Kappa Opioid Receptor Signaling Research Articles

Effects of kappa-opioid agonist U-50488 and p38 MAPK inhibitor SB203580 on the spike activity of pyramidal neurons in the basolateral amygdala

Introduction: Kappa-opioid receptor (KOR) signaling in the basolateral amygdala (BLA) underlies KOR agonist-induced aversion. In this study, we aimed to understand the individual and combined effects of KOR agonist U-50488 and p38 MAPK inhibitor SB203580 on the spiking activity of pyramidal neurons in the BLA to shed light on the complex interplay between KORs, the p38 MAPK, and neuronal excitability. Materials and Methods: Electrophysiological experiments were performed using the patch-clamp technique in the whole-cell configuration. Rat brain slices containing the amygdala were prepared, and pyramidal neurons within the BLA were visually patched and recorded in the current clamp mode. The neurons were identified by their accommodation properties and neural activity signals were amplified and analyzed. Using local perfusion, we obtained three dose-response curves for: (a) U-50488 (0.001–10 μM); (b) U-50488 (0.001–10 μM) in the presence of SB203580 (1 μM); and (c) U-50488 (0.01–10 μM) in the presence of SB203580 (5 μM). Results: After the application of U-50488, pyramidal neurons had a higher action potential firing rate in response to a current injection than control neurons (p<0.001). The dose-dependent curves we obtained indicate that the combination of U-50488 and SB203580 results in non-competitive antagonism. This conclusion is supported by the observed change in the curve’s slope with reduction in the maximum effect of U-50488. Thus, it can be assumed that the increase in spike activity of pyramidal neurons of the amygdala is mediated through the beta-arrestin pathway. When this pathway is blocked, the spike activity reverts to its baseline level. Conclusion: Our study found that the KOR agonist-induced spiking activity of the BLA pyramidal neurons is mediated by the beta-arrestin pathway and can be suppressed by the application of the p38 MAPK inhibitor SB203580.

Dynorphin / kappa-opioid receptor regulation of excitation-inhibition balance toggles afferent control of prefrontal cortical circuits in a pathway-specific manner

The medial prefrontal cortex (mPFC) controls behavior via connections with limbic excitatory afferents that engage various inhibitory motifs to shape mPFC circuit function. The dynorphin (Dyn) / kappa-opioid receptor (KOR) system is highly enriched in the mPFC, and its dysregulation is implicated in neuropsychiatric disorders. However, it is unclear how the Dyn / KOR system modulates excitatory and inhibitory circuits that are integral for mPFC information processing and behavioral control. Here, we provide a circuit-based framework wherein mPFC Dyn / KOR signaling regulates excitation-inhibition balance by toggling which afferents drive mPFC neurons. Dyn / KOR regulation of afferent inputs is pathway-specific. Dyn acting on presynaptic KORs inhibits glutamate release from afferent inputs to the mPFC, including the basolateral amygdala (BLA), paraventricular nucleus of the thalamus, and contralateral cortex. The majority of excitatory synapses to mPFC neurons, including those from the ventral hippocampus (VH), do not express presynaptic KOR, rendering them insensitive to Dyn / KOR modulation. Dyn / KOR signaling also suppresses afferent-driven recruitment of specific inhibitory sub-networks, providing a basis for Dyn to disinhibit mPFC circuits. Specifically, Dyn / KOR signaling preferentially suppresses SST interneuron- relative to PV interneuron-mediated inhibition. Selective KOR action on afferents or within mPFC microcircuits gates how distinct limbic inputs drive spiking in mPFC neurons. Presynaptic Dyn / KOR signaling decreases KOR-positive input-driven (e.g. BLA) spiking of mPFC neurons. In contrast, KOR-negative input recruitment of mPFC neurons is enhanced by Dyn / KOR signaling via suppression of mPFC inhibitory microcircuits. Thus, by acting on distinct circuit elements, Dyn / KOR signaling shifts KOR-positive and negative afferent control of mPFC circuits, providing mechanistic insights into the role of neuropeptides in shaping mPFC function. Together, these findings highlight the utility of targeting the mPFC Dyn / KOR system as a means to treat neuropsychiatric disorders characterized by dysregulation in mPFC integration of long-range afferents with local inhibitory microcircuits.

Kappa opioid receptor activation increases thermogenic energy expenditure which drives increased feeding

Opioid receptors, including the kappa opioid receptor (KOR), exert control over thermoregulation and feeding behavior. Notably, activation of KOR stimulates food intake, leading to postulation that KOR signaling plays a central role in managing energy intake. KOR has also been proposed as a target for treating obesity. Herein, we report studies examining how roles for KOR signaling in regulating thermogenesis, feeding, and energy balance may be interrelated using pharmacological interventions, genetic tools, quantitative thermal imaging, and metabolic profiling. Our findings demonstrate that activation of KOR in the central nervous system causes increased energy expenditure via brown adipose tissue activation. Importantly, pharmacologic, or genetic inhibition of brown adipose tissue thermogenesis prevented the elevated food intake triggered by KOR activation. Furthermore, our data reveal that KOR-mediated thermogenesis elevation is reversibly disrupted by chronic high-fat diet, implicating KOR signaling as a potential mediator in high-fat diet-induced weight gain.

Studies on diketopiperazine and dipeptide analogs as opioid receptor ligands

Using the structure of gliotoxin as a starting point, we have prepared two different chemotypes with selective affinity to the kappa opioid receptor (KOR). Using medicinal chemistry approaches and structure-activity relationship (SAR) studies, structural features required for the observed affinity were identified, and advanced molecules with favorable Multiparameter Optimization (MPO) and Ligand Lipophilicity (LLE) profiles were prepared. Using the Thermal Place Preference Test (TPPT), we have shown that compound2 blocks the antinociceptive effect of U50488, a known KOR agonist. Multiple reports suggest that modulation of KOR signaling is a promising therapeutic strategy in treating neuropathic pain (NP). As a proof-of-concept study, we tested compound 2 in a rat model of NP and recorded its ability to modulate sensory and emotional pain-related behaviors. Observed in vitro and in vivo results suggest that these ligands can be used to develop compounds with potential application as pain therapeutics.

Recapitulating phenotypes of alcohol dependence via overexpression of Oprk1 in the ventral tegmental area of non-dependent TH::Cre rats

The dynorphin (DYN)/kappa-opioid receptor (KOR) system is involved in dysphoria and negative emotional states. Dysregulation of KOR function promotes maladaptive behavioral regulation during withdrawal associated with alcohol dependence. Mesolimbic dopaminergic (DA) projections from the ventral tegmental area (VTA) innervate the extended amygdala circuitry and presynaptic KORs attenuate DA in these regions leading to an excessive alcohol consumption and negative affective-like behavior, whereas mesocortical KOR-regulated DA projections have been implicated in executive function and decision-making. Thus, the neuroadaptations occurring in DYN/KOR systems are important aspects to consider for the development of personalized therapeutic solutions. Herein, we study the contribution of the VTA DA neuron Oprk1 (KOR gene) in excessive alcohol consumption, negative emotional state, and executive function. To do so, Oprk1 mRNA expression and KOR function were characterized to confirm alcohol dependence-induced dysregulation in the VTA. Then, a transgenic Cre-Lox rat model (male and female TH::Cre rats) was used to allow for conditional and inducible overexpression of Oprk1 in VTA DA neurons. The effect of this overexpression was evaluated on operant alcohol self-administration, negative emotional states, and executive function. We found that VTA Oprk1 overexpression recapitulates some phenotypes of alcohol dependence including escalated alcohol self-administration and depressive-like behavior. However, working memory performance was not impacted following VTA Oprk1 overexpression in TH::Cre rats. This supports the hypothesis that dysregulated KOR signaling within the mesolimbic DA system is an important contributor to symptoms of alcohol dependence and shows that understanding Oprk1-mediated contributions to alcohol use disorder (AUD) should be an important future goal.

Dichotomous regulation of striatal plasticity by dynorphin.

Modulation of corticostriatal plasticity alters the information flow throughout basal ganglia circuits and represents a fundamental mechanism for motor learning, action selection, and reward. Synaptic plasticity in the striatal direct- and indirect-pathway spiny projection neurons (dSPNs and iSPNs) is regulated by two distinct networks of GPCR signaling cascades. While it is well-known that dopamine D2 and adenosine A2a receptors bi-directionally regulate iSPN plasticity, it remains unclear how D1 signaling modulation of synaptic plasticity is counteracted by dSPN-specific Gi signaling. Here, we show that striatal dynorphin selectively suppresses long-term potentiation (LTP) through Kappa Opioid Receptor (KOR) signaling in dSPNs. Both KOR antagonism and conditional deletion of dynorphin in dSPNs enhance LTP counterbalancing with different levels of D1 receptor activation. Behaviorally, mice lacking dynorphin in D1 neurons show comparable motor behavior and reward-based learning, but enhanced flexibility during reversal learning. These findings support a model in which D1R and KOR signaling bi-directionally modulate synaptic plasticity and behavior in the direct pathway.

Conditional Deletion of KOR (Oprk1) in Kisspeptin Cells Does Not Alter LH Pulses, Puberty, or Fertility in Mice.

Classic pharmacological studies suggested that endogenous dynorphin-KOR signaling is important for reproductive neuroendocrine regulation. With the seminal discovery of an interconnected network of hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons), the KNDy hypothesis was developed to explain how gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) pulses are generated. Key to this hypothesis is dynorphin released from KNDy neurons acting in a paracrine manner on other KNDy neurons via kappa opioid receptor (KOR) signaling to terminate neural "pulse" events. While in vitro evidence supports this aspect of the KNDy hypothesis, a direct in vivo test of the necessity of KOR signaling in kisspeptin neurons for proper LH secretion has been lacking. We therefore conditionally knocked out KOR selectively from kisspeptin neurons of male and female mice and tested numerous reproductive measures, including in vivo LH pulse secretion. Surprisingly, despite validating successful knockout of KOR in kisspeptin neurons, we found no significant effect of kisspeptin cell-specific deletion of KOR on any measure of puberty, LH pulse parameters, LH surges, follicle-stimulating hormone (FSH) levels, estrous cycles, or fertility. These outcomes suggest that the KNDy hypothesis, while sufficient normally, may not be the only neural mechanism for sculpting GnRH and LH pulses, supported by recent findings in humans and mice. Thus, besides normally acting via KOR in KNDy neurons, endogenous dynorphin and other opioids may, under some conditions, regulate LH and FSH secretion via KOR in non-kisspeptin cells or perhaps via non-KOR pathways. The current models for GnRH and LH pulse generation should be expanded to consider such alternate mechanisms.

14-3-3γ mediates the long-term inhibition of peripheral kappa opioid receptor antinociceptive signaling by norbinaltorphimine

Long-term inhibition of kappa opioid receptor (KOR) signaling in peripheral pain-sensing neurons is a potential obstacle for development of peripherally-restricted KOR agonists that produce analgesia. Such a long-term inhibitory mechanism is invoked from activation of c-Jun N-terminal kinase (JNK) that follows a single injection of the KOR antagonist norbinaltorphimine (norBNI). This effect requires protein synthesis of an unknown mediator in peripheral pain-sensing neurons. Using 2D difference gel electrophoresis with tandem mass spectrometry, we have identified that the scaffolding protein 14-3-3γ is upregulated in peripheral sensory neurons following activation of JNK with norBNI. Knockdown of 14-3-3γ by siRNA eliminates the long-term reduction in KOR-mediated cAMP signaling by norBNI in peripheral sensory neurons in culture. Similarly, knockdown of 14-3-3γ in the rat hind paw abolished the norBNI-mediated long-term reduction in peripheral KOR-mediated antinociception. Further, overexpression of 14-3-3γ in KOR expressing CHO cells prevented KOR-mediated inhibition of cAMP signaling. These long-term effects are selective for KOR as heterologous regulation of other receptor systems was not observed. These data suggest that 14-3-3γ is both necessary and sufficient for the long-term inhibition of KOR by norBNI in peripheral sensory neurons.

Daily Δ9-Tetrahydrocannabinol and Withdrawal Increase Dopamine D1-D2 Receptor Heteromer to Mediate Anhedonia- and Anxiogenic-like Behavior Through a Dynorphin and Kappa Opioid Receptor Mechanism

BackgroundFrequent cannabis use is associated with a higher risk of developing cannabis use disorder and other adverse consequences. However, rodent models studying the underlying mechanisms of the reinforcing and withdrawal effects of the primary constituent of cannabis, Δ9-tetrahydrocannabinol (THC), have been limited. MethodsThis study investigated the effects of daily THC (1 mg/kg, intraperitoneal, 9 days) and spontaneous withdrawal (7 days) on hedonic and aversion-like behaviors in male rats. In parallel, underlying neuroadaptive changes in dopaminergic, opioidergic, and cannabinoid signaling in the nucleus accumbens were evaluated, along with a candidate peptide designed to reverse altered signaling. ResultsChronic THC administration induced anhedonic- and anxiogenic-like behaviors not attributable to altered locomotor activity. These effects persisted after drug cessation. In the nucleus accumbens, THC treatment and withdrawal catalyzed increased cannabinoid CB1 receptor activity without modifying receptor expression. Dopamine D1-D2 receptor heteromer expression rose steeply with THC, accompanied by increased calcium-linked signaling, activation of BDNF/TrkB (brain-derived neurotrophic factor/tropomyosin receptor kinase B) pathway, dynorphin expression, and kappa opioid receptor signaling. Disruption of the D1-D2 heteromer by an interfering peptide during withdrawal reversed the anxiogenic-like and anhedonic-like behaviors as well as the neurochemical changes. ConclusionsChronic THC increases nucleus accumbens dopamine D1-D2 receptor heteromer expression and function, which results in increased dynorphin expression and kappa opioid receptor activation. These changes plausibly reduce dopamine release to trigger anxiogenic- and anhedonic-like behaviors after daily THC administration that persist for at least 7 days after drug cessation. These findings conceivably provide a therapeutic strategy to alleviate negative symptoms associated with cannabis use and withdrawal.

Self-Administration of Entactogen Psychostimulants Dysregulates Gamma-Aminobutyric Acid (GABA) and Kappa Opioid Receptor Signaling in the Central Nucleus of the Amygdala of Female Wistar Rats.

Male rats escalate intravenous self-administration of entactogen psychostimulants, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxymethamphetamine (MDMA) under extended access conditions, as with typical psychostimulants. Here, we investigated whether female rats escalate self-administration of methylone, 3,4-methylenedioxypentedrone (pentylone), and MDMA and then studied consequences of MDMA and pentylone self-administration on GABAA receptor and kappa opioid receptor (KOR) signaling in the central nucleus of the amygdala (CeA), a brain area critically dysregulated by extended access self-administration of alcohol or cocaine. Adult female Wistar rats were trained to self-administer methylone, pentylone, MDMA (0.5 mg/kg/infusion), or saline-vehicle using a fixed-ratio 1 response contingency in 6-h sessions (long-access: LgA) followed by progressive ratio (PR) dose-response testing. The effects of pentylone-LgA, MDMA-LgA and saline on basal GABAergic transmission (miniature post-synaptic inhibitory currents, mIPSCs) and the modulatory role of KOR at CeA GABAergic synapses were determined in acute brain slices using whole-cell patch-clamp. Methylone-LgA and pentylone-LgA rats similarly escalated their drug intake (both obtained more infusions compared to MDMA-LgA rats), however, pentylone-LgA rats reached higher breakpoints in PR tests. At the cellular level, baseline CeA GABA transmission was markedly elevated in pentylone-LgA and MDMA-LgA rats compared to saline-vehicle. Specifically, pentylone-LgA was associated with increased CeA mIPSC frequency (GABA release) and amplitude (post-synaptic GABAA receptor function), while mIPSC amplitudes (but not frequency) was larger in MDMA-LgA rats compared to saline rats. In addition, pentylone-LgA and MDMA-LgA profoundly disrupted CeA KOR signaling such as both KOR agonism (1 mM U50488) and KOR antagonism (200 nM nor-binaltorphimine) decreased mIPSC frequency suggesting recruitment of non-canonical KOR signaling pathways. This study confirms escalated self-administration of entactogen psychostimulants under LgA conditions in female rats which is accompanied by increased CeA GABAergic inhibition and altered KOR signaling. Collectively, our study suggests that CeA GABA and KOR mechanisms play a critical role in entactogen self-administration like those observed with escalation of alcohol or cocaine self-administration.

Data-driven analysis of kappa opioid receptor binding in major depressive disorder measured by positron emission tomography

Preclinical studies have implicated kappa opioid receptors (KORs) in stress responses and depression-related behaviors, but evidence from human studies is limited. Here we present results of a secondary analysis of data acquired using positron emission tomography (PET) with the KOR radiotracer [11C]GR103545 in 10 unmedicated, currently depressed individuals with major depressive disorder (MDD; 32.6 ± 6.5 years, 5 women) and 13 healthy volunteers (34.8 ± 10 years, 6 women). Independent component analysis was performed to identify spatial patterns of coherent variance in KOR binding (tracer volume of distribution, VT) across all subjects. Expression of each component was compared between groups and relationships to symptoms were explored using the 17-item Hamilton Depression Rating Scale (HDRS). Three components of variation in KOR availability across ROIs were identified, spatially characterized by [11C]GR103545 VT in (1) bilateral frontal lobe; (2) occipital and parietal cortices, right hippocampus, and putamen; and (3) right anterior cingulate, right superior frontal gyrus and insula, coupled to negative loading in left middle cingulate. In MDD patients, component 3 was negatively associated with symptom severity on the HDRS (r = −0.85, p = 0.0021). There were no group-wise differences in expression of any component between patients and controls. These preliminary findings suggest that KOR signaling in cortical regions relevant to depression, particularly right anterior cingulate, could reflect MDD pathophysiology.

Retraction: Alcohol drinking alters stress response to predator odor via BNST kappa opioid receptor signaling in male mice.

Maladaptive responses to stress are a hallmark of alcohol use disorder, but the mechanisms that underlie this are not well characterized. Here, we show that kappa opioid receptor signaling in the bed nucleus of the stria terminalis (BNST) is a critical molecular substrate underlying abnormal stress responses to predator odor following heavy alcohol drinking. Exposure to predator odor during protracted withdrawal from intermittent alcohol drinking resulted in enhanced prefrontal cortex (PFC)-driven excitation of prodynorphin-containing neurons in the BNST. Furthermore, deletion of prodynorphin in the BNST and chemogenetic inhibition of the PFC-BNST pathway restored abnormal responses to predator odor in alcohol-exposed mice. These findings suggest that increased corticolimbic drive may promote abnormal stress behavioral responses to predator odor during protracted withdrawal. Various nodes of this PFC-BNST dynorphin-related circuit may serve as potential targets for potential therapeutic mediation as well as biomarkers of negative responses to stress following heavy alcohol drinking.

Input‐specific regulation of discrete populations of Lateral Habenula neurons by Kappa opioid receptors

The lateral habenula (LHb) is an epithalamic brain region associated with value-based decision making and stress evasion through its modulation of reward circuitry. Increased activity of the LHb is associated with drug addiction and stress-related mood disorders. Dynorphin (DYN)/Kappa opioid receptor (KOR) signaling is an endogenous mediator of stress response in reward circuitry. Previously, we have shown a novel functional role of DYN/KOR signaling within LHb and its dysregulation by a severe early life stress (maternal deprivation, MD). Specifically, KOR stimulation has distinct effects on LHb neuronal excitability in distinct neuronal populations identified by the presence of hyperpolarizing current (Ih) that were absent following MD. Namely, KOR stimulation through the KOR agonist, U50,488, significantly decreases excitability of Ih negative (Ih-) LHb neurons while simultaneously increasing excitability of Ih positive (Ih+) LHb neurons. Additionally, KOR-induced alteration in LHb excitability is dependent on both presynaptic glutamatergic and GABAergic signaling likely through presynaptic KORs on inputs to LHb neurons. Given that LHb inputs from the entopeduncular nucleus (EP) and lateral hypothalamus (LH) have been implicated in stress-induced mood disorders through modulation of LHb excitability, we hypothesize that the presynaptic effects of KOR stimulation on LHb neuronal activity may be driven by one or both of these inputs. Using selective in vitro optical stimulation of either EP or LH inputs to LHb neurons in brain slices from rats, our pilot study suggests that KOR activation of EP and LH inputs may differentially affect optically evoked action potential generation across Ih- and Ih+ LHb subpopulations. Moreover, our data from MD rats suggest that there may be an imbalance of excitation to inhibition at EP synapses onto LHb neurons. Given that MD increases immunolabeled DYN (the endogenous KOR agonist) labeling in neuronal fibers in LHb while significantly decreasing mRNA levels of KORs in LHb, we also investigated MD-induced modulation of a KOR-specific downstream signaling (p38 MAPK). Our molecular data indicate that decreased KOR expression may be a homeostatic response to higher basal activation of KORs by MD-induced increases in DYN and p38 expression in the LHb. Ih- and Ih+ neurons molecular identification is further investigated following electrophysiological recordings by combining ionophoresis labeling during electrophysiology experiments and post-hoc immunohistochemistry to verify the glutamatergic (VGLUT+) and GABAergic (somatostatin, SST+ or parvalbumin, PV) nature of LHb neurons. Collectively, our current studies provide evidence for input- and cell-specific regulation of LHb neurons by KOR signaling that could be modulated by MD and highlights a future hotspot for pharmacological intervention of KOR-mediated mood disorders through dysregulation of specific LHb neural circuits.

Kappa‐Opioid Receptors Modulate Innate and Learned Responses to Positive and Negative Reinforcers

There are few effective and approved treatments for Substance Use Disorders (SUDs) but clinical and preclinical research has shown therapeutic potential for Kappa-opioid receptor (KOR) antagonists. The leading theory of the KOR system's role in SUD posits that chronic substance use augments expression and release of the aversive KOR ligand, dynorphin, which produces a dysphoric state. In this model, upregulation of dynorphin/KOR activity drives continued substance use through a negative reinforcement mechanism whereby substances are consumed to alleviate the negative affective state. While systemic KOR antagonists have been shown to reduce drug consumption in several preclinical models, direct effects on positive and negative reinforcement schedules have not been systematically investigated. In the present study we investigate the effects of systemic antagonism of KORs by nor-Binaltorphimine (NorBNI) on acquisition and maintenance of operant responding reinforced by presentation of sucrose (positive reinforcement) or removal of footshock (negative reinforcement). Based on the prevailing theory that KOR activation is aversive and drives negative reinforcement in addiction, we hypothesized that antagonism of the system would result in decreased responding under the negative reinforcement contingency and have no effect on positive reinforcement. Male C57BL/6J mice received 10 mg/kg intraperitoneal injections of the long-acting KOR antagonist NorBNI or 10 ml/kg saline 24 hours prior to their first session. Animals acquired a discriminated operant response for a positive or negative reinforcer (separate cohorts) whereby a cue light above the active nose-poke (presented on a variable time schedule) served as a discriminative stimulus indicating when a nose-poke would result in the receipt of sucrose or the removal of a series of shocks. Contrary to our hypothesis, mice that received NorBNI showed increased performance during acquisition of both positive and negative reinforcement contingencies compared to saline control mice. These results suggest a novel role of the KOR system in learning that is independent of the valence of the stimulus. We also found differential innate responses to sucrose and footshock prior to operant conditioning suggesting a key role of the system in encoding information about stimuli both aversive and non-aversive. These results call for a reevaluation of the role of KOR signaling in behavioral control and how this system influences drug taking behaviors.

Compartment-specific opioid receptor signaling is selectively modulated by different dynorphin peptides.

Many signal transduction systems have an apparent redundancy built into them, where multiple physiological agonists activate the same receptors. Whether this is true redundancy, or whether this provides an as-yet unrecognized specificity in downstream signaling, is not well understood. We address this question using the kappa opioid receptor (KOR), a physiologically relevant G protein-coupled receptor (GPCR) that is activated by multiple members of the Dynorphin family of opioid peptides. We show that two related peptides, Dynorphin A and Dynorphin B, bind and activate KOR to similar extents in mammalian neuroendocrine cells and rat striatal neurons, but localize KOR to distinct intracellular compartments and drive different post-endocytic fates of the receptor. Strikingly, localization of KOR to the degradative pathway by Dynorphin A induces sustained KOR signaling from these compartments. Our results suggest that seemingly redundant endogenous peptides can fine-tune signaling by regulating the spatiotemporal profile of KOR signaling.

Kappa Opioid Receptor Ligands and Pharmacology: Diphenethylamines, a Class of Structurally Distinct, Selective Kappa Opioid Ligands.

The kappa opioid receptor (KOR), a G protein-coupled receptor, and its endogenous ligands, the dynorphins, are prominent members of the opioid neuromodulatory system. The endogenous kappa opioid system is expressed in the central and peripheral nervous systems, and has a key role in modulating pain in central and peripheral neuronal circuits and a wide array of physiological functions and neuropsychiatric behaviors (e.g., stress, reward, emotion, motivation, cognition, epileptic seizures, itch, and diuresis). We review the latest advances in pharmacology of the KOR, chemical developments on KOR ligands with advances and challenges, and therapeutic and potential applications of KOR ligands. Diverse discovery strategies of KOR ligands targeting natural, naturally derived, and synthetic compounds with different scaffolds, as small molecules or peptides, with short or long-acting pharmacokinetics, and central or peripheral site of action, are discussed. These research efforts led to ligands with distinct pharmacological properties, as agonists, partial agonists, biased agonists, and antagonists. Differential modulation of KOR signaling represents a promising strategy for developing pharmacotherapies for several human diseases, either by activating (treatment of pain, pruritus, and epilepsy) or blocking (treatment of depression, anxiety, and addiction) the receptor. We focus on the recent chemical and pharmacological advances on diphenethylamines, a new class of structurally distinct, selective KOR ligands. Design strategies and investigations to define structure-activity relationships together with in vivo pharmacology of diphenethylamines as agonists, biased agonists, and antagonists and their potential use as therapeutics are discussed.

Biosensors Monitor Ligand-Selective Effects at Kappa Opioid Receptors.

The kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to β-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor-biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.

Early life stress dysregulates kappa opioid receptor signaling within the lateral habenula

The lateral habenula (LHb) is an epithalamic brain region associated with value-based decision making and stress evasion through its modulation of dopamine (DA)-mediated reward circuitry. Specifically, increased activity of the LHb is associated with drug addiction, schizophrenia and stress-related disorders such as depression, anxiety and posttraumatic stress disorder. Dynorphin (Dyn)/Kappa opioid receptor (KOR) signaling is a mediator of stress response in reward circuitry. Previously, we have shown that maternal deprivation (MD), a severe early life stress, increases LHb spontaneous neuronal activity and intrinsic excitability while blunting the response of LHb neurons to extrahypothalamic corticotropin-releasing factor (CRF) signaling, another stress mediator. CRF pathways also interact with Dyn/KOR signaling. Surprisingly, there has been little study of direct KOR regulation of the LHb despite its distinct role in stress, reward and aversion processing. To test the functional role of Dyn/KOR signaling in the LHb, we utilized ex-vivo electrophysiology combined with pharmacological tools in rat LHb slices. We show that activation of KORs by a KOR agonist (U50,488) exerted differential effects on the excitability of two distinct sub-populations of LHb neurons that differed in their expression of hyperpolarization-activated cation currents (HCN, Ih). Specifically, KOR stimulation increased neuronal excitability in LHb neurons with large Ih currents (Ih+) while decreasing neuronal excitability in small/negative Ih (Ih-) neurons. We found that an intact fast-synaptic transmission was required for the effects of U50,488 on the excitability of both Ih- and Ih+ LHb neuronal subpopulations. While AMPAR-, GABAAR-, or NMDAR-mediated synaptic transmission alone was sufficient to mediate the effects of U50,488 on excitability of Ih- neurons, either GABAAR- or NMDAR-mediated synaptic transmission could mediate these effects in Ih+ neurons. Consistently, KOR activation also altered both glutamatergic and GABAergic synaptic transmission where stimulation of presynaptic KORs uniformly suppressed glutamate release onto LHb neurons while primarily decreased or in some cases increased GABA release. We also found that MD significantly increased immunolabeled Dyn (the endogenous KOR agonist) labeling in neuronal fibers in LHb while significantly decreasing mRNA levels of KORs in LHb tissues compared to those from non-maternally deprived (non-MD) control rats. Moreover, the U50,488-mediated increase in LHb neuronal firing observed in non-MD rats was absent following MD. Altogether, this is the first demonstration of the existence of functional Dyn/KOR signaling in the LHb that can be modulated in response to severe early life stressors such as MD.

Unique Pharmacological Properties of the Kappa Opioid Receptor Signaling Through Gαz as Shown with Bioluminescence Resonance Energy Tranfer.

Opioid receptors (ORs) convert extracellular messages to signaling events by coupling to the heterotrimeric G proteins, Gα•βγ Classic pharmacological methods, such as [35S]GTPγS binding and inhibition of cyclic AMP production, allow for general opioid characterization, but they are subject to the varying endogenous Gα proteins in a given cell type. Bioluminescence resonance energy transfer (BRET) technology offers new insight by allowing the direct observation of Gα subunit-specific effects on opioid pharmacology. Using a Venus-tagged Gβγ and nanoluciferase-tagged truncated G protein receptor kinase 3, an increase in BRET signal correlated with OR activation mediated by a specific Gα protein. The magnitude of the BRET signal was normalized to the maximum response obtained with 10 µM 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50,488) for the kappa OR (KOR). Opioids reached equilibrium with the KOR, and concentration-response curves were generated. Although the full agonists U50,488, salvinorin A, nalfurafine, and dynorphin peptides were equally efficacious regardless of the Gα subunit present, the concentration-response curves were leftward shifted when the KOR was signaling through Gαz compared with other Gαi/o subunits. In contrast, the Gα subunit distinctly affected both the efficacy and potency of partial kappa agonists, such as the benzomorphans, and the classic mu opioid antagonists, naloxone, naltrexone, and nalmefene. For example, (-)pentazocine had EC50 values of 7.3 and 110 nM and maximal stimulation values of 79% and 35% when the KOR signaled through Gαz and Gαi1, respectively. Together, these observations suggest KOR pharmacology varies based on the specific Gα subunit coupled to the KOR. SIGNIFICANCE STATEMENT: Opioid receptors couple to various heterotrimeric Gαβγ proteins to convert extracellular cues to precise intracellular events. This paper focuses on how the various inhibitory Gα subunits influence the pharmacology of full and partial agonists at the kappa opioid receptor. Using a bioluminescent assay, the efficacy and potency of kappa opioids was determined. Opioid signaling was more potent through Gαz compared with other Gα proteins. These observations suggest that Gαz may impact opioid pharmacology and cellular physiology more than previously thought.

Sex differences in the modulation of mouse nest building behavior by kappa opioid receptor signaling

Emerging evidence suggests that females are less sensitive than males to the effects of kappa opioid receptor (KOR) ligands across multiple behavioral measures. The effects of the KOR agonist U50,488 and the KOR antagonist aticaprant were assessed on nest building behavior, an ethologically relevant indicator of overall well-being and affect, in adult male and female C57BL/6J mice. Females required a higher dose of U50,488 to suppress nesting, and a higher dose of aticaprant to restore U50,488-induced impairment of nesting. Females also required a higher dose of aticaprant to decrease immobility scores in the forced swim test. Pretreatment with the estrogen receptor modulator tamoxifen, at a dose which blocked estrogen receptors, augmented the effect of U50,488 on nesting in female mice, suggesting that estrogen receptors play a key role in attenuating the effects of KOR ligands in female mice. Together, these results suggest that females are less sensitive to KOR mediation, requiring a higher dose to achieve comparable results to males. This behavioral sensitivity, as measured by nesting, may be mediated by estrogen receptors. Together these studies highlight the importance of comparing sex differences in response to KOR regulation on behaviors related to affective states.