Sex Differences in Brain Region-Specific Activation of c-Fos following Kappa Opioid Receptor Stimulation or Acute Stress in Mice.

Kappa opioid receptors (KOPr) are involved in the response to stress. KOPr are also targets for the treatment of stress-related psychiatric disorders including depression, anxiety, and addiction although effects of KOPr are often sex-dependent. Here we investigated c-Fos expression in a range of brain regions in male and female mice following an acute stressor, and a single injection of KOPr agonist. Using adult C57BL/6 c-Fos-GFP transgenic mice and quantitative fluorescence microscopy, we identified brain regions activated in response to a challenge with the KOPr agonist U50,488 (20 mg/kg) or an acute stress (15 min forced swim stress, FSS). In male mice, U50,488 increased expression of c-Fos in the prelimbic area of the prefrontal cortex (PFCx), nucleus accumbens (NAcc), and basolateral nuclei of the amygdala (BLA). In contrast, in female mice U50,488 only activated the BLA but not the PFCx or the NAcc. FSS increased activation of PFCx, NAcc, and BLA in males while there was no activation of the PFCx in female mice. In both sexes, the KOPr antagonist norBNI significantly blocked U50,488-induced, but not stress-induced activation of brain regions. In separate experiments, activated cells were confirmed as non-GABAergic neurons in the PFCx and NAcc. Together these data demonstrate sex differences in activation of brain regions that are key components of the 'reward' circuitry. These differential responses may contribute to sex differences in stress-related psychiatric disorders and in the treatment of depression, anxiety, and addiction.

Kappa opioid receptor agonist U50,488 inhibits dopamine more in caudal than rostral nucleus accumbens core.

The nucleus accumbens (NAc) core is involved in regulating stress and shaping reward seeking behaviours. Multiple neuromodulators, including dynorphin/kappa opioid receptor (KOR) and dopamine systems, converge in this area to influence behavioural outcomes. KOR activation acutely inhibits dopamine release and chronically depresses overall dopamine transmission. Recently, studies in the NAc shell have revealed that the impact of KOR activation on behaviour is regionally specific, and these rostro-caudal differences are likely driven by greater control of KORs over dopamine inhibition in the caudal compared with rostral subregion. Given the importance of NAc core, particularly the interaction between KORs and dopamine in regulating reward seeking behaviours, we examined the impact of KOR activation on dopamine release and uptake along the rostro-caudal axis in the NAc core of male and female mice. Using ex vivo fast scan cyclic voltammetry, we observed that KOR mediated inhibition of dopamine release was significantly greater in caudal compared with rostral NAc core with no significant sex differences observed. These data suggest that KORs regulate dopamine release differentially along the rostro-caudal axis, providing a new axis on which to examine the process by which the KOR/dopamine system controls reward encoding.

Kappa Opioid Receptors Reduce Serotonin Uptake and Escitalopram Efficacy in the Mouse Substantia Nigra Pars Reticulata.

The serotonin and kappa opioid receptor (KOR) systems are strongly implicated in disorders of negative affect, such as anxiety and depression. KORs expressed on axon terminals inhibit the release of neurotransmitters, including serotonin. The substantia nigra pars reticulata (SNr) is involved in regulating affective behaviors. It receives the densest serotonergic innervation in the brain and has high KOR expression; however, the influence of KORs on serotonin transmission in this region is yet to be explored. Here, we used ex vivo fast-scan cyclic voltammetry (FSCV) to investigate the effects of a KOR agonist, U50, 488 (U50), and a selective serotonin reuptake inhibitor, escitalopram, on serotonin release and reuptake in the SNr. U50 alone reduced serotonin release and uptake, and escitalopram alone augmented serotonin release and slowed reuptake, while pretreatment with U50 blunted both the release and uptake effects of escitalopram. Here, we show that the KOR influences serotonin signaling in the SNr in multiple ways and short-term activation of the KOR alters serotonin responses to escitalopram. These interactions between KORs and serotonin may contribute to the complexity in the responses to treatments for disorders of negative affect. Ultimately, the KOR system may prove to be a promising pharmacological target, alongside traditional antidepressant treatments.

Kappa Opioid Receptor Expression and Function in Cells of the Immune System.

The kappa opioid receptor (KOR) is expressed on a number of hematopoietic cell populations, based on both protein binding analysis and the detection of kappa opioid receptor gene (Oprk1) transcripts. There are prominent Oprk1 splice variants that are expressed in the mouse and human brain cells and leukocytes. The activation of KOR results in reduced antibody production, an inhibition of phagocytic cell activity, an inhibition of T cell development, alterations in the production of various pro-inflammatory cytokines, chemokines, and the receptors for these mediators. Finally, the activation of KOR also leads to the regulation of receptor functional activity of chemokine receptors through the process of heterologous desensitization. The functional activity of KOR is important for the regulation of inflammatory responses and may provide opportunities for the development of therapeutics for the treatment of inflammatory disease states.

Novel selective κ agonists SLL-039 and SLL-1206 produce potent antinociception with fewer sedation and aversion.

SLL-039 (N-cyclopropylmethyl-7α-4'-(N'-benzoyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) and SLL-1206 (N-cyclopropylmethyl-7α-3'-(p-methoxybenzyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) are two 4,5-epoxymorphinan-based high selective κ receptor agonists that we recently discovered. In the present study we characterized their pharmacological properties in comparison with arylacetamide-based typical κ agonist U50,488H. We showed that both SLL-039 and SLL-1206 produced potent and long-lasting antinociceptive actions in three different rodent models of pain via activation of κ opioid receptor. In hot-plate assay, the antinociceptive potency of SLL-039 and SLL-1206 increased about 11-and 17.3-fold compared to U50,488H and morphine, respectively, with ED50 values of 0.4 mg/kg. Following repeated administration, SLL-1206, SLL-039, and U50,488H all developed analgesic tolerance tested in hot-plate assay. U50,488H and SLL-039 produced antipruritic effects in a dose-dependent manner, whereas SLL-1206 displayed some antipruritic effects only at very low doses. In addition, SLL-1206 was capable of decreasing morphine-induced physical dependence. More importantly, SLL-039 and SLL-1206 at effective analgesic doses did not cause sedation and conditioned place aversion (CPA), whereas U50,488H did. In comparison with SLL-039, SLL-1206 caused similar antinociceptive responses, but fewer sedation and CPA. In conclusion, our results suggest that SLL-039 and SLL-1206 have potential to be developed as novel analgesic agents, and 4,5-expoxymorphinan scaffold is an attractive structure for the development of selective κ agonists with fewer side effects.

The kappa-opioid receptor agonist, triazole 1.1, reduces oxycodone self-administration and enhances oxycodone-induced thermal antinociception in male rats.

RATIONALE: Triazole 1.1 is a novel kappa-opioid receptor (KOR) agonist reported to produce antinociception without KOR-typical adverse effects. When combined with the mu-opioid receptor (MOR) agonist, oxycodone, triazole 1.1 blocks oxycodone-induced pruritis without producing sedation-like effects in nonhuman primates. However, it is unknown if triazole 1.1 can reduce the abuse-related effects or enhance the antinociceptive effects of oxycodone similarly to other KOR agonists. OBJECTIVES: The aim of the present study was to quantitatively compare the behavioral effects of triazole 1.1 to the KOR agonists, U50,488h and nalfurafine, on oxycodone self-administration and oxycodone-induced thermal antinociception when administered as mixtures with oxycodone. METHODS: In the self-administration study, male Sprague-Dawley (SD) rats (n = 6) self-administered intravenous (i.v.) oxycodone alone (0.056 mg/kg/inj) or combined with U50,488 h (0.032-0.32 mg/kg/inj), nalfurafine (0.00032-0.0032 mg/kg/inj), or triazole 1.1 (0.32-1.8 mg/kg/inj) under a progressive-ratio schedule of reinforcement. In a hot plate assay, male SD rats (n = 6) received i.v. injections of oxycodone (1.0-5.6 mg/kg), U50,488h (1.0-18.0 mg/kg), nalfurafine (0.01-1.0 mg/kg), or triazole 1.1 (3.2-32.0 mg/kg) alone or in combinations of fixed proportion with oxycodone based on the relative potencies of the single drugs. Each study concluded with administration of the KOR antagonist nor-BNI and some degree of retesting of the previous conditions to verify that the behavioral effects were mediated by KOR activation. RESULTS: All KOR agonists reduced oxycodone self-administration in a dose-dependent manner. Moreover, all single drugs and drug combinations produced dose-dependent, fully efficacious thermal antinociception. All KOR agonist:oxycodone combinations produced either additive or super-additive thermal antinociception. Finally, each KOR agonist was blocked in effect by nor-BNI in both behavioral measures. CONCLUSION: This study demonstrates that triazole 1.1 reduces oxycodone's reinforcing effects and enhances oxycodone-induced antinociception to degrees that are comparable to typical KOR agonists. Given triazole 1.1's mild adverse-effect profile, developing MOR-KOR agonist combinations from the triazole 1.1 series may render new pain therapeutics with reduced abuse liability.

Kappa opioid receptor modulation of endometriosis pain in mice.

The kappa opioid receptor is a constituent of the endogenous opioid analgesia system widely expressed in somatosensory nervous pathways and also in endometrial tissues. This work investigates the possible involvement of kappa opioid receptor on the nociceptive, behavioral and histopathological manifestations of endometriosis in a murine model. Female mice receiving endometrial implants develop a persistent mechanical hypersensitivity in the pelvic area that is stronger during the estrus phase of the estrous cycle. The kappa opioid receptor agonist U50,488H produces a dose-dependent relief of this mechanical hypersensitivity, regardless of the cycle phase. Repeated exposure to a low dose of U50,488H (1 mg/kg/day s.c. for one month) provides sustained relief of mechanical hypersensitivity, without tolerance development or sedative side effects. Interestingly, this treatment also inhibits a decreased rearing behavior associated with spontaneous pain or discomfort in endometriosis mice. This KOR-mediated pain relief does not prevent the anxiety-like behavior or the cognitive impairment exhibited by endometriosis mice, and the growth of endometriotic cysts is also unaltered. These data provide evidence of strong pain-relieving properties of kappa opioid receptor stimulation in female mice with endometriosis pain. The persistence of affective and cognitive manifestations suggests that these comorbidities are independent of pelvic pain and simultaneous treatment of these comorbidities may be necessary for successful management of endometriosis.

Evaluation of Antinociceptive Effects of Chitosan-Coated Liposomes Entrapping the Selective Kappa Opioid Receptor Agonist U50,488 in Mice.

Background and Objectives: The selective kappa opioid receptor agonist U50,488 was reported to have analgesic, cough suppressant, diuretic and other beneficial properties. The aim of our study was to analyze the effects of some original chitosan-coated liposomes entrapping U50,488 in somatic and visceral nociceptive sensitivity in mice. Materials and Methods: The influence on the somatic pain was assessed using a tail flick test by counting the tail reactivity to thermal noxious stimulation. The nociceptive visceral estimation was performed using the writhing test in order to evaluate the behavioral manifestations occurring as a reaction to the chemical noxious peritoneal irritation with 0.6% acetic acid (10 mL/kbw). The animals were treated orally, at the same time, with a single dose of: distilled water 0.1 mL/10 gbw; 50 mg/kbw U50,488; 50 mg/kbw U50,488 entrapped in chitosan-coated liposomes, according to the group they were randomly assigned. Results: The use of chitosan-coated liposomesas carriers for U50,488 induced antinociceptive effects that began to manifest after 2 h, andwere prolonged but with a lower intensity than those caused by the free selective kappa opioid in both tests. Conclusion: In this experimental model, the oral administration of nanovesicles containing the selective kappa opioid agonist U50,488 determined a prolonged analgesic outcome in the tail flick test, as well as in the writhing test.

Nalfurafine reduces neuroinflammation and drives remyelination in models of CNS demyelinating disease.

OBJECTIVES: Multiple sclerosis (MS) is a neurodegenerative disease characterised by inflammation and damage to the myelin sheath, resulting in physical and cognitive disability. There is currently no cure for MS, and finding effective treatments to prevent disease progression has been challenging. Recent evidence suggests that activating kappa opioid receptors (KOR) has a beneficial effect on the progression of MS. Although many KOR agonists like U50,488 are not suitable for clinical use because of a poor side-effect profile, nalfurafine is a potent, clinically used KOR agonist with a favorable side-effect profile. METHODS: Using the experimental autoimmune encephalomyelitis (EAE) model, the effect of therapeutically administered nalfurafine or U50,488 on remyelination, CNS infiltration and peripheral immune responses were compared. Additionally, the cuprizone model was used to compare the effects on non-immune demyelination. RESULTS: Nalfurafine enabled recovery and remyelination during EAE. Additionally, it was more effective than U50,488 and promoted disease reduction when administered after chronic demyelination. Blocking KOR with the antagonist, nor-BNI, impaired full recovery by nalfurafine, indicating that nalfurafine mediates recovery from EAE in a KOR-dependent fashion. Furthermore, nalfurafine treatment reduced CNS infiltration (especially CD4+ and CD8+ T cells) and promoted a more immunoregulatory environment by decreasing Th17 responses. Finally, nalfurafine was able to promote remyelination in the cuprizone demyelination model, supporting the direct effect on remyelination in the absence of peripheral immune cell invasion. CONCLUSIONS: Overall, our findings support the potential of nalfurafine to promote recovery and remyelination and highlight its promise for clinical use in MS.

Kappa-opioid receptor-dependent changes in dopamine and anxiety-like or approach-avoidance behavior occur differentially across the nucleus accumbens shell rostro-caudal axis.

Neural circuit engagement within the nucleus accumbens (NAc) shell is implicated in the regulation of both negative and positive affect. Classically, the dynorphin/kappa opioid receptor (KOR) system in the NAc was believed to promote aversion, while dopamine was viewed as interacting with reward behavior, and KOR activation was known to inhibit dopamine release. Recently, however, both the KOR and dopamine systems have, separately, been shown to have differential effects across the rostro-caudal axis of the NAc shell on hedonic responses. Whether or not this is due to interactions between KORs and dopamine, and if it extends to anxiety-like or approach-avoidance behaviors, remains to be determined. In this study, we examined in rats the relationship between the KOR and dopamine systems in both the rostral and caudal NAc shell using ex vivo fast scan cyclic voltammetry and the impact of KOR activation on affective behavior using exploration-based tasks. We report here that activation of KORs in the caudal NAc shell significantly inhibits dopamine release, stimulates rearing behavior in a novel environment, increases anxiety-like or avoidance behavior, and reduces locomotor activity. In contrast, activation of KORs in the rostral NAc shell inhibits dopamine release to a lesser extent and instead reduces anxiety-like behavior or increases approach behavior. Taken together, these results indicate that there is heterogeneity across the rostro-caudal axis of the NAc shell in the effects of KOR stimulation on affective behaviors, and they suggest that this might be due to differences in KOR control over dopamine release.

The kappa opioid receptor agonist U50,488H did not affect brain-stimulation reward while it elicited conditioned place aversion in mice.

OBJECTIVE: Selective kappa opioid receptor (KOR) agonists were shown to produce a dose-dependent depression of brain-stimulation reward (BSR) in the rat intracranial self-stimulation (ICSS) tests. However, limited studies using mice produced less conclusive results. Here the effects of U50,488H were re-examined on BSR in mice with a larger cohort of animals. RESULTS: Forty C57BL/6J male mice were implanted with the electrodes in medial forebrain bundle. About a week after surgery, mice were subject to ICSS training. Only eighteen passed the two-phase procedures, at which point they readily spun the wheels to obtain reinforcing effect of BSR, and were used for the ICSS tests. Compared with saline (s.c.), U50,488H (2 mg/kg, s.c.) did not have effects on the BSR thresholds within 1 h post-treatment, while it decreased the maximum wheel-spinning rates in a time-dependent manner. In contrast, cocaine (5 mg/kg, s.c.) decreased the BSR thresholds time-dependently without affecting the maximum wheel-spinning rates in the same cohort of mice, demonstrating the validity of our mouse ICSS models. For comparison, U50,488H (2 mg/kg, s.c.) induced significant conditioned place aversion (CPA) in a different cohort of mice without surgeries. Thus, ICSS may not be an appropriate test for KOR agonist-induced aversion in mice.

mTORC1 pathway is involved in the kappa opioid receptor activation-induced increase in excessive alcohol drinking in mice.

KOP-r agonist U50,488H produces strong aversion and anxiety/depression-like behaviors that enhance alcohol intake and promote alcohol seeking and relapse-like drinking in rodents. Mammalian target of rapamycin complex 1 (mTORC1) pathway in mouse striatum is highly involved in excessive alcohol intake and seeking, and in the U50,488H-induced conditioned place aversion. Therefore, we hypothesized that KOP-r activation increases alcohol consumption through the mTORC1 activation. This study focuses on: (1) how chronic excessive alcohol drinking (4-day drinking-in-the-dark paradigm followed by 3-week chronic intermittent access drinking paradigm [two-bottle choice, 24-h access every other day]) affected nuclear transcript levels of the mTORC1 pathway genes in mouse nucleus accumbens shell (NAcs), using transcriptome-wide RNA sequencing analysis; and (2) whether selective mTORC1 inhibitor rapamycin could alter excessive alcohol drinking and prevent U50,488H-promoted alcohol intake. Thirteen nuclear transcripts of mTORC1 pathway genes showed significant up-regulation in the NAcs, with two genes down-regulated, after excessive alcohol drinking, suggesting the mTORC1 pathway was profoundly disrupted. Single administration of rapamycin decreased alcohol drinking in a dose-dependent manner. U50,488H increased alcohol drinking, and pretreatment with rapamycin, at a dose lower than effective doses, blocked the U50,488H-promoted alcohol intake in a dose-dependent manner, indicating a mTORC1-mediated mechanism. Our results provide supportive and direct evidence relevant to the transcriptional profiling of the critical mTORC1 genes in mouse NAc shell: with functional and pharmacological effects of rapamycin, altered nuclear transcripts in the mTORC1 signaling pathway after excessive alcohol drinking may contribute to increased alcohol intake triggered by KOP-r activation.

Kappa opioid receptors in the bed nucleus of the stria terminalis regulate binge-like alcohol consumption in male and female mice.

Binge drinking is the most common pattern of excessive alcohol consumption and is a significant contributor to the development of Alcohol Use Disorder and dependence. Previous studies demonstrated involvement of kappa opioid receptors (KOR) in binge-like drinking in mice using the Drinking-in-the-Dark model. The current studies examined the role of KOR specifically in the bed nucleus of the stria terminals (BNST) in binge-like alcohol consumption in male and female mice. Direct administration of the long lasting KOR antagonist, nor-BNI, into the BNST decreased binge-like alcohol consumption and blood alcohol concentrations in male and female C57BL/6J mice. Similarly, direct nor-BNI administration into the BNST modestly reduced sucrose consumption and the suppression of fluid intake was not related to reduced locomotor activity. To further determine the role of KOR within the BNST on binge-like alcohol consumption, the KOR agonist U50,488 was administered systemically which resulted in a robust increase in alcohol intake. Microinjection of nor-BNI into the BNST blocked the high level of alcohol intake after systemic U50,488 challenge reducing intake and resultant blood alcohol concentrations. Together, these data suggest that KOR activity in the BNST contributes to binge-like alcohol consumption in both male and female mice. This article is part of the special issue on 'Neuropeptides'.

Behavioral Pharmacology of Novel Kappa Opioid Receptor Antagonists in Rats.

BACKGROUND: New treatments for stress-related disorders including depression, anxiety, and substance use disorder are greatly needed. Kappa opioid receptors are expressed in the central nervous system, including areas implicated in analgesia and affective state. Although kappa opioid receptor agonists share the antinociceptive effects of mu opioid receptor agonists, they also tend to produce negative affective states. In contrast, selective kappa opioid receptor antagonists have antidepressant- and anxiolytic-like effects, stimulating interest in their therapeutic potential. The prototypical kappa opioid receptor antagonists (e.g., norBNI, JDTic) have an exceptionally long duration of action that complicates their use in humans, particularly in tests to establish safety. This study was designed to test dose- and time-course effects of novel kappa opioid receptor antagonists with the goal of identifying short-acting lead compounds for future medication development. METHODS: We screened 2 novel, highly selective kappa opioid receptor antagonists (CYM-52220 and CYM-52288) with oral efficacy in the warm water tail flick assay in rats to determine initial dose and time course effects. For comparison, we tested existing kappa opioid receptor antagonists JDTic and LY-2456302 (also known as CERC-501 or JNJ-67953964). RESULTS: In the tail flick assay, the rank order of duration of action for the antagonists was LY-2456302 < CYM-52288 < CYM-52220 << JDTic. Furthermore, LY-2456302 blocked the depressive (anhedonia-producing) effects of the kappa opioid receptor agonist U50,488 in the intracranial self-stimulation paradigm, albeit at a higher dose than that needed for analgesic blockade in the tail flick assay. CONCLUSIONS: These results suggest that structurally diverse kappa opioid receptor antagonists can have short-acting effects and that LY-2456302 reduces anhedonia as measured in the intracranial self-stimulation test.

Females are less sensitive than males to the motivational- and dopamine-suppressing effects of kappa opioid receptor activation.

The neuropeptide dynorphin (DYN) activates kappa opioid receptors (KORs) in the brain to produce depressive-like states and decrease motivation. KOR-mediated suppression of dopamine release in the nucleus accumbens (NAc) is considered one underlying mechanism. We previously showed that, regardless of estrous cycle stage, female rats are less sensitive than males to KOR agonist-mediated decreases in motivation to respond for brain stimulation reward, measured with intracranial self-stimulation (ICSS). However, the explicit roles of KORs, circulating gonadal hormones, and their interaction with dopamine signaling in motivated behavior are not known. As such, we measured the effects of the KOR agonist U50,488 on ICSS stimulation thresholds before and after gonadectomy (or sham surgery). We found that ovariectomized females remained less sensitive than sham or castrated males to KOR-mediated decreases in brain stimulation reward, indicating that circulating gonadal hormones do not play a role. We used qRT-PCR to examine whether sex differences in gene expression in limbic brain regions are associated with behavioral sex differences. We found no sex differences in Pdyn or Oprk1 mRNA in the NAc and ventral tegmental area (VTA), but tyrosine hydroxylase (Th) mRNA was significantly higher in female compared to male VTA. To further explore sex-differences in KOR-mediated suppression of dopamine, we used fast scan cyclic voltammetry (FSCV) and demonstrated that U50,488 was less effective in suppressing evoked NAc dopamine release in females compared to males. These data raise the possibility that females are protected from KOR-mediated decreases in motivation by an increased capacity to produce and release dopamine.

κ-opioid receptor activation protects against myocardial ischemia-reperfusion injury via AMPK/Akt/eNOS signaling activation.

This study aims to investigate the effect of κ-opioid receptor activation on myocardial ischemia and reperfusion(I/R) injury and elucidate the underlying mechanisms. Myocardial I/R rat model and simulated I/R cardiomyocytes model were established. In vivo study showed that U50,488 H improved cardiac function, reduced myocardial infarct size and serum cTnT significantly. The effect of U50,488 H was abolished by nor-BNI(a κ-opioid receptor antagonist), Compound C(an AMPK inhibitor), Akt inhibitor and L-NAME(an eNOS inhibitor). AICAR, an AMPK activator, mimicked the effect of U50,488 H. U50,488 H up-regulated p-AMPK, p-Akt, and p-eNOS, which were abolished by nor-BNI. AICAR increased p-Akt and p-eNOS, which was abolished by Compound C. In vitro study showed that U50,488 H increased p-AMPK, p-Akt, and p-eNOS via κ-OR activation. The effect of U50,488 H on p-AMPK was abolished by compound C, but not Akt inhibitor and L-NAME. The effect of U50,488 H on p-Akt was abolished by compound C and Akt inhibitor, but not L-NAME. AICAR increased p-Akt and p-eNOS, which was abolished by Akt inhibitor, but not L-NAME. U50,488 H and AICAR also increased the viability of cardiomyocytes subjected to simulated I/R, the effects of U50,488 H and AICAR were blocked by nor-BNI, Compound C, Akt inhibitor, and L-NAME, respectively. In conclusion, κ-OR activation confers cardioprotection via AMPK/Akt/eNOS signaling.

Activation of kappa opioid receptors in the dorsal raphe have sex dependent effects on social behavior in California mice.

Kappa opioid receptor activation has been linked to stress and anxiety behavior, thus leading to kappa antagonists being popularized in research as potential anxiolytics. However, while these findings may hold true in standard models, the neuromodulatory effects of social defeat may change the behavioral outcome of kappa opioid receptor activation. Previous research has shown that social defeat can lead to hyperactivity of serotonergic neurons in the dorsal raphe nucleus, and that inhibition of this increase blocks the social deficits caused by defeat. Kappa opioid receptor activation in the dorsal raphe nucleus works to decrease serotonergic activity. We injected the kappa opioid receptor U50,488 directly into the dorsal raphe nucleus of male and female, defeat and control adult California mice. Here we show evidence that U50,488 induces anxiety behavior in control male California mice, but helps relieve it in defeated males. Consistent with previous literature, we find little effect in females adding evidence that there are marked and important sex differences in the kappa opioid system.

Quaternary ammonium salt of U50,488H elicits protective effects against hypoxic pulmonary hypertension.

The present study aimed to investigate the role of quaternary ammonium salt of U50,488H (Q-U50,488H) in hypoxic pulmonary hypertension (HPH) and underlying mechanisms involved. A HPH animal model was established in rats under hypoxia and the mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) were measured. Relaxation of the pulmonary artery in response to Q-U50,488H was determined. In addition, expression and activity of endothelial nitric oxide (NO) synthase (eNOS) and inducible NO synthase (iNOS) with NO content, Akt expression, total antioxidant capacity (T-AOC), and gp91phox were evaluated. Cell viability was determined by the cell counting kit-8 (CCK-8) assay. We demonstrated that both the molecular weight and solubility of Q-U50,488H were higher than that of U50,488H. Q-U50,488H reduced mPAP and RVP and prevented the development of HPH. Moreover, Q-U50,488H relaxed the pulmonary arteries from both normal and HPH rats in a time-dependent manner. Under hypoxic conditions, Q-U50,488H significantly increased Akt phosphorylation, eNOS phosphorylation, NO content in serum, and T-AOC in pulmonary arteries of HPH rats. In addition, the activity of eNOS was elevated, but the activity of iNOS was reduced when Q-U50,488H was given under hypoxia. Q-U50,488H significantly counteracted the increase of gp91phox expression in pulmonary arteries under hypoxia. In addition, in vitro studies suggested that Q-U50,488H inhibited pulmonary artery smooth muscle cells (PASMCs) proliferation under hypoxic conditions and that the effects of Q-U50,488H were blocked by nor-binaltorphimine (nor-BNI). Thus, our results provided evidence that Q-U50,488H plays a protective role against HPH via κ-opioid receptor stimulation.

U50: A New Metric for Measuring Assembly Output Based on Non-Overlapping, Target-Specific Contigs.

Advances in next-generation sequencing technologies enable routine genome sequencing, generating millions of short reads. A crucial step for full genome analysis is the de novo assembly, and currently, performance of different assembly methods is measured by a metric called N50. However, the N50 value can produce skewed, inaccurate results when complex data are analyzed, especially for viral and microbial datasets. To provide a better assessment of assembly output, we developed a new metric called U50. The U50 identifies unique, target-specific contigs by using a reference genome as baseline, aiming at circumventing some limitations that are inherent to the N50 metric. Specifically, the U50 program removes overlapping sequence of multiple contigs by utilizing a mask array, so the performance of the assembly is only measured by unique contigs. We compared simulated and real datasets by using U50 and N50, and our results demonstrated that U50 has the following advantages over N50: (1) reducing erroneously large N50 values due to a poor assembly, (2) eliminating overinflated N50 values caused by large measurements from overlapping contigs, (3) eliminating diminished N50 values caused by an abundance of small contigs, and (4) allowing comparisons across different platforms or samples based on the new percentage-based metric UG50%. The use of the U50 metric allows for a more accurate measure of assembly performance by analyzing only the unique, non-overlapping contigs. In addition, most viral and microbial sequencing have high background noise (i.e., host and other non-targets), which contributes to having a skewed, misrepresented N50 value-this is corrected by U50. Also, the UG50% can be used to compare assembly results from different samples or studies, the cross-comparisons of which cannot be performed with N50.

Nicotine Increases Alcohol Intake in Adolescent Male Rats.

Background: Use of alcohol and tobacco, the two most concurrently abused drugs, typically first occurs during adolescence. Yet, there have been no systematic analyses of ethanol (EtOH) and nicotine (Nic) interactions during adolescence. Recent animal studies report that kappa-opioid (KOR) receptor activation mediates age differences in drug reinforcement. Our hypothesis is that concurrent self-administration of EtOH and Nic will be greater in adolescent rats because of age differences in KOR function. Furthermore, exposure to alcohol and nicotine during adolescence has been reported to increase EtOH intake in adulthood. We performed a longitudinal animal study and hypothesized adolescent rats allowed to self-administer nicotine would drink more alcohol as adults. Methods: Adolescent, postnatal day (P)32, and adult (P90) male and female Sprague-Dawley rats were allowed to self-administer EtOH, Nic, or a combination of both, EtOH+Nic, in an intravenous self-administration paradigm. The role of KOR was pharmacologically evaluated with the KOR antagonist, norbinaltorphamine (norBNI) and with the KOR agonist, U50,488H. Alcohol drinking was subsequently evaluated with male rats in a drinking in the dark (DID), 2-bottle choice test. Results: Concurrent Nic increased EtOH intake in adolescent males, but not in adults or females. Pharmacological blockade of KOR with norBNI robustly increased EtOH+Nic self-administration in adult male rats, but had no effect with female rats. Lastly, in our longitudinal study with male rats, we found prior self-administration of Nic or EtOH+Nic during adolescence increased subsequent oral EtOH intake, whereas prior self-administration of EtOH alone in adults increased subsequent EtOH drinking. Conclusions: There are major age- and sex-differences in the reinforcing effects of EtOH+Nic. Adolescent males are sensitive to the reinforcing interactions of the two drugs, whereas this effect is inhibited by KOR activation in male adults. Nicotine self-administration in adolescent males also increased subsequent oral EtOH intake. These findings suggest that brain mechanisms underlying the reinforcing effects of EtOH and nicotine are both age- and sex-dependent, and that tobacco or e-cigarette use may increase the vulnerability of teenage boys to alcohol abuse.