Does GEC1 Enhance Expression and Forward Trafficking of the Kappa Opioid Receptor (KOR) via Its Ability to Interact with NSF Directly?

We reported previously that GEC1 (glandular epithelial cell 1), a member of microtubule-associated proteins (MAPs), interacted directly with the C-tail of KOR (KCT) and tubulin and enhanced cell surface expression of KOR in CHO cells by facilitating its trafficking along the export pathway. Two GEC1 analogs (GABARAP and GATE16) were also shown to increase KOR expression. In addition, to understand the underlying mechanism, we demonstrated that N-ethylmaleimide-sensitive factor (NSF), an essential component for membrane fusion, co-immunoprecipitated with GEC1 from brain extracts. In this study, using pull-down techniques, we have found that (1) GEC1 interacts with NSF directly and prefers the ADP-bound NSF to the ATP-bound NSF; (2) D1 and/or D2 domain(s) of NSF interact with GEC1, but the N domain of NSF does not; (3) NSF does not interact with KCT directly, but forms a protein complex with KCT via GEC1; (4) NSF and/or α-SNAP do not affect KCT-GEC1 interaction. Thus, GEC1 (vs the α-SNAP/SNAREs complex) binds to NSF in distinctive ways in terms of the ADP- or ATP-bound form and domains of NSF involved. In conclusion, GEC1 may, via its direct interactions with KOR, NSF, and tubulin, enhance trafficking and fusion of KOR-containing vesicles selectively along the export pathway, which leads to increase in surface expression of KOR. GABARAP and GATE16 may enhance KOR expression in a similar way.

Fundamentals of the Dynorphins/Kappa Opioid Receptor System: From Distribution to Signaling and Function.

This chapter provides a general introduction to the dynorphins (DYNs)/kappa opioid receptor (KOR) system, including DYN peptides, neuroanatomy of the DYNs/KOR system, cellular signaling, and in vivo behavioral effects of KOR activation and inhibition. It is intended to serve as a primer for the book and to provide a basic background for the chapters in the book.

Considerations on Using Antibodies for Studying the Dynorphins/Kappa Opioid Receptor System.

Antibodies are important tools for protein and peptide research, including for the kappa opioid receptor (KOR) and dynorphins (Dyns). Well-characterized antibodies are essential for rigorous and reproducible research. However, lack of validation of antibody specificity has been thought to contribute significantly to the reproducibility crisis in biomedical research. Since 2003, many scientific journals have required documentation of validation of antibody specificity and use of knockout mouse tissues as a negative control is strongly recommended. Lack of specificity of antibodies against many G protein-coupled receptors (GPCRs) after extensive testing has been well-documented, but antibodies generated against partial sequences of the KOR have not been similarly investigated. For the dynorphins, differential processing has been described in distinct brain areas, resulting in controversial findings in immunohistochemistry (IHC) when different antibodies were used. In this chapter, we summarized accepted approaches for validation of antibody specificity. We discussed two KOR antibodies most commonly used in IHC and described generation and characterization of KOR antibodies and phospho-KOR specific antibodies in western blotting or immunoblotting (IB). In addition, applying antibodies targeting prodynorphin or mature dynorphin A illustrates the diversity of results obtained regarding the distribution of dynorphins in distinct brain areas.

Verifying the role of 3-hydroxy of 17-cyclopropylmethyl-4,5α-epoxy-3,14ß-dihydroxy-6ß-[(4'-pyridyl) carboxamido]morphinan derivatives via their binding affinity and selectivity profiles on opioid receptors.

In the present study, the role of 3-hydroxy group of a series of epoxymorphinan derivatives in their binding affinity and selectivity profiles toward the opioid receptors (ORs) has been investigated. It was found that the 3-hydroxy group was crucial for the binding affinity of these derivatives for all three ORs due to the fact that all the analogues 1a-e exhibited significantly higher binding affinities compared to their counterpart 3-dehydroxy ones 6a-e. Meanwhile most compounds carrying the 3-hydroxy group possessed similar selectivity profiles for the kappa opioid receptor over the mu opioid receptor as their corresponding 3-dehydroxy derivatives. [35S]-GTPγS functional assay results indicated that the 3-hydroxy group of these epoxymorphinan derivatives was important for maintaining their potency on the ORs with various effects. Further molecular modeling studies helped comprehend the remarkably different binding affinity and functional profiles between compound 1c (NCP) and its 3-dehydroxy analogue 6c.

Agonist-Dependent and -Independent κ Opioid Receptor Phosphorylation: Distinct Phosphorylation Patterns and Different Cellular Outcomes.

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse κ opioid receptor (KOPR) at residues S356, T357, T363, and S369. Here, we found that agonist (U50,488H)-dependent KOPR phosphorylation at all the residues was mediated by Gi/o α proteins and multiple protein kinases [GRK2, GRK3, GRK5, GRK6 and protein kinase C (PKC)]. In addition, PKC activation by phorbol ester induced agonist-independent KOPR phosphorylation. Compared with U50,488H, PKC activation promoted much higher S356/T357 phosphorylation, much lower T363 phosphorylation, and similar levels of S369 phosphorylation. After U50,488H treatment, GRKs, but not PKC, were involved in agonist-induced KOPR internalization. In contrast, PKC activation caused a lower level of agonist-independent KOPR internalization, compared with U50,488H. U50,488H-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was G protein-, but not ß-arrestin-, dependent. After U50,488H treatment, GRK-mediated, but not PKC-mediated, KOPR phosphorylation followed by ß-arrestin recruitment desensitized U50,488H-induced ERK1/2 response. Therefore, agonist-dependent (GRK- and PKC-mediated) and agonist-independent (PKC-promoted) KOPR phosphorylations show distinct phosphorylation patterns, leading to diverse cellular outcomes.

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated following U50,488H and etorphine respectively. Thus KOPR internalization requires receptor phosphorylation above a certain threshold, and higher-order KOPR phosphorylation may be disproportionally important.

14-3-3ζ Protein regulates anterograde transport of the human κ-opioid receptor (hKOPR).

By proteomic analysis, we found that 14-3-3ζ was one of the proteins co-immunoprecipitated with human κ-opioid receptor (hKOPR) from extracts of solubilized Neuro2A cells stably expressing FLAG-hKOPR (N2A-FLAG-hKOPR cells). 14-3-3 proteins are a family of conserved regulatory molecules in eukaryotic cells, where they participate in signal transduction, metabolism, and membrane protein transport. 14-3-3ζ co-localized with the hKOPR in N2A cells. The hKOPR C-tail interacted with 14-3-3ζ in rat brain extracts and bound directly to purified 14-3-3ζ as demonstrated by pulldown techniques. 14-3-3ζ siRNA decreased expression of the hKOPR in N2A-FLAG-hKOPR cells and cultured primary cortical neurons of E19 rats by ~25% as determined by immunoblotting, ligand binding, and flow cytometry. The effect of 14-3-3ζ siRNA was reversed by overexpression of 14-3-3ζ. Expression of the 14-3-3 scavenger protein pGpLI-R18 also decreased hKOPR expression. 14-3-3ζ siRNA did not change expressions of the hDOPR and rMOPR in N2A cells. Pulse-chase study showed that 14-3-3ζ siRNA decreased the amount of mature hKOPR but did not change the rate of maturation or stability of hKOPR protein. Mutations of R354A/S358A in the putative 14-3-3 interaction motif (354)RQSTS(358) in the hKOPR C-tail reduced interaction of the hKOPR with 14-3-3ζ and abolished the effect of 14-3-3ζ knockdown on hKOPR expression. Mutation of the endoplasmic reticulum retention motif (359)RVR adjacent to the 14-3-3 interaction motif in the hKOPR C-tail decreased interaction of coatomer protein I (COPI) with the hKOPR and abolished 14-3-3ζ-mediated regulation of hKOPR expression. 14-3-3ζ knockdown increased association of COPI with the hKOPR. These results suggest that 14-3-3ζ promotes expression of the hKOPR by inhibiting COPI and RVR motif-mediated endoplasmic reticulum localization machinery.

A common single nucleotide polymorphism A118G of the µ opioid receptor alters its N-glycosylation and protein stability.

The A118G SNP (single nucleotide polymorphism) of the hMOPR [human MOPR (µ opioid receptor)] gene OPRM1 results in an amino acid substitution (N40D). Subjects homozygous for the 118G allele have been reported to require higher morphine doses to achieve adequate analgesia, and the 118G allele is more prevalent among drug abusers. However, changes in the MOPR protein associated with this SNP are unknown. Using a knockin mouse model (G/G mice; mice homozygous for the 112G allele of MOPR) that possesses the equivalent nucleotide/amino acid substitution (A112G; N38D) of the A118G SNP in the hMOPR gene, we investigated the N-linked glycosylation status of thalamic and striatal MOPR in G/G mice compared with A/A mice (wild-type mice homozygous for the 112A allele of MOPR). The molecular mass of MOPR determined by immunoblotting was lower in G/G mice than in A/A mice. Following treatment with peptide N-glycosidase F, which removes all N-linked glycans, both MOPR variants had an identical molecular mass, indicating that this discrepancy was due to a lower level of N-glycosylation of the MOPR in G/G mice. In Chinese-hamster ovary cells stably expressing hMOPRs, 118G/Asp40-hMOPR had a lower molecular mass than 118A/Asn40-hMOPR, which was similarly due to differential N-glycosylation. Pulse-chase studies revealed that the half-life of the mature form of 118G/Asp40-hMOPR (~12 h) was shorter than that of 118A/Asn40-hMOPR (~28 h). Thus the A118G SNP reduces MOPR N-glycosylation and protein stability.

Paternal morphine exposure enhances morphine self-administration and induces region-specific neural adaptations in reward-related brain regions of male offspring.

Background: A growing body of preclinical studies report that preconceptional experiences can have a profound and long-lasting impact on adult offspring behavior and physiology. However, less is known about paternal drug exposure and its effects on reward sensitivity in the next generation. Methods: Adult male rats self-administered morphine for 65 days; controls received saline. Sires were bred to drug-naïve dams to produce first-generation (F1) offspring. Morphine, cocaine, and nicotine self-administration were measured in adult F1 progeny. Molecular correlates of addiction-like behaviors were measured in reward-related brain regions of drug naïve F1 offspring. Results: Male, but not female offspring produced by morphine-exposed sires exhibited dose-dependent increased morphine self-administration and increased motivation to earn morphine infusions under a progressive ratio schedule of reinforcement. This phenotype was drug-specific as self-administration of cocaine, nicotine, and sucrose were not altered by paternal morphine history. The male offspring of morphine-exposed sires also had increased expression of mu-opioid receptors in the ventral tegmental area but not in the nucleus accumbens. Conclusions: Paternal morphine exposure increased morphine addiction-like behavioral vulnerability in male but not female progeny. This phenotype is likely driven by long-lasting neural adaptations within the reward neural brain pathways.

A suite of engineered mice for interrogating psychedelic drug actions.

Psychedelic drugs like lysergic acid diethylamide (LSD) and psilocybin have emerged as potentially transformative therapeutics for many neuropsychiatric diseases, including depression, anxiety, post-traumatic stress disorder, migraine, and cluster headaches. LSD and psilocybin exert their psychedelic effects via activation of the 5-hydroxytryptamine 2A receptor (HTR2A). Here we provide a suite of engineered mice useful for clarifying the role of HTR2A and HTR2A-expressing neurons in psychedelic drug actions. We first generated Htr2a-EGFP-CT-IRES-CreERT2 mice (CT:C-terminus) to independently identify both HTR2A-EGFP-CT receptors and HTR2A-containing cells thereby providing a detailed anatomical map of HTR2A and identifying cell types that express HTR2A. We also generated a humanized Htr2a mouse line and an additional constitutive Htr2A-Cre mouse line. Psychedelics induced a variety of known behavioral changes in our mice validating their utility for behavioral studies. Finally, electrophysiology studies revealed that extracellular 5-HT elicited a HTR2A-mediated robust increase in firing of genetically-identified pyramidal neurons--consistent with a plasma membrane localization and mode of action. These mouse lines represent invaluable tools for elucidating the molecular, cellular, pharmacological, physiological, behavioral, and other actions of psychedelic drugs in vivo.

Effects of C-terminal modifications of GEC1 protein and gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), two microtubule-associated proteins, on kappa opioid receptor expression.

We demonstrated previously that GEC1, a member of the microtubule-associated protein (MAP) family, bound to the human κ opioid receptor (hKOPR) and promoted hKOPR cell surface expression by facilitating its trafficking along the secretory pathway. GABA(A) receptor-associated protein (GABARAP), a GEC1 analog, also enhanced KOPR expression, but to a lesser extent. The MAP family proteins undergo cleavage of their C-terminal residue(s), and the exposed conserved glycine forms conjugates with phosphatidylethanolamine, which associate with membranes. Here, we examined whether such modifications were required for GEC1 and GABARAP to enhance hKOPR expression. When transiently transfected into CHO or Neuro2A cells, GEC1 and GABARAP were cleaved at the C termini. G116A mutation alone or combined with deletion of Lys(117) in GEC1 (GEC1-A) or Leu(117) in GABARAP (GABARAP-A) blocked their C-terminal cleavage, indicating that the conserved Gly(116) is necessary for C-terminal modification. The two GEC1 mutants enhanced hKOPR expression to similar extents as the wild-type GEC1; however, the two GABARAP mutants did not. Immunofluorescence studies showed that HA-GEC1, HA-GEC1-A, and HA-GABARAP were distributed in a punctate manner and co-localized with KOPR-EGFP in the Golgi apparatus, whereas HA-GABARAP-A did not. Pulldown assay of GST-KOPR-C-tail with HA-GEC1 or HA-GABARAP revealed that GEC1 had stronger association with KOPR-C-tail than GABARAP. These results suggest that because of its stronger binding for hKOPR, GEC1 is able to be recruited by hKOPR sufficiently without membrane association via its C-terminal modification; however, due to its weaker affinity for the hKOPR, GABARAP appears to require C-terminal modifications to enhance KOPR expression.

Agonist-Promoted Phosphorylation and Internalization of the Kappa Opioid Receptor in Mouse Brains: Lack of Connection With Conditioned Place Aversion.

Selective kappa opioid receptor (KOR) agonists are promising antipruritic agents and analgesics. However, clinical development of KOR agonists has been limited by side effects, including psychotomimetic effects, dysphoria, and sedation, except for nalfurafine, and recently. CR845 (difelikefalin). Activation of KOR elicits G protein- and ß-arrestin-mediated signaling. KOR-induced analgesic and antipruritic effects are mediated by G protein signaling. However, different results have been reported as to whether conditioned place aversion (CPA) induced by KOR agonists is mediated by ß-arrestin signaling. In this study, we examined in male mice if there was a connection between agonist-promoted CPA and KOR phosphorylation and internalization, proxies for ß-arrestin recruitment in vivo using four KOR agonists. Herein, we demonstrated that at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, promoted KOR phosphorylation at T363 and S369 in mouse brains, as detected by immunoblotting with phospho-KOR-specific antibodies. In addition, at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, caused KOR internalization in the ventral tegmental area of a mutant mouse line expressing a fusion protein of KOR conjugated at the C-terminus with tdTomato (KtdT). We have reported previously that the KOR agonists U50,488H and methoxymethyl salvinorin B (MOM-SalB) cause CPA, whereas nalfurafine and 42B do not, at doses effective for analgesic and antiscratch effects. Taken together, these data reveal a lack of connection between agonist-promoted KOR-mediated CPA with agonist-induced KOR phosphorylation and internalization in male mice.

Agonist-promoted kappa opioid receptor (KOR) phosphorylation has behavioral endpoint-dependent and sex-specific effects.

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (mKOR) in vitro at four residues in the C-terminal domain. In this study, we generated a mutant mouse line in which all the four residues were mutated to Ala (K4A) to examine the in vivo functional significance of agonist-induced KOR phosphorylation. U50,488H promoted KOR phosphorylation in brains of the wildtype (WT), but not K4A, male and female mice. Autoradiography of [3H] 69,593 binding to KOR in brain sections showed that WT and K4A mice had similar KOR distribution and expression levels in brain regions without sex differences. In K4A mice, U50,488H inhibited compound 48/80-induced scratching and attenuated novelty-induced hyperlocomotion to similar extents as in WT mice without sex differences. Interestingly, repeated pretreatment with U50,488H (80 mg/kg, s.c.) resulted in profound tolerance to the anti-scratch effects of U50,488H (5 mg/kg, s.c.) in WT mice of both sexes and female K4A mice, while in male K4A mice tolerance was attenuated. Moreover, U50,488H (2 mg/kg) induced conditioned place aversion (CPA) in WT mice of both sexes and male K4A mice, but not in female K4A mice. In contrast, U50,488H (5 mg/kg) caused CPA in male, but not female, mice, regardless of genotype. Thus, agonist-promoted KOR phosphorylation plays important roles in U50,488H-induced tolerance and CPA in a sex-dependent manner, without affecting acute U50,488H-induced anti-pruritic and hypo-locomotor effects. These results are the first to demonstrate sex differences in the effects of GPCR phosphorylation on the GPCR-mediated behaviors.

Reactivation of cocaine contextual memory engages mechanistic target of rapamycin/S6 kinase 1 signaling.

Mechanistic target of rapamycin (mTOR) C1 and its downstream effectors have been implicated in synaptic plasticity and memory. Our prior work demonstrated that reactivation of cocaine memory engages a signaling pathway consisting of Akt, glycogen synthase kinase-3ß (GSK3ß), and mTORC1. The present study sought to identify other components of mTORC1 signaling involved in the reconsolidation of cocaine contextual memory, including eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interactions, p70 S6 kinase polypeptide 1 (p70S6K, S6K1) activity, and activity-regulated cytoskeleton (Arc) expression. Cocaine contextual memory was established in adult CD-1 mice using conditioned place preference. After cocaine place preference was established, mice were briefly re-exposed to the cocaine-paired context to reactivate the cocaine memory and brains examined. Western blot analysis showed that phosphorylation of the mTORC1 target, p70S6K, in nucleus accumbens and hippocampus was enhanced 60 min following reactivation of cocaine memories. Inhibition of mTORC1 with systemic administration of rapamycin or inhibition of p70S6K with systemic PF-4708671 after reactivation of cocaine contextual memory abolished the established cocaine place preference. Immunoprecipitation assays showed that reactivation of cocaine memory did not affect eIF4E-eIF4G interactions in nucleus accumbens or hippocampus. Levels of Arc mRNA were significantly elevated 60 and 120 min after cocaine memory reactivation and returned to baseline 24 h later. These findings demonstrate that mTORC1 and p70S6K are required for reconsolidation of cocaine contextual memory.

A novel Oprm1-Cre mouse maintains endogenous expression, function and enables detailed molecular characterization of µ-opioid receptor cells.

Key targets of both the therapeutic and abused properties of opioids are µ-opioid receptors (MORs). Despite years of research investigating the biochemistry and signal transduction pathways associated with MOR activation, we do not fully understand the cellular mechanisms underlying opioid addiction. Given that addictive opioids such as morphine, oxycodone, heroin, and fentanyl all activate MORs, and current therapies such as naloxone and buprenorphine block this activation, the availability of tools to mechanistically investigate opioid-mediated cellular and behavioral phenotypes are necessary. Therefore, we derived, validated, and applied a novel MOR-specific Cre mouse line, inserting a T2A cleavable peptide sequence and the Cre coding sequence into the MOR 3'UTR. Importantly, this line shows specificity and fidelity of MOR expression throughout the brain and with respect to function, there were no differences in behavioral responses to morphine when compared to wild type mice, nor are there any alterations in Oprm1 gene expression or receptor density. To assess Cre recombinase activity, MOR-Cre mice were crossed with the floxed GFP-reporters, RosaLSLSun1-sfGFP or RosaLSL-GFP-L10a. The latter allowed for cell type specific RNA sequencing via TRAP (Translating Ribosome Affinity Purification) of striatal MOR+ neurons following opioid withdrawal. The breadth of utility of this new tool will greatly facilitate the study of opioid biology under varying conditions.

Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the mouse brain, we observed PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways at 5 min; stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1 at 30 min. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.

Comparison of Pharmacological Properties between the Kappa Opioid Receptor Agonist Nalfurafine and 42B, Its 3-Dehydroxy Analogue: Disconnect between in Vitro Agonist Bias and in Vivo Pharmacological Effects.

Nalfurafine, a moderately selective kappa opioid receptor (KOR) agonist, is used in Japan for treatment of itch without causing dysphoria or psychotomimesis. Here we characterized the pharmacology of compound 42B, a 3-dehydroxy analogue of nalfurafine and compared with that of nalfurafine. Nalfurafine and 42B acted as full KOR agonists and partial µ opioid receptor (MOR) agonists, but 42B showed much lower potency for both receptors and lower KOR/MOR selectivity, different from previous reports. Molecular modeling revealed that water-mediated hydrogen-bond formation between 3-OH of nalfurafine and KOR accounted for its higher KOR potency than 42B. The higher potency of both at KOR over MOR may be due to hydrogen-bond formation between nonconserved Y7.35 of KOR and their carbonyl groups. Both showed modest G protein signaling biases. In mice, like nalfurafine, 42B produced antinociceptive and antiscratch effects and did not cause conditioned place aversion (CPA) in the effective dose ranges. Unlike nalfurafine, 42B caused motor incoordination and hypolocomotion. As both agonists showed G protein biases, yet produced different effects on locomotor activity and motor incoordination, the findings and those in the literature suggest caution in correlating in vitro biochemical data with in vivo behavior effects. The factors contributing to the disconnect, including pharmacodynamic and pharmacokinetic issues, are discussed. In addition, our results suggest that among the KOR-induced adverse behaviors, CPA can be separated from motor incoordination and hypolocomotion.

Characterization of a Knock-In Mouse Line Expressing a Fusion Protein of κ Opioid Receptor Conjugated with tdTomato: 3-Dimensional Brain Imaging via CLARITY.

Activation of κ opioid receptor (KOR) produces analgesia, antipruritic effect, sedation and dysphoria. To characterize neuroanatomy of KOR at high resolutions and circumvent issues of specificity of KOR antibodies, we generated a knock-in mouse line expressing KOR fused at the C terminus with the fluorescent protein tdTomato (KtdT). The selective KOR agonist U50,488H caused anti-scratch effect and hypolocomotion, indicating intact KOR neuronal circuitries. Clearing of brains with CLARITY revealed three-dimensional (3-D) images of distribution of KOR, and any G-protein-coupled receptors, for the first time. 3-D brain images of KtdT and immunohistochemistry (IHC) on brain sections with antibodies against tdTomato show similar distribution to that of autoradiography of [3H]U69,593 binding to KOR in wild-type mice. KtdT was observed in regions involved in reward and aversion, pain modulation, and neuroendocrine regulation. KOR is present in several areas with unknown roles, including the claustrum (CLA), dorsal endopiriform nucleus, paraventricular nucleus of the thalamus (PVT), lateral habenula (LHb), and substantia nigra pars reticulata (SNr), which are discussed. Prominent KtdT-containing fibers were observed to project from caudate putamen (CP) and nucleus accumbens (ACB) to substantia innominata (SI) and SNr. Double IHC revealed co-localization of KtdT with tyrosine hydroxylase (TH) in brain regions, including CP, ACB, and ventral tegmental area (VTA). KOR was visualized at the cellular level, such as co-localization with TH and agonist-induced KOR translocation into intracellular space in some VTA neurons. These mice thus represent a powerful and heretofore unparalleled tool for neuroanatomy of KOR at both the 3-D and cellular levels.

Structure and functions of the ventral tube of the clover springtail Sminthurus viridis (Collembola: Sminthuridae).

Springtails (Collembola) are unique in Hexapoda for bearing a ventral tube (collophore) on the first abdominal segment. Although numerous studies have been conducted on the functions of the ventral tube, its fine structure has not been thoroughly elucidated to date. In this paper, we observed the jumping behavior of the clover springtail Sminthurus viridis (Linnaeus, 1758) and dissected the ventral tube using light microscopy to elucidate the fine structure and the possible function of the ventral tube. The results show that a pair of eversible vesicles can be extended from the apical opening of the ventral tube. The eversible vesicles are furnished with numerous small papillae, and can be divided into a basal part and a distal part. The eversible vesicles have a central lumen connected to the tiny papillae and leading to the body cavity. The eversible vesicles can reach any part of the body, and may serve as following functions: (a) absorbing moisture; (b) uptaking water; (c) cleaning the body surface; and (d) fastening the body on a smooth surface.

Coumarin-based Hg2+ fluorescent probe: Fluorescence turn-on detection for Hg2+ bioimaging in living cells and zebrafish.

The need in developing fluorescent probes for trace metal ion detection in biological samples has been an important issue. Herein, a reaction-based fluorescent probe PIC containing a perimidine moiety was designed and synthesized for Hg2+ detection. The probe can selectively distinguish Hg2+ with 42-fold fluorescent enhancement from the other metal ions at physiological pH. This probe can detect Hg2+ with the detection limit of 1.08 µM. The sensor PIC can be applied to real-time detection of Hg2+ in cells with blue emission.