Molecular recognition of morphine and fentanyl by the human µ-opioid receptor.

Morphine and fentanyl are among the most used opioid drugs that confer analgesia and unwanted side effects through both G protein and arrestin signaling pathways of µ-opioid receptor (µOR). Here, we report structures of the human µOR-G protein complexes bound to morphine and fentanyl, which uncover key differences in how they bind the receptor. We also report structures of µOR bound to TRV130, PZM21, and SR17018, which reveal preferential interactions of these agonists with TM3 side of the ligand-binding pocket rather than TM6/7 side. In contrast, morphine and fentanyl form dual interactions with both TM3 and TM6/7 regions. Mutations at the TM6/7 interface abolish arrestin recruitment of µOR promoted by morphine and fentanyl. Ligands designed to reduce TM6/7 interactions display preferential G protein signaling. Our results provide crucial insights into fentanyl recognition and signaling of µOR, which may facilitate rational design of next-generation analgesics.

A Neural Circuit for the Suppression of Pain by a Competing Need State.

Hunger and pain are two competing signals that individuals must resolve to ensure survival. However, the neural processes that prioritize conflicting survival needs are poorly understood. We discovered that hunger attenuates behavioral responses and affective properties of inflammatory pain without altering acute nociceptive responses. This effect is centrally controlled, as activity in hunger-sensitive agouti-related protein (AgRP)-expressing neurons abrogates inflammatory pain. Systematic analysis of AgRP projection subpopulations revealed that the neural processing of hunger and inflammatory pain converge in the hindbrain parabrachial nucleus (PBN). Strikingly, activity in AgRP → PBN neurons blocked the behavioral response to inflammatory pain as effectively as hunger or analgesics. The anti-nociceptive effect of hunger is mediated by neuropeptide Y (NPY) signaling in the PBN. By investigating the intersection between hunger and pain, we have identified a neural circuit that mediates competing survival needs and uncovered NPY Y1 receptor signaling in the PBN as a target for pain suppression.

Fragile-like regulatory T cells modulate opioid withdrawal in humans and mice.

Opioid use alters peripheral immune functions via unknown mechanisms. In a recent issue of Cell, Zhu et al. report increased fragile-like regulatory T cells in patients with opioid use disorder and in morphine-treated mice. In mice, Treg cell-derived interferon-γ within the brain promotes withdrawal-associated alterations in synapses.

Tuning Biased GPCR Signaling for Physiological Gain.

Effective and safe doses of opiate painkillers, like morphine, can be limited by respiratory depression. Schmid et al. (2017) now present a quantitative method to design ligands and correlate GPCR signaling bias to the dose separation between therapeutic and adverse effects in animals.

Bias Factor and Therapeutic Window Correlate to Predict Safer Opioid Analgesics.

Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit ßarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that ßarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.

Myelin plasticity in the ventral tegmental area is required for opioid reward.

All drugs of abuse induce long-lasting changes in synaptic transmission and neural circuit function that underlie substance-use disorders1,2. Another recently appreciated mechanism of neural circuit plasticity is mediated through activity-regulated changes in myelin that can tune circuit function and influence cognitive behaviour3-7. Here we explore the role of myelin plasticity in dopaminergic circuitry and reward learning. We demonstrate that dopaminergic neuronal activity-regulated myelin plasticity is a key modulator of dopaminergic circuit function and opioid reward. Oligodendroglial lineage cells respond to dopaminergic neuronal activity evoked by optogenetic stimulation of dopaminergic neurons, optogenetic inhibition of GABAergic neurons, or administration of morphine. These oligodendroglial changes are evident selectively within the ventral tegmental area but not along the axonal projections in the medial forebrain bundle nor within the target nucleus accumbens. Genetic blockade of oligodendrogenesis dampens dopamine release dynamics in nucleus accumbens and impairs behavioural conditioning to morphine. Taken together, these findings underscore a critical role for oligodendrogenesis in reward learning and identify dopaminergic neuronal activity-regulated myelin plasticity as an important circuit modification that is required for opioid reward.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids.

Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the µ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCß3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.

Oxycodone: A Current Perspective on Its Pharmacology, Abuse, and Pharmacotherapeutic Developments.

Oxycodone, a semisynthetic derivative of naturally occurring thebaine, an opioid alkaloid, has been available for more than 100 years. Although thebaine cannot be used therapeutically due to the occurrence of convulsions at higher doses, it has been converted to a number of other widely used compounds that include naloxone, naltrexone, buprenorphine, and oxycodone. Despite the early identification of oxycodone, it was not until the 1990s that clinical studies began to explore its analgesic efficacy. These studies were followed by the pursuit of several preclinical studies to examine the analgesic effects and abuse liability of oxycodone in laboratory animals and the subjective effects in human volunteers. For a number of years oxycodone was at the forefront of the opioid crisis, playing a significant role in contributing to opioid misuse and abuse, with suggestions that it led to transitioning to other opioids. Several concerns were expressed as early as the 1940s that oxycodone had significant abuse potential similar to heroin and morphine. Both animal and human abuse liability studies have confirmed, and in some cases amplified, these early warnings. Despite sharing a similar structure with morphine and pharmacological actions also mediated by the µ-opioid receptor, there are several differences in the pharmacology and neurobiology of oxycodone. The data that have emerged from the many efforts to analyze the pharmacological and molecular mechanism of oxycodone have generated considerable insight into its many actions, reviewed here, which, in turn, have provided new information on opioid receptor pharmacology. SIGNIFICANCE STATEMENT: Oxycodone, a µ-opioid receptor agonist, was synthesized in 1916 and introduced into clinical use in Germany in 1917. It has been studied extensively as a therapeutic analgesic for acute and chronic neuropathic pain as an alternative to morphine. Oxycodone emerged as a drug with widespread abuse. This article brings together an integrated, detailed review of the pharmacology of oxycodone, preclinical and clinical studies of pain and abuse, and recent advances to identify potential opioid analgesics without abuse liability.

Chronic Morphine Induces Adaptations in Opioid Receptor Signaling in a Thalamostriatal Circuit That Are Location Dependent, Sex Specific, and Regulated by µ-Opioid Receptor Phosphorylation.

Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) via activity at µ-opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in the DMS. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation) in male, but not female, mice. At MThal cell bodies, chronic morphine treatment decreased subsequent morphine activation of potassium conductance (morphine tolerance) in both male and female mice. In knock-in mice expressing phosphorylation-deficient MORs, chronic morphine treatment resulted in tolerance to, rather than facilitation of, subsequent morphine signaling at MThal-DMS terminals, suggesting phosphorylation deficiency unmasks adaptations that counter the facilitation observed at presynaptic terminals in wild-type mice. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry, and sex.

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Different projection neurons of basolateral amygdala participate in the retrieval of morphine withdrawal memory with diverse molecular pathways.

Context-induced retrieval of drug withdrawal memory is one of the important reasons for drug relapses. Previous studies have shown that different projection neurons in different brain regions or in the same brain region such as the basolateral amygdala (BLA) participate in context-induced retrieval of drug withdrawal memory. However, whether these different projection neurons participate in the retrieval of drug withdrawal memory with same or different molecular pathways remains a topic for research. The present results showed that (1) BLA neurons projecting to the prelimbic cortex (BLA-PrL) and BLA neurons projecting to the nucleus accumbens (BLA-NAc) participated in context-induced retrieval of morphine withdrawal memory; (2) there was an increase in the expression of Arc and pERK in BLA-NAc neurons, but not in BLA-PrL neurons during context-induced retrieval of morphine withdrawal memory; (3) pERK was the upstream molecule of Arc, whereas D1 receptor was the upstream molecule of pERK in BLA-NAc neurons during context-induced retrieval of morphine withdrawal memory; (4) D1 receptors also strengthened AMPA receptors, but not NMDA receptors, -mediated glutamatergic input to BLA-NAc neurons via pERK during context-induced retrieval of morphine withdrawal memory. These results suggest that different projection neurons of the BLA participate in the retrieval of morphine withdrawal memory with diverse molecular pathways.

Tiam1-mediated maladaptive plasticity underlying morphine tolerance and hyperalgesia.

Opioid pain medications, such as morphine, remain the mainstay for treating severe and chronic pain. Prolonged morphine use, however, triggers analgesic tolerance and hyperalgesia (OIH), which can last for a long period after morphine withdrawal. How morphine induces these detrimental side effects remains unclear. Here, we show that morphine tolerance and OIH are mediated by Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network. Tiam1 is a Rac1 GTPase guanine nucleotide exchange factor that promotes excitatory synaptogenesis by modulating actin cytoskeletal dynamics. We found that prolonged morphine treatment activated Tiam1 in the spinal dorsal horn and Tiam1 ablation from spinal neurons eliminated morphine antinociceptive tolerance and OIH. At the same time, the pharmacological blockade of Tiam1-Rac1 signalling prevented the development and reserved the established tolerance and OIH. Prolonged morphine treatment increased dendritic spine density and synaptic NMDA receptor activity in spinal dorsal horn neurons, both of which required Tiam1. Furthermore, co-administration of the Tiam1 signalling inhibitor NSC23766 was sufficient to abrogate morphine tolerance in chronic pain management. These findings identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause for the development and maintenance of morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

Low-dose ketamine safely reduces acute pain in the ED with a more rapid and shorter effect than morphine.

SOURCE CITATION: Guo J, Zhao F, Bian J, et al. Low-dose ketamine versus morphine in the treatment of acute pain in the emergency department: a meta-analysis of 15 randomized controlled trials. Am J Emerg Med. 2024;76:140-149. 38071883.

Differential Patterns of Synaptic Plasticity in the Nucleus Accumbens Caused by Continuous and Interrupted Morphine Exposure.

Opioid exposure and withdrawal both cause adaptations in brain circuits that may contribute to abuse liability. These adaptations vary in magnitude and direction following different patterns of opioid exposure, but few studies have systematically manipulated the pattern of opioid administration while measuring neurobiological impact. In this study, we compared cellular and synaptic adaptations in the nucleus accumbens shell caused by morphine exposure that was either continuous or interrupted by daily bouts of naloxone-precipitated withdrawal. At the behavioral level, continuous morphine administration caused psychomotor tolerance, which was reversed when the continuity of morphine action was interrupted by naloxone-precipitated withdrawal. Using ex vivo slice electrophysiology in female and male mice, we investigated how these patterns of morphine administration altered intrinsic excitability and synaptic plasticity of medium spiny neurons (MSNs) expressing the D1 or D2 dopamine receptor. We found that morphine-evoked adaptations at excitatory synapses were predominately conserved between patterns of administration, but there were divergent effects on inhibitory synapses and the subsequent balance between excitatory and inhibitory synaptic input. Overall, our data suggest that continuous morphine administration produces adaptations that dampen the output of D1-MSNs, which are canonically thought to promote reward-related behaviors. Interruption of otherwise continuous morphine exposure does not dampen D1-MSN functional output to the same extent, which may enhance behavioral responses to subsequent opioid exposure. Our findings support the hypothesis that maintaining continuity of opioid administration could be an effective therapeutic strategy to minimize the vulnerability to opioid use disorders.SIGNIFICANCE STATEMENT Withdrawal plays a key role in the cycle of addiction to opioids like morphine. We studied how repeated cycles of naloxone-precipitated withdrawal from otherwise continuous opioid exposure can change brain function of the nucleus accumbens, which is an important brain region for reward and addiction. Different patterns of opioid exposure caused unique changes in communication between neurons in the nucleus accumbens, and the nature of these changes depended on the type of neuron being studied. The specific changes in communication between neurons caused by repeated cycles of withdrawal may increase vulnerability to opioid use disorders. This highlights the importance of reducing or preventing the experience of withdrawal during opioid treatment.

Opioid Receptors Modulate Firing and Synaptic Transmission in the Paraventricular Nucleus of the Thalamus.

The paraventricular nucleus of the thalamus (PVT) is involved in drug addiction-related behaviors, and morphine is a widely used opioid for the relief of severe pain. Morphine acts via opioid receptors, but the function of opioid receptors in the PVT has not been fully elucidated. Here, we used in vitro electrophysiology to study neuronal activity and synaptic transmission in the PVT of male and female mice. Activation of opioid receptors suppresses the firing and inhibitory synaptic transmission of PVT neurons in brain slices. On the other hand, the involvement of opioid modulation is reduced after chronic morphine exposure, probably because of desensitization and internalization of opioid receptors in the PVT. Overall, the opioid system is essential for the modulation of PVT activities.SIGNIFICANCE STATEMENT Opioid receptors modulate the activities and synaptic transmission in the PVT by suppressing the firing rate and inhibitory synaptic inputs. These modulations were largely diminished after chronic morphine exposure.

Selective TLR4 Antagonism Prevents and Reverses Morphine-Induced Persistent Postoperative Cognitive Dysfunction, Dysregulation of Synaptic Elements, and Impaired BDNF Signaling in Aged Male Rats.

Perioperative neurocognitive disorders (PNDs) are characterized by confusion, difficulty with executive function, and episodic memory impairment in the hours to months following a surgical procedure. Postoperative cognitive dysfunction (POCD) represents such impairments that last beyond 30 d postsurgery and is associated with increased risk of comorbidities, progression to dementia, and higher mortality. While it is clear that neuroinflammation plays a key role in PND development, what factors underlie shorter self-resolving versus persistent PNDs remains unclear. We have previously shown that postoperative morphine treatment extends POCD from 4 d (without morphine) to at least 8 weeks (with morphine) in aged male rats, and that this effect is likely dependent on the proinflammatory capabilities of morphine via activation of toll-like receptor 4 (TLR4). Here, we extend these findings to show that TLR4 blockade, using the selective TLR4 antagonist lipopolysaccharide from the bacterium Rhodobacter sphaeroides (LPS-RS Ultrapure), ameliorates morphine-induced POCD in aged male rats. Using either a single central preoperative treatment or a 1 week postoperative central treatment regimen, we demonstrate that TLR4 antagonism (1) prevents and reverses the long-term memory impairment associated with surgery and morphine treatment, (2) ameliorates morphine-induced dysregulation of the postsynaptic proteins postsynaptic density 95 and synaptopodin, (3) mitigates reductions in mature BDNF, and (4) prevents decreased activation of the BDNF receptor TrkB (tropomyosin-related kinase B), all at 4 weeks postsurgery. We also reveal that LPS-RS Ultrapure likely exerts its beneficial effects by preventing endogenous danger signal HMGB1 (high-mobility group box 1) from activating TLR4, rather than by blocking continuous activation by morphine or its metabolites. These findings suggest TLR4 as a promising therapeutic target to prevent or treat PNDs.SIGNIFICANCE STATEMENT With humans living longer than ever, it is crucial that we identify mechanisms that contribute to aging-related vulnerability to cognitive impairment. Here, we show that the innate immune receptor toll-like receptor 4 (TLR4) is a key mediator of cognitive dysfunction in aged rodents following surgery and postoperative morphine treatment. Inhibition of TLR4 both prevented and reversed surgery plus morphine-associated memory impairment, dysregulation of synaptic elements, and reduced BDNF signaling. Together, these findings implicate TLR4 in the development of postoperative cognitive dysfunction, providing mechanistic insight and novel therapeutic targets for the treatment of cognitive impairments following immune challenges such as surgery in older individuals.

mGluR5 from Primary Sensory Neurons Promotes Opioid-Induced Hyperalgesia and Tolerance by Interacting with and Potentiating Synaptic NMDA Receptors.

Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with µ-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance.SIGNIFICANCE STATEMENT Opioids are essential analgesics for managing severe pain caused by cancer, surgery, and tissue injury. However, these drugs paradoxically induce pain hypersensitivity and tolerance, which can cause rapid dose escalation and even overdose mortality. This study demonstrates, for the first time, that opioids promote trafficking of mGluR5, a G protein-coupled glutamate receptor, from peripheral sensory neurons to the spinal cord; there, mGluR5 proteins dimerize and physically interact with NMDARs to augment their synaptic expression and activity. Through dynamic interactions, the two distinct glutamate receptors mutually amplify and sustain nociceptive input from peripheral sensory neurons to the spinal cord. Thus, inhibiting mGluR5 activity or disrupting mGluR5-NMDAR interactions could reduce opioid-induced hyperalgesia and tolerance and potentiate opioid analgesic efficacy.

Biasing Gßγ Downstream Signaling with Gallein Inhibits Development of Morphine Tolerance and Potentiates Morphine-Induced Nociception in a Tolerant State.

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gßγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gßγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gßγ and inhibit interactions of Gßγ with phospholipase-Cß3 (PLCß3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCß3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gßγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gßγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cß is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.

Exploring neuroadaptive cellular pathways in chronic morphine exposure: An in-vitro analysis of cabergoline and Mdivi-1 co-treatment effects on the autophagy-apoptosis axis.

The complex impacts of prolonged morphine exposure continue to be a significant focus in the expanding area of addiction studies. This research investigates the effectiveness of a combined treatment using Cabergoline and Mdivi-1 to counteract the neuroadaptive changes caused by in vitro morphine treatment. The impact of Methadone, Cabergoline, and a combination of Cabergoline and Mdivi-1 on the cellular and molecular responses associated with Morphine-induced changes was studied in human Neuroblastoma (SK-N-MC) and Glioblastoma (U87-MG) cell lines that were exposed to prolong Morphine treatment. Cabergoline and Mdivi-1 combined treatment effectively influenced the molecular alterations associated with neuroadaptation in chronic morphine-exposed neural cells. This combination therapy normalized autophagy and reduced oxidative stress by enhancing total-antioxidant capacity, mitigating apoptosis, restoring BDNF expression, and balancing apoptotic elements. Our research outlines morphine's dual role in modulating mitochondrial dynamics via the dysregulation of the autophagy-apoptosis axis. This emphasizes the significant involvement of DRP1 activity in neurological adaptation processes, as well as disturbances in the dopaminergic pathway during in vitro chronic exposure to morphine in neural cells. This study proposes a novel approach by recommending the potential effectiveness of combining Cabergoline and Mdivi-1 to modulate the neuroadaptations caused by morphine. Additionally, we identified BDNF and PCNA in neural cells as potential neuroprotective markers for assessing the effectiveness of drugs against opioid toxicity, emphasizing the need for further validation. The study uncovers diverse effects observed in pretreated morphine glioblastoma cells under treatment with Cabergoline and methadone. This highlights the potential for new treatments in the DRD2 pathway and underscores the importance of investigating the interplay between autophagy and apoptosis to advance research in managing cancer-related pain. The study necessitates an in-depth investigation into the relationship between autophagy and apoptosis, with a specific emphasis on protein interactions and the dynamics of cell signaling.