sites and further incorporated the dimethoxy-nitrobenzyl moiety to improve sensitivity to ultraviolet light-emitting diodes (LEDs). Residual activity was characterized using an in vitro functional assay, and the power dependence and kinetics of the uncaging response to 355 nm laser irradiation were assayed using electrophysiological recordings of mu opioid receptor-mediated potassium currents in brain slices of rat locus coeruleus. These experiments identified N-MNVOC-LE as an optimal compound. Using ultraviolet LED illumination to photoactivate N-MNVOC-LE in the CA1 region of hippocampus, we found that enkephalin engages both mu and delta opioid receptors to suppress inhibitory synaptic transmission.

The ability of G protein-coupled receptors (GPCRs) to initiate complex cascades of cellular signaling is governed by the sequential coupling of three main transducer proteins, G protein, GPCR kinase (GRK), and β-arrestin. Mounting evidence indicates these transducers all have distinct conformational preferences and binding modes. However, interrogating each transducer's mechanism of interaction with GPCRs has been complicated by the interplay of transducer-mediated signaling events. For example, GRK-mediated receptor phosphorylation recruits and induces conformational changes in β-arrestin, which facilitates coupling to the GPCR transmembrane core. Here we compare the allosteric interactions of G proteins and β-arrestins with GPCRs' transmembrane cores by using the enzyme sortase to ligate a synthetic phosphorylated peptide onto the carboxyl terminus of three different receptors. Phosphopeptide ligation onto the β2-adrenergic receptor (β2AR) allows stabilization of a high-affinity receptor active state by β-arrestin1, permitting us to define elements in the β2AR and β-arrestin1 that contribute to the receptor transmembrane core interaction. Interestingly, ligation of the identical phosphopeptide onto the β2AR, the muscarinic acetylcholine receptor 2 and the μ-opioid receptor reveals that the ability of β-arrestin1 to enhance agonist binding relative to G protein differs substantially among receptors. Furthermore, strong allosteric coupling of β-arrestin1 correlates with its ability to attenuate, or "desensitize," G protein activation in vitro. Sortase ligation thus provides a versatile method to introduce complex, defined phosphorylation patterns into GPCRs, and analogous strategies could be applied to other classes of posttranslationally modified proteins. These homogeneously phosphorylated GPCRs provide an innovative means to systematically study receptor-transducer interactions.

Cellular interactions between delta and mu opioid receptors (DORs and MORs), including heteromerization, are thought to regulate opioid analgesia. However, the identity of the nociceptive neurons in which such interactions could occur in vivo remains elusive. Here we show that DOR-MOR co-expression is limited to small populations of excitatory interneurons and projection neurons in the spinal cord dorsal horn and unexpectedly predominates in ventral horn motor circuits. Similarly, DOR-MOR co-expression is rare in parabrachial, amygdalar, and cortical brain regions processing nociceptive information. We further demonstrate that in the discrete DOR-MOR co-expressing nociceptive neurons, the two receptors internalize and function independently. Finally, conditional knockout experiments revealed that DORs selectively regulate mechanical pain by controlling the excitability of somatostatin-positive dorsal horn interneurons. Collectively, our results illuminate the functional organization of DORs and MORs in CNS pain circuits and reappraise the importance of DOR-MOR cellular interactions for developing novel opioid analgesics.

Systemic fentanyl induces hyperalgesic priming, long-lasting neuroplasticity in nociceptor function characterized by prolongation of inflammatory mediator hyperalgesia. To evaluate priming at both nociceptor terminals, we studied, in male Sprague Dawley rats, the effect of local administration of agents that reverse type I (protein translation) or type II [combination of Src and mitogen-activated protein kinase (MAPK)] priming. At the central terminal, priming induced by systemic, intradermal, or intrathecal fentanyl was reversed by the combination of Src and MAPK inhibitors, but at the peripheral terminal, it was reversed by the protein translation inhibitor. Mu-opioid receptor (MOR) antisense prevented fentanyl hyperalgesia and priming. To determine whether type I and II priming occur in the same population of neurons, we used isolectin B4-saporin or [Sar9, Met(O2)11]-substance P-saporin to deplete nonpeptidergic or peptidergic nociceptors, respectively. Following intrathecal fentanyl, central terminal priming was prevented by both saporins, whereas that in peripheral terminal was not attenuated even by their combination. However, after intradermal fentanyl, priming in the peripheral terminal requires both peptidergic and nonpeptidergic nociceptors, whereas that in the central terminal is dependent only on peptidergic nociceptors. Pretreatment with dantrolene at either terminal prevented fentanyl-induced priming in both terminals, suggesting communication between central and peripheral terminals mediated by intracellular Ca2+ signaling. In vitro application of fentanyl increased cytoplasmic Ca2+ concentration in dorsal root ganglion neurons, which was prevented by pretreatment with dantrolene and naloxone. Therefore, acting at MOR in the nociceptor, fentanyl induces hyperalgesia and priming rapidly at both the central (type II) and peripheral (type I) terminal and this is mediated by Ca2+ signaling.SIGNIFICANCE STATEMENT Fentanyl, acting at the μ-opioid receptor (MOR), induces hyperalgesia and hyperalgesic priming at both the central and peripheral terminal of nociceptors and this is mediated by endoplasmic reticulum Ca2+ signaling. Priming in the central terminal is type II, whereas that in the peripheral terminal is type I. Our findings may provide useful information for the design of drugs with improved therapeutic profiles, selectively disrupting individual MOR signaling pathways, to maintain an adequate long-lasting control of pain.

The μ-opioid receptor (MOR) is the primary target of methadone and buprenorphine. The primary neuronal transcript of the OPRM1 gene, MOR-1, contains a ~13 kb 3' untranslated region with five common haplotypes in European-Americans. We analyzed the effects of these haplotypes on the percentage of opioid positive urine tests in European-Americans (n=582) during a 24-week, randomized, open-label trial of methadone or buprenorphine/naloxone (Suboxone) for the treatment of opioid dependence. A single haplotype, tagged by rs10485058, was significantly associated with patient urinalysis data in the methadone treatment group. Methadone patients with the A/A genotype at rs10485058 were less likely to have opioid-positive urine drug screens than those in the combined A/G and G/G genotypes group (relative risk=0.76, 95% confidence intervals=0.73-0.80, P=0.0064). Genotype at rs10485058 also predicted self-reported relapse rates in an independent population of Australian patients of European descent (n=1215) who were receiving opioid substitution therapy (P=0.003). In silico analysis predicted that miR-95-3p would interact with the G, but not the A allele of rs10485058. Luciferase assays indicated miR-95-3p decreased reporter activity of constructs containing the G, but not the A allele of rs10485058, suggesting a potential mechanism for the observed pharmacogenetic effect. These findings suggest that selection of a medication for opioid dependence based on rs10485058 genotype might improve outcomes in this ethnic group.

Opioid receptors are the sites of action for morphine and most other clinically used opioid drugs. Abundant evidence now demonstrates that different opioid receptor types can physically associate to form heteromers. Owing to their constituent monomers' involvement in analgesia, mu/delta opioid receptor (M/DOR) heteromers have been a particular focus of attention. Understandings of the physiological relevance and indisputable proof of M/DOR formation in vivo are still evolving. This aspect of the field has been slow to progress in large part by the limitations of most available experimental models; recently however, promising progress is being made. As a result, the long-repeated promise of opioid receptor heteromers as selective therapeutic targets is now being realized.

Mu‘tezile ve Şîa (Zeydiyye-İmâmiyye) imâmet dışındaki birçok itikâdî meselede müşterek görüşlere sahiptir. Bu husus, Şiî olmayan İslâm âlimleri ve Batılı araştırmacılar tarafından onların birbirlerinden etkilenmeleriyle açıklanmıştır. Mu‘tezile ve Şîa arasındaki bu etkileşimde, etkilenen kesim daha çok Şîa tarafıdır. Ancak Şîa’nın da Hz Ali’yi diğer sahabeden üstün tutmada Mu‘tezile’yi kısmen etkilediği gözden kaçırılmamalıdır. Nitekim Bağdat Mu‘tezilesinden Bişr b. Mu‘temir (öl. 210/825), Ca‘fer b. Mübeşşir (öl. 234/848), Ca‘fer b. Harb (öl. 236/850) ve el-İskâfî (öl. 240/854) gibi isimler, Hz. Ali’yi Hz. Peygamber’den sonra insanların en faziletlisi olarak kabul etmekle birlikte mefdûl (daha az faziletli) olan Hz. Ebû Bekir’in imâmetini câiz görmüşlerdir. Ayrıca Basra Mu‘tezilesinin reislerinden Ebû Ali el-Cübbâî (öl. 303/916), Ebû Hâşim el-Cübbâî (öl. 321/933), el-Allaf (öl. 235/849-850), en-Nazzâm (öl. 231/845), Ebû Abdullah el-Basrî (öl. 369/979-80) ve Kâdî Abdülcebbâr (öl. 415/1025) gibi isimler de hilafet sıralamasını fazilet sıralaması olarak kabul etmemişlerdir. Bu çalışma, Mu‘tezile ile Şîa arasındaki bu etkileşimin, özellikle Hz. Ali ile ilişkilendirilen âyetlerin tefsiri noktasında Mu‘tezile’yi etkileyip etkilemediği meselesine odaklanmaktadır. Çalışmada söz konusu problem, Mu‘tezilî müfessir Zemahşerî’nin (öl. 538/1144) el-Keşşâf tefsiri üzerinden incelenmiştir. Bu meselenin el-Keşşâf üzerinden incelenmesinde; tefsirin öneminin yanı sıra, Zemahşerî’nin Şiî/Zeydî isimlerden ders almış olması ve tefsirini Mekke’de Zeydî emir el-Vehhâs’ın (öl. 506/1112) desteği ile yazması etkili olmuştur. Çalışmanın ilk bölümünde Mu‘tezile ile Şîa’nın en önemli iki kolu olan İmâmiyye ve Zeydiyye etkileşimine kısaca değinilmekte, ardından genelde Mu‘tezile’nin özelde Zemahşerî’nin Hz. Ali’nin diğer halifeler arasındaki konumuna dair yaklaşımına yer verilmektedir. Mu‘tezile’nin Basra ekolünden olan Zemahşerî, bu ekolün önde gelen isimleri olan el-Allâf, Ebû Ali el-Cübbâî, Ebû Hâşim el-Cübbâî ve Kâdî Abdülcebbâr’ın aksine Hz. Ali’yi Hz. Ebu Bekir’den daha üstün görmemektedir. Hatta onun hulefâ-i râşidînin faziletinden bahsederken daima hilafet sırasını gözetmesi, seleflerinin aksine hilafet sıralamasını fazilet sırası olarak kabul etmiş olma ihtimalini desteklemektedir. İkinci bölümde ise Zemahşerî’nin Hz. Ali ile ilişkilendirilen âyetlerin tefsirinde aktardığı rivayetlerin, Şiî/Zeydî/İmâmî rivayetlerle benzeşip benzeşmediği ve âyetleri bu rivayetlere paralel olarak yorumlayıp yorumlanmadığı sorgulanmaktadır. el-Keşşâf tefsiri, bu açıdan kendisinden önceki Sünnî ve Şiî tefsirlerle mukayeseli olarak incelenmektedir. Sonuçta Zemahşerî’nin, Ehl-i beyt ile ilişkilendirilen bu âyetleri yorumlarken, Hz. Ali ve evlâdının faziletini öne çıkaran rivayetleri tercih ettiği görülmüştür. Ancak o, Şîa’nın imâmet inancına uygun düşecek yorum yapmaktan da kaçınmıştır. Özellikle Ahzâb 33/33. âyetini bağlama uygun olarak, tamamen Hz. Peygamber’in hanımları özelinde tefsir etmesi bunun en büyük göstergesidir. Zemahşerî’nin Hz. Ali ve evlâdının faziletini öne çıkaran rivayetlere yer verirken, daha çok Sünnî müfessir Sa‘lebî’yi takip ettiği tespit edilmiştir. Özellikle Zemahşerî’nin ilgili âyetlerin tefsirindeki rivayet malzemesini hemen hemen aynı şekilde Sa‘lebî’nin tefsirinden aktardığı görülmüştür. Ancak Zemahşerî’nin Âl-i İmrân 3/61 ve Mâide 5/55 gibi bazı âyetlerde bu rivayetleri dil açısından desteklemesi gibi bazı detaylarda ondan ayrıldığı dikkat çekmektedir. Sa‘lebî’nin de Sünnî-Şiî ihtilafının olduğu yerlerde Şiîleri destekleyen rivayetleri sıkça nakletmesi nedeniyle dolaylı bir etkilenmeden bahsedilebilir. Ayrıca bu rivayetlerin önemli bir kısmının da ilk dönem tefsirlerinden Mukâtil’in tefsirinde de yer aldığı belirtilmelidir. Dolayısıyla Zemahşerî’nin bu rivayetleri Şiî kaynaklardan aldığına dair bir bulguya rastlanılmamış olmakla birlikte Sünnî kaynaklardaki Hz. Ali eksenli rivayetleri ön plana çıkardığı görülmüştür. Hatta bazen Hz. Ali ile ilgili rivayet, diğer müfessirler tarafından tercih edilen görüş olmamasına rağmen onun yalnızca bu rivayete yer vermesi onun bu konudaki tutumunun Hz. Ali’ye olan meyliyle ilgisi olduğunu düşündürmektedir. Hatta Zemahşerî’nin Zeydî/Mu‘tezilî çizgideki hocası Cüşemî’nin tefsirinde dahi söz konusu âyetlerin yorumunda onun kadar Hz. Ali ve Ehl-i beyt vurgusunun olmaması bu görüşümüzü desteklemektedir.