Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 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 H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 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 H2O2 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 Aß-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. Lastly, we demonstrated acute opioid-induced generation of H2O2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 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.

Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 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 H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 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 H2O2 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 Aß-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 H2O2 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.

Optogenetic stimulation of dynorphinergic neurons within the dorsal raphe activate kappa opioid receptors in the ventral tegmental area and ablation of dorsal raphe prodynorphin or kappa receptors in dopamine neurons blocks stress potentiation of cocaine reward.

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 prodynorphinCre 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.

Stress decreases serotonin tone in the nucleus accumbens in male mice to promote aversion and potentiate cocaine preference via decreased stimulation of 5-HT1B receptors.

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-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

Release of endogenous dynorphin opioids in the prefrontal cortex disrupts cognition.

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 pdynCre 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.

Sequencing the serotonergic neuron translatome reveals a new role for Fkbp5 in stress.

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.

Insidious Transmission of a Stress-Related Neuroadaptation.

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.

Regulation of Kappa Opioid Receptor Inactivation Depends on Sex and Cellular Site of Antagonist Action.

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.

Synergy of Distinct Dopamine Projection Populations in Behavioral Reinforcement.

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.

Reactive oxygen species (ROS) generation is stimulated by κ opioid receptor activation through phosphorylated c-Jun N-terminal kinase and inhibited by p38 mitogen-activated protein kinase (MAPK) activation.

Activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) by the Gi/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 Gαi 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.

Repeated Administration of Norbinaltorphimine Produces Cumulative Kappa Opioid Receptor Inactivation.

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.

Estrogen Regulation of GRK2 Inactivates Kappa Opioid Receptor Signaling Mediating Analgesia, But Not Aversion.

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 Gßγ-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 Gßγ-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 Gßγ. These studies suggest that estradiol, through increased phosphorylation of GRK2 and possible sequestration of Gßγ 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.

κ-Opioid Receptor Activation in Dopamine Neurons Disrupts Behavioral Inhibition.

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.

Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase.

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 G-protein biased agonist having significantly greater bias at the human than rodent form of the kappa opioid receptor.

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.

Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons.

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.