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Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.

The harmful side effects of opioid drugs such as respiratory depression, tolerance, dependence, and abuse potential have limited the therapeutic utility of opioids for their entire clinical history. However, no previous attempt to develop effective pain drugs that substantially ameliorate these effects has succeeded, and the current opioid epidemic affirms that they are a greater hindrance to the field of pain management than ever. Recent attempts at new opioid development have sought to reduce these side effects by minimizing engagement of the regulatory protein arrestin-3 at the mu-opioid receptor, but there is significant controversy around this approach. Here, we discuss the ongoing effort to develop safer opioids and its relevant historical context. We propose a new model that reconciles results previously assumed to be in direct conflict to explain how different signaling profiles at the mu-opioid receptor contribute to opioid tolerance and dependence. Our goal is for this framework to inform the search for a new generation of lower liability opioid analgesics.

BackgroundSecond-generation antipsychotics (SGAs) are frontline treatments for serious mental illness. Often, individual patients benefit only from some SGAs and not others. The mechanisms underlying this unpredictability in treatment efficacy remain unclear. All SGAs bind the dopamine D3 receptor (D3R) and are traditionally considered antagonists for dopamine receptor signaling.MethodsHere, we used a combination of two-photon calcium imaging, in vitro signaling assays, and mouse behavior to assess signaling by SGAs at D3R.ResultsWe report that some clinically important SGAs function as arrestin-3 agonists at D3R, resulting in modulation of calcium channels localized to the site of action potential initiation in prefrontal cortex pyramidal neurons. We further show that chronic treatment with an arrestin-3 agonist SGA, but not an antagonist SGA, abolishes D3R function through postendocytic receptor degradation by GASP1 (G protein-coupled receptor-associated sorting protein-1).ConclusionsThese results implicate D3R-arrestin-3 signaling as a source of SGA variability, highlighting the importance of including arrestin-3 signaling in characterizations of drug action. Furthermore, they suggest that postendocytic receptor trafficking that occurs during chronic SGA treatment may contribute to treatment efficacy.

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.

Introduction: Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the µ-opioid receptor. Opioid use is limited by side effects, including significant risk of opioid use disorder. Improvement of the effect/side effect profile of opioid medications is a key pursuit of opioid research, yet there is no consensus on how to achieve this goal. One hypothesis is that the degree of arrestin-3 recruitment to the µ-opioid receptor impacts therapeutic utility. However, it is not clear whether increased or decreased interaction of the µ-opioid receptor with arrestin-3 would reduce compulsive drug-seeking. Methods: We utilized three genotypes of mice with varying abilities to recruit arrestin-3 to the µ-opioid receptor in response to morphine in a novel longitudinal operant self-administration model. We also created a quantitative method to define compulsivity in drug-seeking based on a multi-variate analysis of several operant response variables. Results: We demonstrate that arrestin-3 knockout and wild type mice have highly variable drug-seeking behavior with few genotype differences. In contrast, in mice where the µ-opioid receptor strongly recruits arrestin-3, drug-seeking behavior is much less varied. We found that mice lacking arrestin-3 were more likely to meet the criteria for compulsivity whereas mice with enhanced arrestin-3 recruitment did not develop a compulsive phenotype. Conclusion: These experiments show that a lack of arrestin-3 is not protective against the abuse liability of morphine in an operant self-administration context. Our data also suggest that opioids that engage both G protein and arrestin-3, recapitulating the endogenous signaling pattern, will reduce abuse liability. [ABSTRACT FROM AUTHOR]

Opioid therapeutics are excellent analgesics, whose utility is compromised by dependence. Morphine (1) and its clinically relevant derivatives such as OxyContin (2), Vicodin (3), and Dilaudid (4) are "biased" agonists at the μ opioid receptor (OR), wherein they engage G protein signaling but poorly engage β-arrestin and the endocytic machinery. In contrast, endorphins, the endogenous peptide agonists for ORs, are potent analgesics, show reduced liability for tolerance and dependence, and engage both G protein and β-arrestin pathways as "balanced" agonists. We set out to determine if marine-derived alkaloids could serve as novel OR agonist chemotypes with a signaling profile distinct from morphine and more similar to the endorphins. Screening of 96 sponge-derived extracts followed by LC-MS-based purification to pinpoint the active compounds and subsequent evaluation of a mini library of related alkaloids identified two structural classes that modulate the ORs. These included the following: aaptamine (10), 9-demethyl aaptamine (11), demethyl (oxy)-aaptamine (12) with activity at the δ-OR (EC50: 5.1, 4.1, 2.3 μM, respectively) and fascaplysin (17), and 10-bromo fascaplysin (18) with activity at the μ-OR (EC50: 6.3, 4.2 μM respectively). An in vivo evaluation of 10 using δ-KO mice indicated its previously reported antidepressant-like effects are dependent on the δ-OR. Importantly, 17 functioned as a balanced agonist promoting both G protein signaling and β-arrestin recruitment along with receptor endocytosis similar to the endorphins. Collectively these results demonstrate the burgeoning potential for marine natural products to serve as novel lead compounds for therapeutic targets in neuroscience research.

Neural circuits involving midbrain dopaminergic (DA) neurons regulate reward and goal-directed behaviors. Although local GABAergic input is known to modulate DA circuits, the mechanism that controls excitatory/inhibitory synaptic balance in DA neurons remains unclear. Here, we show that DA neurons use autocrine transforming growth factor β (TGF-β) signaling to promote the growth of axons and dendrites. Surprisingly, removing TGF-β type II receptor in DA neurons also disrupts the balance in TGF-β1 expression in DA neurons and neighboring GABAergic neurons, which increases inhibitory input, reduces excitatory synaptic input, and alters phasic firing patterns in DA neurons. Mice lacking TGF-β signaling in DA neurons are hyperactive and exhibit inflexibility in relinquishing learned behaviors and re-establishing new stimulus-reward associations. These results support a role for TGF-β in regulating the delicate balance of excitatory/inhibitory synaptic input in local microcircuits involving DA and GABAergic neurons and its potential contributions to neuropsychiatric disorders.

G-protein-coupled receptors (GPCRs) initiate a variety of signaling cascades, depending on effector coupling. β-arrestins, which were initially characterized by their ability to "arrest" GPCR signaling by uncoupling receptor and G protein, have recently emerged as important signaling effectors for GPCRs. β-arrestins engage signaling pathways that are distinct from those mediated by G protein. As such, arrestin-dependent signaling can play a unique role in regulating cell function, but whether neuromodulatory GPCRs utilize β-arrestin-dependent signaling to regulate neuronal excitability remains unclear. Here, we find that D3 dopamine receptors (D3R) regulate axon initial segment (AIS) excitability through β-arrestin-dependent signaling, modifying CaV3 voltage dependence to suppress high-frequency action potential generation. This non-canonical D3R signaling thereby gates AIS excitability via pathways distinct from classical GPCR signaling pathways.

UnlabelledRemyelinating therapies seek to promote restoration of function and normal cellular architecture following demyelination in diseases, such as multiple sclerosis (MS). Functional screening for small molecules or novel targets for remyelination is a major hurdle to the identification and development of rational therapeutics for MS. Recent findings and technical advances provide us with a unique opportunity to provide insight into the cell autonomous mechanisms for remyelination and address this unmet need. Upon screening a G-protein-coupled receptor small-molecule library, we report the identification of a cluster of κ-opioid receptor (KOR) agonists that significantly promotes oligodendrocyte differentiation and myelination. KOR agonists were validated in purified rat oligodendroglial cultures, and the (±)U-50488 compound proved to be most effective for differentiation. (±)U-50488 treatment significantly enhances differentiation and myelination in purified oligodendroglial cocultures and greatly accelerates the kinetics of remyelination in vivo after focal demyelination with lysolecithin. The effect of (±)U-50488 is attenuated by KOR antagonists and completely abolished in KOR-null oligodendroglia. Conditional deletion of KOR in murine oligodendrocyte precursor cells (OPCs) greatly inhibits remyelination after focal demyelination lacking any response to (±)U-50488 treatment. To determine whether agonism of KOR represents a feasible therapeutic approach, human induced pluripotent stem cell-derived OPCs were treated with (±)U-50488. Consistent with findings, differentiation of human OPCs into mature oligodendrocytes was significantly enhanced. Together, KOR is a therapeutic target to consider for future remyelination therapy.Significance statementRemyelination represents a promising strategy to achieve functional recovery in demyelinating diseases, like MS. Thus, identification of potent compounds and targets that promote remyelination represents a critically unmet need. This study reports a cluster of compounds that are highly effective in enhancing remyelination and identifies κ-opioid receptor (KOR) as a positive regulator for oligodendroglial differentiation, implicating KOR agonism as a potential strategy to accelerate remyelination.

Kappa opioid receptors (KORs) are involved in a variety of aversive behavioral states, including anxiety. To date, a circuit-based mechanism for KOR-driven anxiety has not been described. Here, we show that activation of KORs inhibits glutamate release from basolateral amygdala (BLA) inputs to the bed nucleus of the stria terminalis (BNST) and occludes the anxiolytic phenotype seen with optogenetic activation of BLA-BNST projections. In addition, deletion of KORs from amygdala neurons results in an anxiolytic phenotype. Furthermore, we identify a frequency-dependent, optically evoked local dynorphin-induced heterosynaptic plasticity of glutamate inputs in the BNST. We also find that there is cell type specificity to the KOR modulation of the BLA-BNST input with greater KOR-mediated inhibition of BLA dynorphin-expressing neurons. Collectively, these results provide support for a model in which local dynorphin release can inhibit an anxiolytic pathway, providing a discrete therapeutic target for the treatment of anxiety disorders.

The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking, and metabolism.

Potentiation of glutamate responses is a critical synaptic response to cocaine exposure in ventral tegmental area (VTA) neurons. However, the mechanism by which cocaine exposure promotes potentiation of NMDA receptors (NMDARs) and subsequently AMPA receptors (AMPARs) is not fully understood. In this study we demonstrate that repeated cocaine treatment causes loss of D2 dopamine receptor functional responses via interaction with lysosome-targeting G-protein-associated sorting protein1 (GASP1). We also show that the absence of D2 downregulation in GASP1-KO mice prevents cocaine-induced potentiation of NMDAR currents, elevation of the AMPA/NMDA ratio, and redistribution of NMDAR and AMPAR subunits to the membrane. As a pharmacological parallel, coadministration of the high-affinity D2 agonist, aripiprazole, reduces not only functional downregulation of D2s in response to cocaine but also potentiation of NMDAR and AMPAR responses in wild-type mice. Together these data suggest that functional loss of D2 receptors is a critical mechanism mediating cocaine-induced glutamate plasticity in VTA neurons.

Delta (DOR) and mu opioid receptors (MOR) can complex as heteromers, conferring functional properties in agonist binding, signaling and trafficking that can differ markedly from their homomeric counterparts. Because of these differences, DOR/MOR heteromers

Opioid receptors, including the mu and delta opioid receptors (MOR and DOR) are important targets for the treatment of pain. Although there is mounting evidence that these receptors form heteromers, the functional role of the MOR/DOR heteromer remains unresolved. We have designed and synthesized bivalent ligands as tools to elucidate the functional role of the MOR/DOR heteromer. Our ligands (L2 and L4) are comprised of a compound with low affinity at the DOR tethered to a compound with high affinity at the MOR, with the goal of producing ligands with "tuned affinity" at MOR/DOR heteromers compared to DOR homomers. Here we show that both L2 and L4 demonstrate enhanced affinity at MOR/DOR heteromers compared to DOR homomers, thereby providing unique pharmacological tools to dissect the role of the MOR/DOR heteromer in pain.

Research indicates that analgesic tolerance effects of the morphine can be counteracted by employing a combination of morphine and opiate analogs in the treatment of pain. Data show that the tolerance is diminished by endocytosis of the mu opioid receptor.

Author(s): Mark A. Ungless [1]; Jennifer L. Whistler [1]; Robert C. Malenka [2]; Antonello Bonci (corresponding author) [1] How do drugs of abuse modify neural circuitry and thereby lead to [...]

[Mu] opioid receptors are targets of native opioid peptides and addictive analgesic drugs. A major clinical liability of opiate drugs is their ability to cause physiological tolerance. Individual opiates, such as morphine and etorphine, differ both in their ability to promote physiological tolerance and in their effects on receptor regulation by endocytosis. Here, we demonstrate that arrestins play a fundamental role in mediating this agonist-selective regulation and that morphine-activated [Mu] receptors fail to undergo arrestin-dependent uncoupling from cognate G proteins. Thus, highly addictive opiate drugs elude a fundamental mode of physiological regulation that is mediated by agonist-specific interaction of opioid receptors with arrestins.

Opioid receptors are components of the G protein-coupled receptor (GPCR) clan, and are stimulated by internally generated opioid peptides and externally injected opiate drugs. The diverse characteristics of opioid receptors are examined with the purpose of deriving more knowledge on the processes of opioid acceptance, craving, and addiction.