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The endogenous dynorphin/kappa opioid receptor (KOR) system in the brain mediates the dysphoric effects of stress, and KOR antagonists may have therapeutic potential for the treatment of drug addiction, depression, and psychosis. One class of KOR antagonists, the long-acting norBNI-like antagonists, have been suggested to act by causing KOR inactivation through a cJun-kinase mechanism rather than by competitive inhibition. In this study, we screened for other opioid ligands that might produce norBNI-like KOR inactivation and found that nalfurafine (a G-biased KOR agonist) and nalmefene (a KOR partial agonist) also produce long-lasting KOR inactivation. Neither nalfurafine nor nalmefene are completely selective KOR ligands, but KOR inactivation was observed at doses 10-100 fold lower than necessary for mu opioid receptor actions. Daily microdosing with nalfurafine or nalmefene blocked KORs responsible for antinociceptive effects, blocked KORs mediating stress-induced aversion, and mitigated the aversion during acute and protracted withdrawal in fentanyl-dependent mice. Both nalfurafine and nalmefene have long histories of safety and use in humans and could potentially be repurposed for the treatment of dynorphin-mediated stress disorders., Competing Interests: Competing Interest Statement: The authors declare no conflicts of interest.

Hydrogen Peroxide (H 2 O 2 ) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H 2 O 2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H 2 O 2 dynamics. We successfully tracked real-time transient and steady-state H 2 O 2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse brain ex vivo and in vivo . These applications demonstrate oROS's capabilities to monitor H 2 O 2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aβ-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. Lastly, we demonstrated acute opioid-induced generation of H 2 O 2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H 2 O 2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for understanding diseases associated with oxidative stress, such as cancer, neurodegenerative, and cardiovascular diseases., Competing Interests: Additional Declarations: There is NO Competing Interest.

Hydrogen Peroxide (H 2 O 2 ) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H 2 O 2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H 2 O 2 dynamics. We successfully tracked real-time transient and steady-state H 2 O 2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROS's capabilities to monitor H 2 O 2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aβ-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H 2 O 2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.

Increases in drug consumption over time, also known as escalation, is a key behavioral component of substance use disorder (SUD) that is related to potential harm to users, such as overdose. Studying escalation also allows researchers to investigate the transition from casual drug use to more SUD-like drug use. Understanding the neurobiological systems that drive this transition will inform therapeutic treatments in the aim to prevent increases in drug use and the development of SUD. The kappa opioid receptor (KOR) system is typically known for its role in negative affect, which is commonly found in SUD as well. Furthermore, the KOR system has also been implicated in drug use and importantly, modulating the negative effects of drug use. However, the specific neuronal subpopulation expressing KOR involved has not been identified. Here, we first demonstrated that pharmacologically inhibiting KOR in the nucleus accumbens core (NAcC), as a whole, blocks cocaine escalation under long-access self-administration conditions. We then demonstrated that KOR expressed on ventral tegmental area (VTA) neurons but not NAcC neurons is sufficient for blocking cocaine escalation by utilizing a novel virally-mediated CRISPR-SaCas9 knock-out of the oprk1 gene. Together, this suggests that activation of KOR on VTA terminals in the NAcC drives the transition to the SUD-like phenotype of escalation of cocaine consumption.

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT + neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3 + neurons blocked subsequent cocaine CPP. SERT + /VGluT3 - expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT + neurons produced place aversion, whereas optical stimulation of SERT + terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT 1B receptors potentiated subsequent cocaine CPP. 5-HT 1B is known to be expressed on 5-HT terminals in NAc, and 5-HT 1B transcript was also detected in Pdyn + , Adora2a + and ChAT + (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT 1B transcript was selectively elevated in Pdyn + cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT 1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Behavioral stress exposure increases the risk of drug-taking in individuals with substance use disorders by mechanisms involving the dynorphins, which are the endogenous neuropeptides for the kappa opioid receptor (KOR). KOR agonists have been shown to encode dysphoria, aversion, and changes in reward valuation, and kappa opioid antagonists are in clinical development for treating substance use disorders. In this study, we confirmed that KORs were expressed in dopaminergic neurons in the ventral tegmental area (VTA) of male C57BL6/J mice. Genetic ablation of KORs from dopamine neurons blocked the potentiating effects of repeated forced swim stress on cocaine conditioned place preference (CPP). KOR activation inhibited dopamine neuron GCaMP6m calcium activity in VTA during swim stress and caused a rebound enhancement during the period after stress exposure. Transient optogenetic inhibition of VTA dopamine neurons with AAV5-DIO-SwiChR was acutely aversive in a real time place preference assay and blunted cocaine CPP when inhibition was administered concurrently with cocaine conditioning. However, when inhibition preceded cocaine conditioning by 30 min, cocaine CPP was enhanced. Retrograde tracing with CAV2-DIO-ZsGreen identified a population of prodynorphin Cre neurons in the dorsal raphe nucleus (DRN) projecting to the VTA. Optogenetic stimulation of dynorphinergic neurons within the DRN by Channelrhodopsin2 activated KOR in VTA and ablation of prodynorphin blocked stress potentiation of cocaine CPP. Together, these studies demonstrate the presence of a dynorphin/KOR midbrain circuit that projects from the DRN to VTA and is involved in altering the dynamic response of dopamine neuron activity to enhance drug reward learning., Competing Interests: Declaration of Competing Interest None.

Following repeated opioid use, some dependent individuals experience persistent cognitive deficits that contribute to relapse of drug-taking behaviors, and one component of this response may be mediated by the endogenous dynorphin/kappa opioid system in neocortex. In C57BL/6 male mice, we find that acute morphine withdrawal evokes dynorphin release in the medial prefrontal cortex (PFC) and disrupts cognitive function by activation of local kappa opioid receptors (KORs). Immunohistochemical analyses using a phospho-KOR antibody confirmed that both withdrawal-induced and optically evoked dynorphin release activated KOR in PFC. Using a genetically encoded sensor based on inert KOR (kLight1.2a), we revealed the in vivo dynamics of endogenous dynorphin release in the PFC. Local activation of KOR in PFC produced multi-phasic disruptions of memory processing in an operant-delayed alternation behavioral task, which manifest as reductions in response number and accuracy during early and late phases of an operant session. Local pretreatment in PFC with the selective KOR antagonist norbinaltorphimine (norBNI) blocked the disruptive effect of systemic KOR activation during both early and late phases of the session. The early, but not late phase disruption was blocked by viral excision of PFC KORs, suggesting an anatomically dissociable contribution of pre- and postsynaptic KORs. Naloxone-precipitated withdrawal in morphine-dependent mice or optical stimulation of pdyn Cre neurons using Channelrhodopsin-2 disrupted delayed alternation performance, and the dynorphin-induced effect was blocked by local norBNI. Our findings describe a mechanism for control of cortical function during opioid dependence and suggest that KOR antagonism could promote abstinence., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Serotonin is a key mediator of stress, anxiety, and depression, and novel therapeutic targets within serotonin neurons are needed to combat these disorders. To determine how stress alters the translational profile of serotonin neurons, we sequenced ribosome-associated RNA from these neurons after repeated stress in male and female mice. We identified numerous sex- and stress-regulated genes. In particular, Fkbp5 mRNA, which codes for the glucocorticoid receptor co-chaperone protein FKBP51, was consistently upregulated in male and female mice following stress. Pretreatment with a selective FKBP51 inhibitor into the dorsal raphe prior to repeated forced swim stress decreased resulting stress-induced anhedonia. Our results support previous findings linking FKBP51 to stress-related disorders and provide the first evidence suggesting that FKBP51 function may be an important regulatory node integrating circulating stress hormones and serotonergic regulation of stress responses., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

The prototypical member of the receptor-inactivating kappa opioid receptor (KOR) antagonists, norbinaltorphimine (norBNI), produces prolonged receptor inactivation by a cJun kinase mechanism. These antagonists have potential therapeutic utility in the treatment of stress disorders; however, additional preclinical characterization is necessary to understand important aspects of their action. In this study, we report that norBNI does not work as effectively in female mice as in males because of estrogen regulation of G protein receptor kinase (GRK); pretreatment of ovary-intact female mice with the selective GRK2/3 inhibitor, Compound 101, made females equally sensitive to norBNI as males. Prior observations suggested that in vivo treatment with norBNI does not produce long-lasting inhibition of KOR regulation of dopamine release in the nucleus accumbens. We assessed the persistence of norBNI receptor inactivation in subcellular compartments. Fast-scan cyclic voltammetry recordings confirmed that presynaptic inhibition of dopamine release by the KOR agonist U69,593 was not blocked by in vivo pretreatment with norBNI under conditions that prevented KOR-mediated aversion and analgesia. We employed a novel in vivo proxy sensor of KOR activation, adenovirus associated double floxed inverted-HyPerRed, and demonstrated that KOR activation stimulates cJun kinase-dependent reactive oxygen species (ROS) production in somatic regions of ventral tegmental area dopamine neurons, but did not activate ROS production in dopamine terminals. The compartment selective action helps explain how dopamine somatic, but not terminally expressed, KORs are inactivated by norBNI. These results further elucidate molecular signaling mechanisms mediating receptor-inactivating KOR antagonist action and advance medication development for this novel class of stress-resilience medications. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) antagonists are being developed as novel proresilience therapeutics for the treatment of mood and substance use disorders. This study showed that the long-acting KOR antagonists are affected by both the sex of the animal and the subcellular compartment in which the receptor is expressed., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Stress is highly pervasive in humans, impacting motivated behaviors with an enormous toll on life quality. Many of the effects of stress are orchestrated by neuropeptides such as corticotropin-releasing factor (CRF). It has previously been shown that in stress-naïve male mice, CRF acts in the core of the nucleus accumbens (NAc) to produce appetitive effects and to increase dopamine release; yet in stress-exposed male mice, CRF loses its capacity to modulate NAc dopamine release and is aversive. In the current research, we tested whether this effect is comparable in females to males and whether the neuroadaptation is susceptible to social transmission. We found that, like in males, CRF increased dopamine release in stress-naïve but not stress-exposed female mice. Importantly, this persistent physiological change was not accompanied by overt behavioral changes that would be indicative of depression- or anxiety-like phenotype. Nonetheless, when these mice were housed for 7 days with stress-naïve conspecifics, the cage mates also exhibited a loss of dopamine potentiation by CRF. These data demonstrate the asymptomatic, yet pervasive transmission of stress-related neuroadaptations in the population., (Copyright © 2020 Steger, Land, Lemos, Chavkin and Phillips.)

Dopamine neurons of the ventral tegmental area (VTA) regulate reward association and motivation. It remains unclear whether there are distinct dopamine populations to mediate these functions. Using mouse genetics, we isolated two populations of dopamine-producing VTA neurons with divergent projections to the nucleus accumbens (NAc) core and shell. Inhibition of VTA-core-projecting neurons disrupted Pavlovian reward learning, and activation of these cells promoted the acquisition of an instrumental response. VTA-shell-projecting neurons did not regulate Pavlovian reward learning and could not facilitate acquisition of an instrumental response, but their activation could drive robust responding in a previously learned instrumental task. Both populations are activated simultaneously by cues, actions, and rewards, and this co-activation is required for robust reinforcement of behavior. Thus, there are functionally distinct dopamine populations in the VTA for promoting motivation and reward association, which operate on the same timescale to optimize behavioral reinforcement., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) by the G i/o protein-coupled κ opioid receptor (KOR), μ opioid, and D2 dopamine receptors stimulates peroxiredoxin 6 (PRDX6)-mediated production of reactive oxygen species (ROS). ROS production by KOR-inactivating antagonists norbinaltorphimine (norBNI) and JDTic blocks Gα i protein activation, but the signaling mechanisms and consequences of JNK activation by KOR agonists remain uncharacterized. Binding of arrestins to KOR causes desensitization of G protein signaling and acts as a scaffold to initiate MAPK activation. Here, we found that the KOR agonists U50,488 and dynorphin B stimulated biphasic JNK activation with an early arrestin-independent phase, requiring the small G protein RAC family small GTPase 1 (RAC1) and protein kinase C (PKC), and a later arrestin-scaffolded phase, requiring RAC1 and Ras homolog family member (RHO) kinase. JNK activation by U50,488 and dynorphin B also stimulated PRDX6-dependent ROS production but with an inverted U-shaped dose-response relationship. KOR agonist-induced ROS generation resulted from the early arrestin-independent phase of JNK activation, and this ROS response was suppressed by arrestin-dependent activation of the MAPK p38. The apparent balance between p38 MAPK and JNK/ROS signaling has important physiological implications for understanding of dynorphin activities during the stress response. To visualize these activities, we monitored KOR agonist-mediated activation of ROS in transfected live cells by two fluorescent sensors, CellROX Green and HyPerRed. These findings establish an important aspect of opioid receptor signaling and suggest that ROS induction may be part of the physiological response to KOR activation., (© 2019 Schattauer et al.)

Kappa receptor activation by dynorphins contributes to the anxiogenic, dysphoric, and cognitive disrupting effects of repeated stress, suggesting that kappa receptor antagonists might have therapeutic utility in the treatment of stress disorders. Three classes of kappa antagonists have been distinguished: non-selective, selective-competitive (readily reversible), and non-competitive (receptor-inactivating); however, which would be the most effective medication has not been established. To assess the utility of receptor inactivating antagonists, we tested the effects of a range of doses in both male and female mice. As previously established, the antinociceptive effects of the kappa agonist U50,488 were blocked by a single injection of the long-acting antagonist norbinatorphimine (norBNI) (10 mg/kg i.p.) in male mice. Ten to 20-fold lower doses of norBNI were ineffective after a single administration, but daily administration of 1.0 or 0.5 mg/kg for 5 days completely blocked U50,488 antinociceptive effects. Daily administration of 0.1 mg/kg norBNI produced slowly accumulating inhibition and completely blocked the antinociceptive effect of U50,488 after 20-30 days. Estrogen reduces female sensitivity to kappa opioid effects, but 30 days of 0.1 mg/kg norBNI completely blocked U50,488 analgesia in ovariectomized mice. Receptor inactivation in both male and female mice treated for 30 days with 0.1 mg/kg norBNI persisted for at least 1-week. These results suggest that receptor-inactivating kappa antagonists are effective in both males and females when given at 100-fold lower doses than typically administered in preclinical studies. The enhanced safety of this low-dosing protocol has important clinical implications if receptor inactivating kappa antagonists advance in medication development.

Activation of κ opioid receptors (KORs) produces analgesia and aversion via distinct intracellular signaling pathways, but whether G protein-biased KOR agonists can be designed to have clinical utility will depend on a better understanding of the signaling mechanisms involved. We found that KOR activation produced conditioned place aversion and potentiated CPP for cocaine in male and female C57BL/6N mice. Consistent with this, males and females both showed arrestin-mediated increases in phospho-p38 MAPK following KOR activation. Unlike in males, however, KOR activation had inconsistent analgesic effects in females and KOR increased Gβγ-mediated ERK phosphorylation in males, but not females. KOR desensitization was not responsible for the lack of response in females because neither Grk3 nor Pdyn gene knock-out enhanced analgesia. Instead, responsiveness was estrous cycle dependent because KOR analgesia was evident during low estrogen phases of the cycle and in ovariectomized (OVX) females. Estradiol treatment of OVX females suppressed KOR-mediated analgesia, demonstrating that estradiol was sufficient to blunt Gβγ-mediated KOR signals. G protein-coupled receptor kinase 2 (GRK2) is known to regulate ERK activation, and we found that the inhibitory, phosphorylated form of GRK2 was significantly higher in intact females. GRK2/3 inhibition by CMPD101 increased KOR stimulation of phospho-ERK in females, decreased sex differences in KOR-mediated inhibition of dopamine release, and enhanced mu opioid receptor and KOR-mediated analgesia in females. In OVX females, estradiol increased the association between GRK2 and Gβγ. These studies suggest that estradiol, through increased phosphorylation of GRK2 and possible sequestration of Gβγ by GRK2, blunts G protein-mediated signals. SIGNIFICANCE STATEMENT Chronic pain disorders are more prevalent in females than males, but opioid receptor agonists show inconsistent analgesic efficacy in females. κ opioid receptor (KOR) agonists have been tested in clinical trials for treating pain disorders based on their analgesic properties and low addictive potential. However, the molecular mechanisms underlying sex differences in KOR actions were previously unknown. Our studies identify an intracellular mechanism involving estradiol regulation of G protein-coupled receptor kinase 2 that is responsible for sexually dimorphic analgesic responses following opioid receptor activation. Understanding this mechanism will be critical for developing effective nonaddictive opioid analgesics for use in women and characterizing sexually dimorphic effects in other inhibitory G protein-coupled receptor signaling responses., (Copyright © 2018 the authors 0270-6474/18/388031-13$15.00/0.)

The dynorphin/κ-opioid receptor (KOR) system has been previously implicated in the regulation of cognition, but the neural circuitry and molecular mechanisms underlying KOR-mediated cognitive disruption are unknown. Here, we used an operational test of cognition involving timing and behavioral inhibition and found that systemic KOR activation impairs performance of male and female C57BL/6 mice in the differential reinforcement of low response rate (DRL) task. Systemic KOR antagonism also blocked stress-induced disruptions of DRL performance. KOR activation increased 'bursts' of incorrect responses in the DRL task and increased marble burying, suggesting that the observed disruptions in DRL performance may be attributed to KOR-induced increases in compulsive behavior. Local inactivation of KOR by injection of the long-acting antagonist nor-BNI in the ventral tegmental area (VTA), but not the infralimbic prefrontal cortex (PFC) or dorsal raphe nucleus (DRN), prevented disruption of DRL performance caused by systemic KOR activation. Cre-dependent genetic excision of KOR from dopaminergic, but not serotonergic neurons, also blocked KOR-mediated disruption of DRL performance. At the molecular level, we found that these disruptive effects did not require arrestin-dependent signaling, because neither global deletion of G-protein receptor kinase 3 (GRK3) nor cell-specific deletion of GRK3/arrestin-dependent p38α MAPK from dopamine neurons blocked KOR-mediated DRL disruptions. We then showed that nalfurafine, a clinically available G-biased KOR agonist, could also produce DRL disruptions. Together, these studies demonstrate that KOR activation in VTA dopamine neurons disrupts behavioral inhibition in a GRK3/arrestin-independent manner and suggests that KOR antagonists could be beneficial for decreasing stress-induced compulsive behaviors.

Inactivation of opioid receptors limits the therapeutic efficacy of morphine-like analgesics and mediates the long duration of kappa opioid antidepressants by an uncharacterized, arrestin-independent mechanism. Here we use an iterative, discovery-based proteomic approach to show that following opioid administration, peroxiredoxin 6 (PRDX6) is recruited to the opioid receptor complex by c-Jun N-terminal kinase (JNK) phosphorylation. PRDX6 activation generates reactive oxygen species via NADPH oxidase, reducing the palmitoylation of receptor-associated Gαi in a JNK-dependent manner. Selective inhibition of PRDX6 blocks Gαi depalmitoylation, prevents the enhanced receptor G-protein association and blocks acute analgesic tolerance to morphine and kappa opioid receptor inactivation in vivo. Opioid stimulation of JNK also inactivates dopamine D2 receptors in a PRDX6-dependent manner. We show that the loss of this lipid modification distorts the receptor G-protein association, thereby preventing agonist-induced guanine nucleotide exchange. These findings establish JNK-dependent PRDX6 recruitment and oxidation-induced Gαi depalmitoylation as an additional mechanism of Gαi-G-protein-coupled receptor inactivation.Opioid receptors are important modulators of nociceptive pain. Here the authors show that opioid receptor activation recruits peroxiredoxin 6 (PRDX6) to the receptor-Gαi complex by c-Jun N-terminal kinase, resulting in Gαi depalmitoylation and enhanced receptor-Gαi association.

Nalfurafine is a moderately selective kappa opioid receptor (KOR) analgesic with low incidence of dysphoric side effects in clinical development for the treatment of uremic pruritis. The basis for its reduced dysphoric effect compared to other KOR agonists is not clear, but prior studies suggest that the aversive properties of KOR agonists require p38α MAPK activation through an arrestin-dependent mechanism. To determine whether nalfurafine is a functionally selective KOR agonist, we measured its potency to activate the G protein-dependent early phase of Extracellular Signal-Regulated Kinase (ERK1/2) phosphorylation and the arrestin-dependent late phase of p38 MAPK signaling. Nalfurafine was approximately 250 fold more potent for ERK1/2 activation as compared to p38 MAPK activation in human KOR (hKOR) expressing HEK293 cells, and approximately 20 fold more potent for ERK1/2 activation than p38 activation in rodent KOR (rKOR) expressing HEK293 cells. The 10-fold greater G-bias at the hKOR than rKOR was unexpected, however the G protein biased effect of nalfurafine is consistent with its reduced dysphoric effects in human and rodent models. Although nalfurafine is reported to have low receptor selectivity in radioligand binding assays, its antinociceptive effect was blocked by the selective KOR antagonist norbinaltorphimine. Nalfurafine pretreatment also resulted in a KOR-dependent and mu opioid receptor-independent reduction in scratching induced by 5'-GNTI. These findings suggest that nalfurafine is a functionally selective KOR agonist and that KOR agonists able to selectively activate G protein signaling without activating p38α MAPK may have therapeutic potential as non-dysphoric antipruritic analgesics., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: The etiology of postmenopausal hot flashes is poorly understood, making it difficult to develop and target ideal therapies. A network of hypothalamic estrogen-sensitive neurons producing kisspeptin, neurokinin B and dynorphin-called KNDy neurons-are located adjacent to the thermoregulatory center. KNDy neurons regulate pulsatile secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). Dynorphin may inhibit this system by binding κ opioid receptors within the vicinity of KNDy neurons. We hypothesize that hot flashes are reduced by KNDy neuron manipulation., Methods: A double-blind, cross-over, placebo-controlled pilot study evaluated the effects of a κ agonist. Hot flash frequency was the primary outcome. Twelve healthy postmenopausal women with moderate to severe hot flashes (aged 48-60 y) were randomized. Eight women with sufficient baseline hot flashes for statistical analysis completed all three interventions: placebo, standard-dose pentazocine/naloxone (50/0.5 mg), or low-dose pentazocine/naloxone (25/0.25 mg). In an inpatient research setting, each participant received the three interventions, in randomized order, on three separate days. On each day, an intravenous catheter was inserted for LH blood sampling, and skin conductance and Holter monitors were placed. Subjective hot flash frequency and severity were recorded., Results: The mean (SEM) hot flash frequency 2 to 7 hours after therapy initiation was lower than that for placebo (standard-dose κ agonist, 4.75 [0.67] hot flashes per 5 h; low-dose κ agonist, 4.50 [0.57] hot flashes per 5 h; placebo, 5.94 [0.78] hot flashes per 5 h; P = 0.025). Hot flash intensity did not vary between interventions. LH pulsatility mirrored objective hot flashes in some--but not all--women., Conclusions: This pilot study suggests that κ agonists may affect menopausal vasomotor symptoms.

The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR(-/-) mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release., Significance Statement: Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood. Understanding how KOR activation produces dysphoria is key to the development of better analgesics and to defining how the endogenous dynorphin opioids produce their depression-like effects. Results in this study show that the aversive effects of κ receptor activation required arrestin-dependent p38α MAPK activation in dopamine neurons but did not require inhibition of dopamine release in the nucleus accumbens. Thus, contrary to the prevailing view, inhibition of mesolimbic dopamine release does not mediate the aversive effects of KOR activation and functionally selective κ opioids that do not activate arrestin signaling may be effective analgesics lacking dysphoric effects., (Copyright © 2015 the authors 0270-6474/15/3512917-15$15.00/0.)

G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G protein-coupled receptor kinase (GRK) and subsequent arrestin binding; morphine, however, was previously found to activate a c-Jun N-terminal kinase (JNK)-dependent, GRK/arrestin-independent pathway to produce mu opioid receptor (MOR) inactivation in spinally-mediated, acute anti-nociceptive responses [Melief et al.] [1]. In the current study, we determined that JNK2 was also required for centrally-mediated analgesic tolerance to morphine using the hotplate assay. We compared JNK activation by morphine and fentanyl in JNK1(-/-), JNK2(-/-), JNK3(-/-), and GRK3(-/-) mice and found that both compounds specifically activate JNK2 in vivo; however, fentanyl activation of JNK2 was GRK3-dependent, whereas morphine activation of JNK2 was GRK3-independent. In MOR-GFP expressing HEK293 cells, treatment with either arrestin siRNA, the Src family kinase inhibitor PP2, or the protein kinase C (PKC) inhibitor Gö6976 indicated that morphine activated JNK2 through an arrestin-independent Src- and PKC-dependent mechanism, whereas fentanyl activated JNK2 through a Src-GRK3/arrestin-2-dependent and PKC-independent mechanism. This study resolves distinct ligand-directed mechanisms of JNK activation by mu opioid agonists and understanding ligand-directed signaling at MOR may improve opioid therapeutics., (Copyright © 2015 Elsevier Inc. All rights reserved.)

There is ongoing debate about the role of G protein-coupled receptor kinases (GRKs) in agonist-induced desensitization of the μ-opioid receptor (MOPr) in brain neurons. In the present paper, we have used a novel membrane-permeable, small-molecule inhibitor of GRK2 and GRK3, Takeda compound 101 (Cmpd101; 3-[[[4-methyl-5-(4-pyridyl)-4H-1,2,4-triazole-3-yl] methyl] amino]-N-[2-(trifuoromethyl) benzyl] benzamidehydrochloride), to study the involvement of GRK2/3 in acute agonist-induced MOPr desensitization. We observed that Cmpd101 inhibits the desensitization of the G protein-activated inwardly-rectifying potassium current evoked by receptor-saturating concentrations of methionine-enkephalin (Met-Enk), [d-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), endomorphin-2, and morphine in rat and mouse locus coeruleus (LC) neurons. In LC neurons from GRK3 knockout mice, Met-Enk-induced desensitization was unaffected, implying a role for GRK2 in MOPr desensitization. Quantitative analysis of the loss of functional MOPrs following acute agonist exposure revealed that Cmpd101 only partially reversed MOPr desensitization. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase C, c-Jun N-terminal kinase, or GRK5 did not inhibit the Cmpd101-insensitive component of desensitization. In HEK 293 cells, Cmpd101 produced almost complete inhibition of DAMGO-induced MOPr phosphorylation at Ser(375), arrestin translocation, and MOPr internalization. Our data demonstrate a role for GRK2 (and potentially also GRK3) in agonist-induced MOPr desensitization in the LC, but leave open the possibility that another, as yet unidentified, mechanism of desensitization also exists., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Cocaine-experienced Wistar and Wistar Kyoto (WKY) rats received four daily repeated forced swim stress sessions (R-FSS), each of which preceded 4-hour cocaine self-administration sessions. Twenty-four hours after the last swim stress, cocaine valuation was assessed during a single-session threshold procedure. Prior exposure to R-FSS significantly altered cocaine responding in Wistar, but not WKY, rats. Behavioral economic analysis of responding revealed that the Wistar rats that had received R-FSS exhibited an increase in the maximum price that they were willing to pay for cocaine (Pmax ). Pre-treatment with the long-lasting kappa opioid receptor (KOR) antagonist norbinaltorphimine prevented the stress-induced increase in Pmax . Thus, R-FSS exposure had strain-dependent effects on cocaine responding during the threshold procedure, and the stress effects on cocaine valuation exhibited by Wistar, but not WKY, required intact KOR signaling., (© 2014 Society for the Study of Addiction.)

Behavioral stressors increase addiction risk in humans and increase the rewarding valence of drugs of abuse including cocaine, nicotine and ethanol in animal models. Prior studies have established that this potentiation of drug reward was mediated by stress-induced release of the endogenous dynorphin opioids and subsequent kappa opioid receptor (KOR) activation. In this study, we used in vivo fast scan cyclic voltammetry to test the hypothesis that KOR activation before cocaine administration might potentiate the evoked release of dopamine from ventral tegmental (VTA) synaptic inputs to the nucleus accumbens (NAc) and thereby increase the rewarding valence of cocaine. The KOR agonist U50488 inhibited dopamine release evoked by either medial forebrain bundle (MFB) or pedunculopontine tegmental nucleus (PPTg) activation of VTA inputs to the shell or core of the mouse NAc. Cocaine administration increased the dopamine response recorded in either the shell or core evoked by either MFB or PPTg stimulation. Administration of U50488 15 min before cocaine blocked the conditioned place preference (CPP) to cocaine, but only significantly reduced the effect of cocaine on the dopamine response evoked by PPTg stimulation to NAc core. In contrast, administration of U50488 60 min before cocaine significantly potentiated cocaine CPP and significantly increased the effects of cocaine on the dopamine response evoked by either MFB or PPTg stimulation, recorded in either NAc shell or core. Results of this study support the concept that stress-induced activation of KOR by endogenous dynorphin opioids may enhance the rewarding valence of drugs of abuse by potentiating the evoked dopamine response.

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the CNS. Characterization of murine CSF proteomes can provide a valuable resource for studying CNS injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through nonterminal CSF extractions in C57Bl/6 mice and high-resolution 2D-LC MS/MS analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. We identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis. The data are available in the ProteomeXchange with identifier PXD000248 (http://proteomecentral.proteomexchange.org/dataset/PXD000248)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Studies of kappa opioid receptor signaling mechanisms during the last decade have demonstrated that agonist activation of the receptor results in Gβγ-dependent signaling and distinct arrestin-dependent signaling events. Gβγ-dependent signaling results in ion channel regulation causing neuronal inhibition, inhibition of transmitter release, and subsequent analgesic responses. In contrast, arrestin-dependent signaling events result in p38 MAPK activation and subsequent dysphoric and proaddictive behavioral responses. Resolution of these two branches of signaling cascades has enabled strategies designed to identify pathway-selective drugs that may have unique therapeutic utilities.

The calcium-activated small conductance potassium channel SK3 plays an essential role in the regulation of dopamine neuron activity patterns. Here we demonstrate that expression of a human disease-related SK3 mutation (hSK3Δ) in dopamine neurons of mice disrupts the balance between tonic and phasic dopamine neuron activity. Expression of hSK3Δ suppressed endogenous SK currents, reducing coupling between SK channels and NMDA receptors (NMDARs) and increasing permissiveness for burst firing. Consistent with enhanced excitability of dopamine neurons, hSK3Δ increased evoked calcium signals in dopamine neurons in vivo and potentiated evoked dopamine release. Specific expression of hSK3Δ led to deficits in attention and sensory gating and heightened sensitivity to a psychomimetic drug. Sensory-motor alterations and psychomimetic sensitivity were recapitulated in a mouse model of transient, reversible dopamine neuron activation. These results demonstrate the cell-autonomous effects of a human ion channel mutation on dopamine neuron physiology and the impact of activity pattern disruption on behavior., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This issue of Molecular Pharmacology is dedicated to Dr. Avram Goldstein, the journal's founding editor and one of the leaders in the development of modern pharmacology. This article focuses on his contributions to the discovery of the dynorphins and evidence that members of this family of opioid peptides are endogenous agonists for the kappa opioid receptor. In his original publication describing the purification and sequencing of dynorphin A, Avram described this peptide as "extraordinarily potent" ("dyn" from the Greek, dynamis = power and "orphin" for endogenous morphine peptide). The name originally referred to its high affinity and great potency in the bioassay that was used to follow its activity during purification, but the name has come to have a second meaning: studies of its physiologic function in brain continue to provide powerful insights to the molecular mechanisms controlling mood disorders and drug addiction. During the 30 years since its discovery, we have learned that the dynorphin peptides are released in brain during stress exposure. After they are released, they activate kappa opioid receptors distributed throughout the brain and spinal cord, where they trigger cellular responses resulting in different stress responses: analgesia, dysphoria-like behaviors, anxiety-like responses, and increased addiction behaviors in experimental animals. Avram predicted that a detailed molecular analysis of opiate drug actions would someday lead to better treatments for drug addiction, and he would be gratified to know that subsequent studies enabled by his discovery of the dynorphins resulted in insights that hold great promise for new treatments for addiction and depressive disorders.

Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance.

KOR activation of Gβγ dependent signaling results in analgesia, whereas the dysphoric effects of KOR agonists are mediated by a different pathway involving G protein receptor kinase and non-visual arrestin. Based on this distinction, a partial KOR agonist that does not efficiently activate arrestin-dependent biased signaling may produce analgesia without dysphoria. No KOR-selective partial agonists are currently available, and preclinical assessment is complicated by sequence differences between rodent (r) and human (h) KOR. In this study, we compared the signaling initiated by the available partial agonists. Pentazocine was significantly more potent at activating p38 MAPK in hKOR than rKOR expressed in HEK293 cells but equally potent at arrestin-independent activation of ERK1/2 in hKOR and rKOR. Similarly, butorphanol increased phospho-p38-ir in hKOR-expressing cells but did not activate p38 in rKOR-HEK293. Like pentazocine, butorphanol was equally efficacious at activating ERK1/2 in rKOR and hKOR. In contrast, levorphanol, nalorphine, and U50,488 did not distinguish between hKOR and rKOR in p38 MAPK activation. Consistent with its low potency at p38 activation, pentazocine did not produce conditioned place aversion in mice. hKOR lacks the Ser-369 phosphorylation site in rKOR required for G protein receptor kinase/arrestin-dependent p38 activation, but mutation of the Ser-358 to asparagine in hKOR blocked p38 activation without affecting the acute arrestin-independent activation of ERK1/2. This study shows that hKOR activates p38 MAPK through a phosphorylation and arrestin-dependent mechanism; however, activation differs between hKOR and rKOR for some ligands. These functional selectivity differences have important implications for preclinical screening of partial KOR agonists.

Activation of the dynorphin/κ-opioid receptor (KOR) system by repeated stress exposure or agonist treatment produces place aversion, social avoidance, and reinstatement of extinguished cocaine place preference behaviors by stimulation of p38α MAPK, which subsequently causes the translocation of the serotonin transporter (SERT, SLC6A4) to the synaptic terminals of serotonergic neurons. In the present study we extend those findings by showing that stress-induced potentiation of cocaine conditioned place preference occurred by a similar mechanism. In addition, SERT knock-out mice did not show KOR-mediated aversion, and selective reexpression of SERT by lentiviral injection into the dorsal raphe restored the prodepressive effects of KOR activation. Kinetic analysis of several neurotransporters demonstrated that repeated swim stress exposure selectively increased the V(max) but not K(m) of SERT without affecting dopamine transport or the high-capacity, low-affinity transporters. Although the serotonergic neurons in the dorsal raphe project throughout the forebrain, a significant stress-induced increase in cell-surface SERT expression was only evident in the ventral striatum, and not in the dorsal striatum, hippocampus, prefrontal cortex, amygdala, or dorsal raphe. Stereotaxic microinjections of the long-lasting KOR antagonist norbinaltorphimine demonstrated that local KOR activation in the nucleus accumbens, but not dorsal raphe, mediated this stress-induced increase in ventral striatal surface SERT expression. Together, these results support the hypothesis that stress-induced activation of the dynorphin/KOR system produces a transient increase in serotonin transport locally in the ventral striatum that may underlie some of the adverse consequences of stress exposure, including the potentiation of the rewarding effects of cocaine.

Stressors motivate an array of adaptive responses ranging from 'fight or flight' to an internal urgency signal facilitating long-term goals. However, traumatic or chronic uncontrollable stress promotes the onset of major depressive disorder, in which acute stressors lose their motivational properties and are perceived as insurmountable impediments. Consequently, stress-induced depression is a debilitating human condition characterized by an affective shift from engagement of the environment to withdrawal. An emerging neurobiological substrate of depression and associated pathology is the nucleus accumbens, a region with the capacity to mediate a diverse range of stress responses by interfacing limbic, cognitive and motor circuitry. Here we report that corticotropin-releasing factor (CRF), a neuropeptide released in response to acute stressors and other arousing environmental stimuli, acts in the nucleus accumbens of naive mice to increase dopamine release through coactivation of the receptors CRFR1 and CRFR2. Remarkably, severe-stress exposure completely abolished this effect without recovery for at least 90 days. This loss of CRF's capacity to regulate dopamine release in the nucleus accumbens is accompanied by a switch in the reaction to CRF from appetitive to aversive, indicating a diametric change in the emotional response to acute stressors. Thus, the current findings offer a biological substrate for the switch in affect which is central to stress-induced depressive disorders.

Repeated stress releases dynorphins and causes subsequent activation of κ-opioid receptors (KORs) in limbic brain regions. The serotonergic dorsal raphe nucleus (DRN) has previously been found to be an important site of action for the dysphoric effects of dynorphin-κ-opioid receptor system activation during stress-evoked behaviors, and KOR-induced activation of p38α mitogen-activated protein kinase (MAPK) in serotonergic neurons was found to be a critical mediator of the aversive properties of stress. Yet, how dynorphins and KORs functionally regulate the excitability of serotonergic DRN neurons both in adaptive and pathological stress states is poorly understood. Here we report that acute KOR activation by the selective agonist U69,593 [(+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide] inhibits serotonergic neuronal excitability within the DRN through both presynaptic inhibition of excitatory synaptic transmission and postsynaptic activation of G-protein-gated inwardly rectifying potassium channels (GIRKs) electrophysiologically recorded in brain slices. C57BL/6 mice subjected to repeated swim, stress sessions had significantly reduced KOR-mediated GIRK currents recorded in serotonergic neurons in DRN postsynaptically, without significantly affecting presynaptic KOR-mediated regulation of excitatory transmission. This effect was blocked by genetic excision of p38α MAPK selectively from serotonergic neurons. An increase in phospho-immunoreactivity suggests that this functional dysregulation may be a consequence of tyrosine phosphorylation of GIRK (K(IR)3.1) channels. These data elucidate a mechanism for stress-induced dysregulation of the excitability of neurons in the DRN and identify a functional target of stress-induced p38α MAPK activation that may underlie some of the negative effects of pathological stress exposure.

Critically ill preterm infants are often exposed to stressors that may affect neurodevelopment and behavior. We reported that exposure of neonatal mice to stressors or morphine produced impairment of adult morphine-rewarded conditioned place preference (CPP) and altered hippocampal gene expression. We now further this line of inquiry by examining both short- and long-term effects of neonatal stress and morphine treatment. Neonatal C57BL/6 mice were treated twice daily from postnatal day (P) 5 to P9 using different combinations of factors. Subsets received saline or morphine injections (2mg/kgs.c.) or were exposed to our neonatal stress protocol (maternal separation 8h/d × 5d+gavage feedings ± hypoxia/hyperoxia). Short-term measures examined on P9 were neuronal fluorojade B and bromodeoxyuridine staining, along with urine corticosterone concentrations. Long-term measures examined in adult mice (>P60) included CPP learning to cocaine reward (± the kappa opioid receptor (KOR) agonist U50,488 injection), and adult hippocampal neurogenesis (PCNA immunolabeling). Neonatal stress (but not morphine) decreased the cocaine-CPP response and this effect was reversed by KOR stimulation. Both neonatal stress or morphine treatment increased hippocampal neurogenesis in adult mice. We conclude that reduced learning and increased hippocampal neurogenesis are both indicators that neonatal stress desensitized mice and reduced their arousal and stress responsiveness during adult CPP testing. Reconciled with other findings, these data collectively support the stress inoculation hypothesis whereby early life stressors prepare animals to tolerate future stress., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Many smokers describe the anxiolytic and stress-reducing effects of nicotine, the primary addictive component of tobacco, as a principal motivation for continued drug use. Recent evidence suggests that activation of the stress circuits, including the dynorphin/κ-opioid receptor system, modulates the rewarding effects of addictive drugs. In the present study, we find that nicotine produced dose-dependent conditioned place preference (CPP) in mice. κ-receptor activation, either by repeated forced swim stress or U50,488 (5 or 10 mg/kg, i.p.) administration, significantly potentiated the magnitude of nicotine CPP. The increase in nicotine CPP was blocked by the κ-receptor antagonist norbinaltorphimine (norBNI) either systemically (10 mg/kg, i.p.) or by local injection in the amygdala (2.5 μg) without affecting nicotine reward in the absence of stress. U50,488 (5 mg/kg, i.p.) produced anxiety-like behaviors in the elevated-plus maze and novel object exploration assays, and the anxiety-like behaviors were attenuated both by systemic nicotine (0.5 mg/kg, s.c.) and local injection of norBNI into the amygdala. Local norBNI injection in the ventral posterior thalamic nucleus (an adjacent brain region) did not block the potentiation of nicotine CPP or the anxiogenic-like effects of κ-receptor activation. These results suggest that the rewarding effects of nicotine may include a reduction in the stress-induced anxiety responses caused by activation of the dynorphin/κ-opioid system. Together, these data implicate the amygdala as a key region modulating the appetitive properties of nicotine, and suggest that κ-opioid antagonists may be useful therapeutic tools to reduce stress-induced nicotine craving.

The κ-opioid receptor is a widely expressed G-protein-coupled receptor that has been implicated in biological responses to pain, stress, anxiety, and depression, and its potential as a therapeutic target in these syndromes is becoming increasingly apparent. However, the prototypical selective κ-opioid antagonists have very long durations of action that have been attributed to c-Jun N-terminal kinase (JNK) 1 activation in vivo. To test generality of this proposed noncompetitive mechanism, we used C57BL/6 wild type mice to determine the durations of antagonist action of novel κ-opioid receptor ligands and examined their efficacies for JNK1 activation compared with conventional competitive antagonists. Of the 12 compounds tested, 5 had long durations of action that positively correlated with JNK activation: RTI-5989-97 [(3S)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylpropyl]-2-methyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide], RTI-5989-194 [(3R)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylbutyl]-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide], RTI-5989-241 [(3R)-7-hydroxy-N-[(1S)-1-{[(3R,4R)-4-(3-methoxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide)], nor-binaltorphimine (nor-BNI); and (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic). Seven had short durations of action and did not increase phospho-JNK-ir: RTI-5989-212[(3R)-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylpropyl]-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide], RTI-5989-240 [(3R)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-(2-methylpropyl]-3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide], JSPA0658 [(S)-3-fluoro-4-(4-((2-(3,5-dimethylphenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide], JSPA071B [(S)-3-fluoro-4-(4-((2-(3,5-bis(trifluoromethyl)phenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide]. PF-4455242 [2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine], PF-4455242 [2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine], FP3FBZ [(S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide], and naloxone. After long-acting antagonist treatment, pJNK-ir did not increase in mice lacking the κ-opioid receptor; increased pJNK-ir returned to baseline by 48 h after treatment; and a second challenge with nor-BNI 72 h after the first did not increase pJNK-ir. Long-lasting antagonism and increased phospho-JNK-ir were not seen in animals lacking the JNK1 isoform. These results support the hypothesis that the duration of action of small molecule κ-opioid receptor antagonists in vivo is determined by their efficacy in activating JNK1 and that persistent inactivation of the κ-receptor does not require sustained JNK activation.

Maladaptive responses to stress adversely affect human behavior, yet the signaling mechanisms underlying stress-responsive behaviors remain poorly understood. Using a conditional gene knockout approach, the α isoform of p38 mitogen-activated protein kinase (MAPK) was selectively inactivated by AAV1-Cre-recombinase infection in specific brain regions or by promoter-driven excision of p38α MAPK in serotonergic neurons (by Slc6a4-Cre or ePet1-Cre) or astrocytes (by Gfap-CreERT2). Social defeat stress produced social avoidance (a model of depression-like behaviors) and reinstatement of cocaine preference (a measure of addiction risk) in wild-type mice, but not in mice having p38α MAPK selectively deleted in serotonin-producing neurons of the dorsal raphe nucleus. Stress-induced activation of p38α MAPK translocated the serotonin transporter to the plasma membrane and increased the rate of transmitter uptake at serotonergic nerve terminals. These findings suggest that stress initiates a cascade of molecular and cellular events in which p38α MAPK induces a hyposerotonergic state underlying depression-like and drug-seeking behaviors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Psychiatric disorders including anxiety, depression, and addiction are both precipitated and exacerbated by severe or chronic stress exposure. While acutely, stress responses are adaptive, repeated exposure to stress can dysregulate the brain in such a way as to predispose the organism to both physiological and mental illness. Understanding the neuronal chemicals, cell types, and circuits involved in both normal and pathological stress responses are essential in developing new therapeutics for psychiatric diseases. Varying degrees of stressor exposure cause the release of a constellation of chemicals, including neuropeptides such as dynorphin. Neuropeptidergic release can be very difficult to directly measure with adequate spatial and temporal resolution. Moreover, the downstream consequences following release and receptor binding are numerous and also difficult to measure with cellular resolution. Following repeated stressor exposure, dynorphin is released, binds to the kappa opioid receptor (KOR), and causes activation of KOR. Agonist-activated KOR becomes a substrate for G protein receptor kinase (GRK), which phosphorylates the Ser369 residue at the C-terminal tail of the receptor in the first step in the β-Arrestin-dependent desensitization cascade. Through the use of phospho--selective antibodies developed and validated in the laboratory, we have the tools, to assess with fine cellular resolution, the strength of behavioral stimulus required for release, time course of the release, and regional location of release. We have gone on to show that following KOR activation, both ERK 1/2 and p38 MAP kinase phosphorylation are increased through use of commercially available phospho-selective antibodies. Finally, we have identified that one effector of KOR/p38MAP kinase is K(IR) 3.1 and have developed a phospho-selective antibody against the Y12 motif of this channel. Much like KOR and p38 MAP kinase, phosphorylation of this potassium channel increases following repeated stress. The following chapter discusses immunohistochemical and quantification methods used for phospho-selective antibodies used in various brain regions following behavioral manipulations.

α(1D)-Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α(1D)-ARs form a "signalosome" with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α(1D)-AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α(1D)-AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α(1D)-AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α(1D)-AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.

Kappa-opioid receptors (κOR) are positioned to modulate pre- and post-synaptic responses of norepinephrine-containing neurons in the rat locus coeruleus (LC). The ability of an acute systemic injection of a long acting κOR agonist, U50,488, to induce trafficking of κOR was assessed in the LC using immunogold-silver detection in male Sprague-Dawley rats. U50,488 administration shifted immunogold-silver labeling indicative of κOR from primarily plasmalemmal sites to intracellular sites when compared to vehicle-treated subjects. This translocation from the plasma membrane to the cytoplasmic compartment was prevented by pre-treatment with the κOR antagonist, norbinaltorphimine (norBNI). To determine whether agonist stimulation could induce adaptations in the expression of the noradrenergic synthesizing enzyme, dopamine beta hydroxylase (DβH), and κOR expression, Western blot analysis was used to compare expression levels of DβH and κOR following U50,488 administration. Expression levels for DβH and κOR were significantly increased following U50,488 administration when compared to controls. These data indicate that a systemic injection of a κOR agonist stimulates internalization of κORs in noradrenergic neurons and can impact κOR and DβH expression levels in this stress-sensitive brain region., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Stress exposure increases the risk of addictive drug use in human and animal models of drug addiction by mechanisms that are not completely understood. Mice subjected to repeated forced swim stress (FSS) before cocaine develop significantly greater conditioned place preference (CPP) for the drug-paired chamber than unstressed mice. Analysis of the dose dependency showed that FSS increased both the maximal CPP response and sensitivity to cocaine. To determine whether FSS potentiated CPP by enhancing associative learning mechanisms, mice were conditioned with cocaine in the absence of stress, then challenged after association was complete with the kappa-opioid receptor (KOR) agonist U50,488 or repeated FSS, before preference testing. Mice challenged with U50,488 60 min before CPP preference testing expressed significantly greater cocaine-CPP than saline-challenged mice. Potentiation by U50,488 was dose and time dependent and blocked by the KOR antagonist norbinaltorphimine (norBNI). Similarly, mice subjected to repeated FSS before the final preference test expressed significantly greater cocaine-CPP than unstressed controls, and FSS-induced potentiation was blocked by norBNI. Novel object recognition (NOR) performance was not affected by U50,488 given 60 min before assay, but was impaired when given 15 min before NOR assay, suggesting that KOR activation did not potentiate CPP by facilitating memory retrieval or expression. The results from this study show that the potentiation of cocaine-CPP by KOR activation does not result from an enhancement of associative learning mechanisms and that stress may instead enhance the rewarding valence of cocaine-associated cues by a dynorphin-dependent mechanism.

Ligand-directed signaling has been suggested as a basis for the differences in responses evoked by otherwise receptor-selective agonists. The underlying mechanisms are not understood, yet clearer definition of this concept may be helpful in the development of novel, pathway-selective therapeutic agents. We previously showed that kappa-opioid receptor activation of JNK by one class of ligand, but not another, caused persistent receptor inactivation. In the current study, we found that the mu-opioid receptor (MOR) could be similarly inactivated by a specific ligand class including the prototypical opioid, morphine. Acute analgesic tolerance to morphine and related opioids (morphine-6-glucuronide and buprenorphine) was blocked by JNK inhibition, but not by G protein receptor kinase 3 knockout. In contrast, a second class of mu-opioids including fentanyl, methadone, and oxycodone produced acute analgesic tolerance that was blocked by G protein receptor kinase 3 knockout, but not by JNK inhibition. Acute MOR desensitization, demonstrated by reduced D-Ala(2)-Met(5)-Glyol-enkephalin-stimulated [(35)S]GTPgammaS binding to spinal cord membranes from morphine-pretreated mice, was also blocked by JNK inhibition; however, desensitization of D-Ala(2)-Met(5)-Glyol-enkephalin-stimulated [(35)S]GTPgammaS binding following fentanyl pretreatment was not blocked by JNK inhibition. JNK-mediated receptor inactivation of the kappa-opioid receptor was evident in both agonist-stimulated [(35)S]GTPgammaS binding and opioid analgesic assays; however, gene knockout of JNK 1 selectively blocked kappa-receptor inactivation, whereas deletion of JNK 2 selectively blocked MOR inactivation. These findings suggest that ligand-directed activation of JNK kinases may generally provides an alternate mode of G protein-coupled receptor regulation.

Background and Rationale: The dynorphin/kappa opioid receptor (KOR) system has been implicated as a critical component of the stress response. Stress-induced activation of dynorphin-KOR is well known to produce analgesia, and more recently, it has been implicated as a mediator of stress-induced responses including anxiety, depression, and reinstatement of drug seeking., Objective: Drugs selectively targeting specific KOR signaling pathways may prove potentially useful as therapeutic treatments for mood and addiction disorders., Results: KOR is a member of the seven transmembrane spanning (7TM) G-protein coupled receptor (GPCR) superfamily. KOR activation of pertussis toxin-sensitive G proteins leads to Galphai/o inhibition of adenylyl cyclase production of cAMP and releases Gbetagamma, which modulates the conductances of Ca(+2) and K(+) channels. In addition, KOR agonists activate kinase cascades including G-protein coupled Receptor Kinases (GRK) and members of the mitogen-activated protein kinase (MAPK) family: ERK1/2, p38 and JNK. Recent pharmacological data suggests that GPCRs exist as dynamic, multi-conformational protein complexes that can be directed by specific ligands towards distinct signaling pathways. Ligand-induced conformations of KOR that evoke beta-arrestin-dependent p38 MAPK activation result in aversion; whereas ligand-induced conformations that activate JNK without activating arrestin produce long-lasting inactivation of KOR signaling., Conclusions: In this review, we discuss the current status of KOR signal transduction research and the data that support two novel hypotheses: (1) KOR selective partial agonists that do not efficiently activate p38 MAPK may be useful analgesics without producing the dysphoric or hallucinogenic effects of selective, highly efficacious KOR agonists and (2) KOR antagonists that do not activate JNK may be effective short-acting drugs that may promote stress-resilience.

The effects of phosphorylation of the tyrosine residue in the highly conserved DRY motif expressed in the putative second cytoplasmic loop of the mu-opioid receptor were assessed after expression in human embryonic kidney (HEK) 293 cells. Tyrosine kinase activation by epidermal growth factor (EGF) or hydrogen peroxide treatment effectively increased phosphorylation of the tyrosine-166 in the mu-opioid receptor (MOR-Tyr166p) as measured by a novel phosphoselective antibody. We were surprised to find that the increase in MOR-Tyr166p immunoreactivity (ir) required coactivation by the opioid agonist [D-Ala(2),methyl-Phe(4),Gly(5)-ol]enkephalin (DAMGO), as demonstrated by both Western blot imaging of membrane proteins and confocal microscopy of transfected cells; MOR-Tyr166p-ir did not significantly increase after either DAMGO, EGF, or H(2)O(2) treatment alone. The increase in MOR-Tyr166p-ir was blocked by pretreatment with the opioid antagonist naloxone or the Src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Consistent with these data, mutation of the tyrosine-166 to phenylalanine blocked the increased immunoreactivity, and untransfected HEK293 cells did not increase MOR-Tyr166p-ir after treatment. DAMGO increased guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding to membranes from cells expressing wild-type MOR or MOR-Y166F receptors in a dose-dependent manner. Pretreatment of the wild-type MOR-expressing cells with the combination of DAMGO and EGF completely blocked subsequent DAMGO stimulation of [(35)S]GTP gamma S binding membranes, whereas [(35)S]GTP gamma S binding to membranes from cells expressing mutated MOR(Y166F) was only partially inhibited. These results suggest that G-protein activation as measured by [(35)S]GTP gamma S binding can be regulated by DAMGO and EGF by convergent mechanisms and support the hypothesis that tyrosine phosphorylation within the DRY motif may reduce mu-opioid receptor-G-protein coupling efficiency.